Differential Gene Expression Analysis and Cytological Evidence Reveal a Sexual Stage of an Amoeba with Multiparental Cellular and Nuclear Fusion

Abstract

25 Sex is a hallmark of eukaryotes but its evolution in microbial eukaryotes is poorly elucidated.26 Recent genomic studies revealed microbial eukaryotes possess genetic toolkit necessary for 27 sexual reproduction. However, the mechanism of sexual development in a majority of microbial 28 eukaryotes including amoebozoans is poorly characterized. The major hurdle in studying sex in 29 microbial eukaryotes is lack of observational evidence, primarily due to its cryptic nature. In this 30 study, we used a tractable fusing amoeba, Cochliopodium, to investigate sexual development 31 using stage specific Differential Gene Expression (DGE) and cytological analyses. Both DGE 32 and cytological results showed that most of the meiosis and sex-related genes are upregulated in 33 Cochliopodium undergoing fusion in laboratory culture. Relative gene ontology (GO) category 34 representations in unfused (single) and fused cells revealed functional skew of the fused 35 transcriptome toward DNA metabolism, nucleus and ligases that are suggestive of a commitment 36 to sexual development. While single cells GO categories were dominated by metabolic pathways 37 and other processes indicative of vegetative phase. Our study provides strong evidence that the 38 fused cells represent a sexual stage in Cochliopodium. Our findings have further implications in 39 understanding the evolution and mechanism of inheritance involving multiparents in other 40 eukaryotes with similar reproductive strategy. 41 42 43 44 45 46 3 47 Introduction 48 Sexual reproduction is ubiquitous and considered to have originated in the last common 49 ancestors of all eukaryotes; however, its evolution still remains a mystery particularly among 50 microbial eukaryotes [1-6]. Sexual reproduction can be defined as a stage in the life cycle 51 involving meiosis -a biological process that reduces the genome complement by one-half 52 (haploid); this is followed by fusion of these haploid cells (gametes), in a process commonly 53 known as fertilization, to form a diploid zygote. This definition is primarily based on 54 observations in macroscopic eukaryotes (e.g. animals and plants) that are usually dimorphic with 55 two distinct sexes. Sexual reproduction in macroscopic eukaryotes is well defined with 56 recognizable cellular and molecular signatures [5, 7]. While some variations of sexual 57 reproduction are generally known [1, 8], the nature or existence of sex in most microbial 58 eukaryotes is poorly documented [1]. Microbial eukaryotes including amoebozoans display 59 diverse quality of life cycles that involve various types of asexual reproduction and sexual stages 60 that are usually cryptic [1, 9, 10]. 61 62Recent comparative genomic studies of microbial eukaryotes including amoebozoans 63 demonstrate these microbes not only possess sex genes in their genomes, but also these genes are 64 actively being expressed [11][12][13][14][15]. These discoveries debunked the long-held view that microbial 65 eukaryotes were strictly asexual, and solidified the ancestral nat...

Comprehensive Comparative Genomics Reveals Over 50 Phyla of Free-living and Pathogenic Bacteria are Associated with Diverse Members of the Amoebozoa

“…Based on preliminary analysis that showed amoebozoan transcriptomes contained large bacterial transcripts and some ribosomal genes, we analyzed RNA-Seq from previous publications collected in a similar manner as above 32,33,35,44 . The whole culture RNA-Seq dataset included a total of 35 species (15 discoseans, 12 evoseans, and 8 tubulinids) with three additional duplicate samples from Discosea sequenced in two different labs 32,33,35 .…”

“…These discosean duplicate samples were included in the analysis to examine the effects of culturing methods and environment on the number and composition bacterial community recovered. The single cell RNA-Seq dataset was represented by 5 samples obtained from Cochliopodium minus 44 . Data collection, sequencing and assembly of transcriptome data of these diverse amoebozoans, representing the three main clades of Amoebozoa (Discosea, Evosea, and Tubulinea) of the whole culture and single cell RNA-Seq datasets, are described in Kang et al 32 and Tekle et al 33,35 , and Tekle et al 44 , respectively.…”

“…The single cell RNA-Seq dataset was represented by 5 samples obtained from Cochliopodium minus 44 . Data collection, sequencing and assembly of transcriptome data of these diverse amoebozoans, representing the three main clades of Amoebozoa (Discosea, Evosea, and Tubulinea) of the whole culture and single cell RNA-Seq datasets, are described in Kang et al 32 and Tekle et al 33,35 , and Tekle et al 44 , respectively. Some good quality transcriptomes whose origin was not certain or is collected using a combination of single cell and whole culture are placed in the whole culture RNA-Seq dataset.…”

“…Some good quality transcriptomes whose origin was not certain or is collected using a combination of single cell and whole culture are placed in the whole culture RNA-Seq dataset. All transcriptomes used for single cell RNA-Seq dataset are collected in our laboratory under similar experimental conditions 44 .…”

Draft Genome of Cochliopodium minus (Amoebozoa): Insights into Its Complex Sexual Behavior, Across Domain Gene Acquisitions and Metazoan Type Signaling

“…Cochliopodium is among the fast-evolving amoeboid lineages and its phylogenetic position within the supergroup has only been recently resolved using phylogenomic analysis 13,19 . Cochliopodium has been a subject of extensive study due to the unusual behavior of cell-to-cell interaction it exhibits in actively growing cultures during its life cycle 6,20,21 . Cochliopodium has long been considered asexual.…”