Characterizing the reproductive transcriptomic correlates of acute dehydration in males in the desert-adapted rodent, Peromyscus eremicus

Background The understanding of genomic and physiological mechanisms related to how organisms living in extreme environments survive and reproduce is an outstanding question facing evolutionary and organismal biologists. One interesting example of adaptation is related to the survival of mammals in deserts, where extreme water limitation is common. Research on desert rodent adaptations has focused predominantly on adaptations related to surviving dehydration, while potential reproductive physiology adaptations for acute and chronic dehydration have been relatively neglected. This study aims to explore the reproductive consequences of acute dehydration by utilizing RNAseq data in the desert-specialized cactus mouse (Peromyscus eremicus). Results We exposed 22 male cactus mice to either acute dehydration or control (fully hydrated) treatment conditions, quasimapped testes-derived reads to a cactus mouse testes transcriptome, and then evaluated patterns of differential transcript and gene expression. Following statistical evaluation with multiple analytical pipelines, nine genes were consistently differentially expressed between the hydrated and dehydrated mice. We hypothesized that male cactus mice would exhibit minimal reproductive responses to dehydration; therefore, this low number of differentially expressed genes between treatments aligns with current perceptions of this species’ extreme desert specialization. However, these differentially expressed genes include Insulin-like 3 (Insl3), a regulator of male fertility and testes descent, as well as the solute carriers Slc45a3 and Slc38a5, which are membrane transport proteins that may facilitate osmoregulation. Conclusions These results suggest that in male cactus mice, acute dehydration may be linked to reproductive modulation via Insl3, but not through gene expression differences in the subset of other a priori tested reproductive hormones. Although water availability is a reproductive cue in desert-rodents exposed to chronic drought, potential reproductive modification via Insl3 in response to acute water-limitation is a result which is unexpected in an animal capable of surviving and successfully reproducing year-round without available external water sources. Indeed, this work highlights the critical need for integrative research that examines every facet of organismal adaptation, particularly in light of global climate change, which is predicted, amongst other things, to increase climate variability, thereby exposing desert animals more frequently to the acute drought conditions explored here. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3840-1) contains supplementary material, which is available to authorized users.

organismal biologists. One interesting example of adaptation is related to the survival of mammals in 23 deserts, where extreme water limitation is common. Research on desert rodent adaptations has focused 24 predominantly on adaptations related to surviving dehydration, while potential reproductive physiology 25 adaptations for acute and chronic dehydration have been relatively neglected. This study aims to explore 26 the reproductive consequences of acute dehydration by utilizing RNAseq data in the desert-specialized 27 cactus mouse (Peromyscus eremicus). Specifically, we exposed 22 male cactus mice to either acute 28 dehydration or control (fully hydrated) treatment conditions, quasimapped testes-derived reads to a 29 cactus mouse testes transcriptome, and then evaluated patterns of differential transcript and gene 30 expression. Following statistical evaluation with multiple analytical pipelines, nine genes were 31 consistently differentially expressed between the hydrated and dehydrated mice. We hypothesized that 32 male cactus mice would exhibit minimal reproductive responses to dehydration; therefore, this low 33 number of differentially expressed genes between treatments aligns with current perceptions of this 34 species' extreme desert specialization. However, these differentially expressed genes include Insulin-35 like 3 (Insl3), a regulator of male fertility and testes descent, as well as the solute carriers Slc45a3 and 36 Slc38a5, which are membrane transport proteins that may facilitate osmoregulation. Together, these 37 results suggest that in male cactus mice, acute dehydration may be linked to reproductive modulation via 38 Insl3, but not through gene expression differences in the subset of other a priori tested reproductive 39 hormones. Although water availability is a reproductive cue in desert-rodents exposed to chronic 40 drought, potential reproductive modification via Insl3 in response to acute water-limitation is a result 41 which is unexpected in an animal capable of surviving and successfully reproducing year-round without 42 We assembled a testes transcriptome from a single reproductively mature male using the de novo 190 transcriptome protocol described previously (MacManes, 2016). The testes transcripts were assembled 191 with alternative methodologies utilizing several optimization procedures to produce a high-quality 192 transcriptome; however, the permutations of this assembly process are described extensively elsewhere 193 (MacManes, 2016; Kordonowy and MacManes, 2016). The testes transcriptome we selected was 194 constructed as described below. The raw reads were error corrected using Rcorrector version 1.0.1 195 (Song & Florea, 2015), then subjected to quality trimming (using a threshold of PHRED <2, as per 196 MacManes 2014) and adapter removal using Skewer version 0.1.127 (Jiang et al, 2014). These reads 197 were then assembled in the de novo transcriptome assembler BinPacker version 1.0 (Liu et al., 2016). 198 We also reduced sequence redundancy to improve the assembly using the sequence clustering software 0.02847). We then evaluated the assembly's structural integrity with Transrate and assessed 202 completeness using the vertebrata database in BUSCO version 1.1b1 (Simão et al., 2015). We 203 quasimapped the raw reads to the assembly with Salmon version 0.7.2 (Patro, Duggal & Kingsford, 204 2015) to confirm that mapping rates were high. Finally, the assembly was also annotated in dammit 205

