bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.061598; this version posted April 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 3 An integrated mosquito small RNA genomics resource reveals 4 dynamic evolution and host responses to viruses and transposons. 5 6 7 Qicheng Ma1† Satyam P. Srivastav1†, Stephanie Gamez2†, Fabiana Feitosa-Suntheimer3, 8 Edward I. Patterson4, Rebecca M. Johnson5, Erik R. Matson1, Alexander S. Gold3, Douglas E. 9 Brackney6, John H. Connor3, Tonya M. Colpitts3, Grant L. Hughes4, Jason L. Rasgon5, Tony 10 Nolan4, Omar S. Akbari2, and Nelson C. Lau1,7* 11 1. Boston University School of Medicine, Department of Biochemistry 12 2. University of California San Diego, Division of Biological Sciences, Section of Cell and 13 Developmental Biology, La Jolla, CA 92093-0335, USA. 14 3. Boston University School of Medicine, Department of Microbiology and the National 15 Emerging Infectious Disease Laboratory 16 4. Departments of Vector Biology and Tropical Disease Biology, Centre for Neglected Tropical 17 Diseases, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK 18 5. Pennsylvania State University, Department of Entomology, Center for Infectious Disease 19 Dynamics, and the Huck Institutes for the Life Sciences 20 6. Department of Environmental Sciences, The Connecticut Agricultural Experiment Station 21 7. Boston University Genome Science Institute 22 23 * Corresponding author: NCL: [email protected] 24 † These authors contributed equally to this study. 25 26 27 28 Running title: Mosquito small RNA genomics 29 30 Keywords: mosquitoes, small RNAs, piRNAs, viruses, transposons microRNAs, siRNAs 31 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.061598; this version posted April 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 ABSTRACT 2 Although mosquitoes are major transmission vectors for pathogenic arboviruses, 3 viral infection has little impact on mosquito health. This immunity is due in part to 4 mosquito RNA interference (RNAi) pathways that generate antiviral small interfering 5 RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome 6 integrity by potently repressing mosquito transposon activity in the germline and soma. 7 However, viral and transposon small RNA regulatory pathways have not been 8 systematically examined together in mosquitoes. Therefore, we developed an integrated 9 Mosquito Small RNA Genomics (MSRG) resource that analyzes the transposon and 10 virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues 11 across four medically important mosquito species. Our resource captures both somatic 12 and gonadal small RNA expression profiles within mosquito cell cultures, and we report 13 the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus 14 (MCpiRCL) composed of satellite DNA repeats. In the larger culicine mosquito genomes 15 we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the 16 expansion of Piwi pathway genes. Finally, our resource enables detection of crosstalk 17 between piRNA and siRNA populations in mosquito cells during a response to virus 18 infection. The MSRG resource will aid efforts to dissect and combat the capacity of 19 mosquitoes to tolerate and spread arboviruses. 20 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.061598; this version posted April 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 INTRODUCTION 2 Mosquitoes are one of the most prevalent vectors of human pathogens, yet there 3 is a wide variation in ability of the subclades to vector different pathogens. For example, 4 all human malaria parasites are exclusively vectored by anopheline mosquitoes yet 5 these same mosquitoes transmit few viruses other than O’Nyong nyong virus (ONNV) 6 and Mayaro virus (Vanlandingham et al. 2006; Brustolin et al. 2018). On the contrary, 7 culicine mosquitoes frequently transmit a range of human viral pathogens, such as 8 dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV) and yellow fever 9 virus (YFV) in tropical climates where AeAlbo and AeAeg thrive; whereas eastern 10 equine encephalitis virus (EEEV) and West Nile Virus (WNV) are spread mainly in 11 Culex mosquitoes that inhabit more temperate climates (Conway et al. 2014; Olson and 12 Blair 2015; Londono-Renteria and Colpitts 2016; Halbach et al. 2017; Lambrechts and 13 Saleh 2019). 14 Since vector-pathogen interactions are complex and variable, no dominant theory 15 yet explains why anopheline mosquitoes are less prolific than culicine mosquitoes in 16 spreading arboviruses. Arbovirus infections in humans lead to devastating symptoms 17 including fever, nausea, bleeding, extreme pain, brain damage and death. However, 18 culicine mosquitoes showing highly active arbovirus replication are practically 19 unaffected (Goic and Saleh 2012; Blair and Olson 2014; Olson and Blair 2015; 20 Lambrechts and Saleh 2019) and therefore are highly competent transmitters of 21 arboviruses to human hosts. 