Differential Gene and Transcript Expression Analyses 209
Several recent studies have critically evaluated alternative methodologies for differential 210 transcript and gene expression to determine the relative merits of these approaches (Gierlinski et al.,  211  significantly expressed transcripts without corresponding gene matches were selected for an additional  258   BLASTn  search  in  the  NCBI  non-redundant  nucleotide  database  259 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). However, these results were not subjected to any additional 260 analyses, because these matches were not consistent across all three differential expression analyses. 261 This list of BLASTn search matches is provided in supplementary materials (DTEno-262 matchBLASTnSequences.md). 263 The third analysis used DESeq2 to conduct an additional gene-level test, using the same methods 264 as described for the previous gene-level analysis, with the exception that data were imported into an 265 alternative software package. We determined the significantly differentially expressed genes (p < 0.05) 266 based on normalized counts and using the Benjamini-Hochburg correction (Benjamini & Hochburg,267 1995) for multiple comparisons. We only retained genes with a -1 < log 2 fold change > 1 in order to 268 filter genes at a conservative threshold for differential expression based on our sample size (Schurch et 269 al., 2016). This filtering was not necessary for either of the edgeR analyses because log 2 fold changes 270 exceeded this threshold for the differentially expressed genes and transcripts (-1.3 < log 2 fold change > 271

1.4, in all cases). 272
We also compared the log 2 fold change values (of treatment differences by mapped count) for 273 each gene from the edgeR and DESeq2 gene-level analyses in a linear regression. This statistical test 274 was performed in order to evaluate the degree of concordance between the two DGE analyses. 275 Furthermore, we constructed a list of genes identified as differentially expressed by all three analyses, 276 which were further evaluated for function as well as chromosomal location. These genes were also 277 explored in STRING version 10.0 (string-db.org) to determine their protein-protein interactions ( The testes transcriptome was assembled from a 45.8 million paired read data set. Additionally, 293 there were 9-20 million paired reads for each of the 22 testes data sets used for the differential 294 expression analysis (Supplemental Table 1), yielding 304,466,486 reads total for this analysis. The raw 295 reads are available at the European Nucleotide Archive under study accession number PRJEB18655. All 296 data files, including the testes un-annotated transcriptome, the dammit annotated transcriptome, and the 297 data generated by the differential gene expression analysis (described below) are available on DropBox 298 (https://www.dropbox.com/sh/ffr9xrmjxj9md1m/AACpxjQNn-Jlf25qNdslfRSCa?dl=0). These files will 299 be posted to Dryad upon manuscript acceptance. All code for these analyses is posted on GitHub 300 (https://github.com/macmanes-lab/testesDGE).
The performance of multiple transcriptome assemblies was evaluated thoroughly, and the 303 selected optimized testes assembly met high quality and completeness standards, and it also contains 304 relatively few contigs and has high read mapping rates (Table 1). Therefore, this transcriptome was used 305 for our differential expression analyses. The transcriptome was also annotated, and the complete 306 statistics for this dammit annotation are provided in Table 1 Figure 1). Specifically, seven genes were more highly expressed in WET 314 individuals, and eight genes were more highly expressed in DRY individuals (Table 2). 315 We also performed an alternative transcript-level analysis using the referenced transcriptome 316 mapped reads exclusively with edgeR. The exact test found 66 differentially expressed transcripts 317 (Supplemental Figure 2), 45 of which were more highly expressed in the WET group, and 21 were 318 more highly expressed in the DRY group ( Table 3). 10 of these differentially expressed transcripts were 319 consistent with differentially expressed genes from the edgeR DGE analysis. In addition, the 320 significantly expressed transcripts without an Ensembl ID match (nine WET and nine DRY) were 321 retrieved for performing an nt all species BLASTn search (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and 322 these results are in the supplementary materials. 323 The gene-level analysis conducted in DESeq2 yielded 215 significantly differentially expressed 324 genes (Supplemental Figure 3), 67 of which were more highly expressed in the WET group, while 148 were highly expressed in the DRY group. However, only 20 of these genes remained when we filtered 326 them with a -1 < log 2 fold change > 1 to retain genes with a conservative threshold difference between 327 treatment groups. This list of 20 genes yielded 16 genes more highly expressed in WET mice and four 328 genes highly expressed in DRY mice (Table 4). Nine of these genes overlapped with those found to be 329 significant in the previous two edgeR analyses. 330 To evaluate the correlation of log 2 fold change results for each gene (Ensembl ID) from the two 331 DGE analyses (EdgeR and DESeq2), we performed a regression of these log values, and they were 332 significantly correlated (Figure 1: Adj-R 2 = 0.6596; F(1,14214) = 2.754x10 4 ; p < 2.2x10 -16 ). This 333 further demonstrates the concordance of the DGE analyses in these two software packages. 334 To evaluate the degree to which the three analyses produced concordant results, we generated a 335 list of genes which were found to be significantly differently expressed by treatment across all three 336 analyses (Supplemental Table 2). There were six genes that were consistently highly-expressed in the 337 WET group and three genes that were highly-expressed in the DRY group. The six highly-expressed 338 WET genes are Insulin-like 3 (Insl3), Free-fatty acid receptor 4 (Ffar4), Solute carrier family 45 member 339 3 (Slc45a3), Solute carrier family 38 member 5 (Slc38a5), Integrin alpha L (Itgal), and Transferrin (Trf). 340 The three highly-expressed DRY genes are Ras and Rab Interactor 2 (Rin2), Insulin-like growth factory 341 binding protein 3 (Igfbp3), and Connective tissue growth factor (Ctgf). Because the patterns of 342 expression of these nine genes were corroborated by multiple methodologies, we are confident that they 343 are differentially expressed between our treatments. Estimates of expression for these genes generated 344 using the gene-level edgeR analysis are plotted in Figure 2. 345 The significantly differently expressed genes were evaluated for gene function and chromosomal 346 location ( Table 5). These genes occur throughout the genome; namely, they are located on different 347 chromosomes. The diverse functions of each gene will be described below. In addition, we generated 348 STRING diagrams (string-db.org) to view the protein-protein interactions for each of these nine genes 349 (Snel et  Ffar4 was also down-regulated in the DRY group. Omega-3 fatty acid receptor 1 (O3Far1) is an 372 alias of Ffar4, and it has roles in metabolism and inflammation (Moniri, 2016). This protein interacts 373 with multiple other free fatty acid receptors and G-protein coupled receptors as well as Stanniocalcin 1 374 (Stc1) (Figure 3d). Stc1 is involved in phosphate and calcium transportation (Wagner and Dimattia, 375 2006); however, this protein's functional role in mice remains enigmatic (Chang et al, 2005). 376 Another of the lower expressed DRY group genes is Itgal (also known as CDa11a), which has 377 This is the first study to evaluate gene expression levels of a reproductive tissue (testes) in 418 response to acute dehydration in a desert-specialized rodent, Peromyscus eremicus (cactus mouse). Our 419 results demonstrate differential expression of Insl3, which is a gene linked to reproduction, but not for a 420 small subset of other reproductive hormone (and hormone receptor) genes. We also found expression 421 differences in two solute carrier proteins, which is consistent with previous findings asserting the 422 importance of this protein family for osmoregulation in desert rodents. Our findings lead us to 423 hypothesize that reproductive function may be modified via Insl3 in acutely dehydrated mice. Any 424 transcriptomic indication of potential reproductive modification in response to acute dehydration is 425 surprising, given that this is not consistent with our understanding of P. eremicus as a desert specialist 426 capable of breeding year-round in the wild. However, future studies must determine the physiological 427 effects of decreased Insl3 expression on acutely dehydrated cactus mice. While acute dehydration is less 428 common than chronic dehydration for desert mammals, given their ecology, it is a selective force they 429 must overcome. Indeed, throughout much of the described range of the cactus mouse, rainfall events 430 may occur several times per year. Cactus mice, and many other rodents, are known to rehydrate during 431 these rainfall events (MacManes, personal observation). Following rehydration, cactus mice experience 432 acute dehydration, followed by a steady state of chronic dehydration. The reproductive responses of 433 cactus mice to these acute and chronic dehydration events are unknown; therefore, this study describes 434 the transcriptomic effects of acute dehydration in testes. While the relationship between these differentially expressed genes and the hormones involved in 487 reproductive function are currently poorly-characterized, our findings that genes integral to sperm 488 development and activation interact with genes differentially expressed in acute dehydration may 489 indicate that, contrary to our expectations, acute dehydration is linked to reproductive modulation in the 490 cactus mouse. However, functional studies will be necessary to elucidate the connection between these 491 genes and physiological responses to dehydration. This is particularly important because many 492 hormones have pleotropic effects, and further mechanisms of action unrelated to reproduction may be 493 elucidated for these proteins in Peromyscus eremicus. 494 In contrast to genes that are down-regulated in dehydration, the genes that were upregulated in 495 the DRY group are known to be responsible for water homeostasis and cellular growth. The significance 496 of Rin2 is notable, because this protein is an effector for Rab5, which as a GTPase involved in 497 vasopressin-regulated water reabsorption, a critical homeostatic process mediated through the Aqp2 498 Emerging from this work is a hypothesis related to the reproductive response to water stress in 530 the cactus mouse, and perhaps other desert rodents. Specifically, we hypothesize that acute dehydration 531 may be related to reproductive mitigation; however, we hypothesize that chronic dehydration is not.
Indeed, it is virtually oxymoronic to suggest that chronic dehydration, which is the baseline condition in 533 desert animals, has negative consequences for reproductive success. Indeed, desert rodents dynamically 534 respond to water-availability to initiate and cease reproductive function. Generating an integrative, 535 systems-level understanding of the reproductive responses to both acute and chronic dehydration across 536 desert-adapted rodent is required for testing our hypothesis. While understanding the renal response to 537 dehydration is critical for making predictions about survival, understanding the reproductive correlates 538 is perhaps even more relevant to evolutionary fitness. This study, to the best of our knowledge, is the 539 first to describe the reproductive correlates of water-limitation in the cactus mouse, and the first to use a 540 differential gene expression approach to evaluate reproductive tissue responses to drought. Furthermore, 541 this study contributes to a research aim to determine whether novel physiological reproductive 542 adaptations are present in male Cactus mouse (Kordonowy and MacManes, 2016). Developing a 543 comprehensive understanding of reproductive responses to drought, and also the mechanisms underlying 544 potential physiological adaptations, is necessary if we are to understand how increasing environmental 545 variability due to climate change may modify the distribution of extant organisms. 546 547