22 The three main classes of animal small regulatory RNAs are microRNAs 23 (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), which range in size 24 between 18-23nt long and are typically bound by Argonaute proteins; and Piwi- 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.061598; this version posted April 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 interacting RNAs (piRNAs) that are bound by Piwi proteins and which range in size 2 between 24-32nt in length. Arguably, the most extensively characterized animal small 3 regulatory RNAs are from Drosophila melanogaster, whose compact genome has 4 decades of careful annotation and pioneering genetics tools. Many groups including 5 ours have characterized D. melanogaster (Dmel) small RNAs comprising 258 miRNA 6 genes (Kozomara et al. 2019), ~20 large intergenic piRNA cluster loci also referred to 7 as master control loci (Brennecke et al. 2007; Malone et al. 2009; Wen et al. 2014), 8 >1000 genic piRNA cluster loci (Robine et al. 2009; Wen et al. 2014; Chirn et al. 2015), 9 and >1000 endogenous loci generating either large fold-back transcripts or sense- 10 antisense pairing transcripts that can give rise to endogenous siRNAs (Czech et al. 11 2008; Ghildiyal et al. 2008; Kawamura et al. 2008; Mirkovic-Hosle and Forstemann 12 2014; Wen et al. 2014; Wen et al. 2015). Finally, arbovirus-specific siRNAs and piRNAs 13 are also detected in systemically infected Dmel cell cultures (Flynt et al. 2009; Wu et al. 14 2010; Vodovar et al. 2011; Goic et al. 2013; Wen et al. 2014; Palmer et al. 2018). 15 Culicidae mosquitoes are relatives of Drosophilid fruit flies as members of the 16 Dipteran insect clade (Figure 1A, (Wiegmann et al. 2011)), yet ~260 Million Years Ago 17 (MYA) of evolutionary distance between Drosophilids and Culicidae imparts 18 physiological and molecular differences in small RNA compositions. Within mosquito 19 phylogeny, the anopheline subclade represented by Anopheles gambiae (AnGam) 20 display a greater degree of chromosome synteny to Drosophilids than the culicine 21 subclade of mosquitoes such as Culex quinquefasciatus (CuQuin), Aedes aegypti 22 (AeAeg) and Aedes albopictus (AeAlbo) (Dudchenko et al. 2017). Indeed, AnGam’s 23 genome (~0.28GB) is as compact in size as Dmel’s genome (~0.18GB), whereas 24 culicine mosquito genomes are an order of magnitude greater in size due to massive 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.061598; this version posted April 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Ma, Srivastav, Gamez et al. Figure 1. A B Samples Sequencing Small RNA genomics pipeline 18-23nt Arboviruses Genic & Intergenic D. melanogaster piRNA Clusters Loci Mosquito cell miRNAs A. gambiae ~260 culture ? ? endo- ~218 C. quinquefasciatus ? siRNAs Transposon piRNA Phasing 3' 5' ~170 A. aegypti Ovary Testis consensus families 24-31nt ~70 MYA A. albopictus 5' 5' 5' 5' Carcass piRNAs Reduced redundancy lists C Anopheles gambiae Culex quinquefasciatus Aedes aegypti Aedes albopictus (AnGam) (CuQuin) (AeAeg) (AeAlbo) Sua5b Hsu Aag2 C6/36 Mos55 CCL-125 C7/10 U4.4 AgamP4 assembly CpipJ2 assembly AaegL5 assembly AalbF2 assembly * 0.28 GB: Chr 2, 3, X, MT 0.58 GB: no Chromosomes 1.87 GB: Chr 1, 2, 3, X, MT 2.54 GB: no Chromosomes Few unassembled contigs only 3172 super-contigs 2306 unassembled contigs ~2197 scaffolds ~13.1K gene models ~19.8K gene models ~14.6K gene models ~31.0K * gene models Figure 1. Overview of the mosquito small RNA genomics resource. (A) Phylogenetic tree of Dipteran insects in this study, with evolutionary distance measured by Million Years Ago (MYA). Blue and red color denote the anopheline and culicine lineages. (B) Organization of this resource that compares mosquito cell cultures to tissue types via determining the small RNA types and their genomic profiles. (C) Overview of the four mosquito species genomes and eight cell culture lines subjected to the small RNA genomics analysis pipeline. The specific genome assembly names are noted with genome configuration statistics below. The asterisk by the AeAlbo AalbF2 assembly indicates the early stage assembly annotation has an redundant list of gene models. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.061598; this version posted April 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.