Conclusions 548
The genetic mechanisms responsible for physiological adaptations for survival and reproduction 549 in deserts remain enigmatic. Desert rodent research has focused primarily on physiological adaptations 550 related to survival, specifically on renal adaptations to combat extreme water-limitation. In contrast, 551 while previous studies have investigated reproductive effects of water-limitation in desert rodents, the 552 underlying mechanisms for physiological adaptations for reproduction during acute and chronic 553 dehydration are unknown. Furthermore, ours is the first study to evaluate reproductive transcriptomic 554 responses to water limitation in a desert-rodent, the cactus mouse. To this end, we characterized the reproductive correlates of acute dehydration in this desert-specialized rodent using a highly replicated 556 RNAseq experiment. In contrast to expectations, we describe a potential signal of reproductive 557 modulation in dehydrated male cactus mouse testes. Specifically, dehydrated mice demonstrated 558 significantly lower expression of Insl3, which is a canonical regulator of fertility (and testes descent). 559 Lower expression was also found in Slc45a3 and Slc38a5, lending further credence to the important role 560 of solute carrier proteins for osmoregulation in the cactus mouse. While the low number of differentially 561 expressed genes between acutely dehydrated and control mice might otherwise have suggested that this 562 species is relatively unaffected by acute water-limitation, the diminished expression of Insl3 in 563 dehydrated mice leads us to propose that acute dehydration may compromise reproductive function via 564 decreased fertility. Indeed, we hypothesize that non-traditional reproductive hormone pathways, such as 565 those involving Insl3 or AVP (which has elicited suppressive reproductive responses in other desert 566 rodent research), warrant further investigation in studies evaluating the reproductive effects of acute and 567 chronic dehydration. Although future research must experimentally evaluate the potential functional 568 relationship between Insl3 expression pattern and reproductive function and fertility, our findings that 569 acute-dehydration alters Insl3 expression may be concerning, particularly with respect to global climate 570 change. Climate change driven increased variabilities in weather patterns may result in a greater 571 frequency of acute water-stress, which could result in reduced reproductive function for the cactus 572 mouse. In addition, because global climate change is predicted to shift habitats toward extremes in 573 temperature, salinity, and aridity, and to alter species ranges, an enhanced understanding of the 574 reproductive consequences of these changes, and of the potential for organisms to rapidly adapt, may 575 enable us to effectively conserve innumerable species facing dramatic habitat changes. 576 Availability of data and materials: 591

List of abbreviations
The raw reads are available at the European Nucleotide Archive under study accession number 592 PRJEB18655. All data files, including the testes un-annotated transcriptome, the dammit annotated 593 transcriptome, and the data generated by the differential gene expression analysis (described below) are 594 available on DropBox (https://www.dropbox.com/sh/ffr9xrmjxj9md1m/AACpxjQNn-595 Jlf25qNdslfRSCa?dl=0). These files will be posted to Dryad upon manuscript acceptance. All code for 596 these analyses is posted on GitHub (https://github.com/macmanes-lab/testesDGE). 597

Competing Interests 598
The authors declare that they have no competing interests. 599