An Exploration of a Body Size Allele in the Fig Worm Caenorhabditis Inopinata

Abstract

As a model organism, the nematode Caenorhabditis elegans has been the subject of prolificbiological research. Since its discovery, the genetic and phenotypic diversity of Caenorhabditis inopinata, the much larger sibling species of C. elegans, has been investigated. Comparative body size studies are ongoing between the two species. Because of the existing knowledge base on how body size in C. elegans has been studied, including its genetic landscape and life history traits, this prior research helps to inform on the ways the body size of C. inopinata may be studied. First discovered in Okinawa, Japan, wild-type C. inopinata lives in the interior of the fresh fig syconia Ficus septica. The genetic cause of the elongated appearance of C. inopinata has been studied. Postembryonic cell size expansion is primarily responsible for C. inopinata’s large size, and gene expression patterns during the developmental stage from larval to adult have been described. As such, post embryonic growth is the period of interest in this body study study. In previous work, a forward genetic screen was utilized on wild type C. inopinata to create body size mutants in order to discover novel body size regulatory genes. Presented here is a study of a novel allele classified as nok3 that presents with a shorter and wider body size in C. inopinata. This allele promotes a characterized as a dumpy phenotype (Dpy). This paper includes a series of genetic and observational studies of the cultivated mutant strain of C. inopinata made in an effort to fill in the gap in knowledge pertaining to this body size variant and adding to the existing knowledge base concerning body size regulation in C. inopinata. Through an initial genetic map, a region believed to harbor a causative gene for the Dpy mutation was determined. Gene editing using Crispr/Cas9 technology was performed on wild type C. inopinata in an attempt to identify a gene that controls the within species body size. A candidate gene known as Sp34_40141100 was targeted. Using Crispr/Cas9 technology, a deletion was generated which removed the entire open reading frame of the gene of interest creating the nok6 allele. The nok6 allele did not express the expected phenotype. Two other genes found in C. inopinata near the candidate region, were sequenced in the mutant strain. These genes are known orthologs to genes dpy-13 and col-34 that produce the dumpy phenotype in C. elegans. Sequencing revealed no differences in these genes compared to wild type. To further characterize the Dpy allele, a comparison of growth rates across lines of cultivated wild type and mutant C. inopinata was performed. Wild type females grew faster in length but not width compared to the mutant. An examination of individual fecundity over a series of 24 hour intervals after a single mating event found no significant difference in the total number of embryos laid between these strains. However, the timing of egg laying shows an oscillating patten within the Dpy strain which is not present within wild type. Dominance of nok3 was determined to be recessive. These findings will aid in future efforts to discover more about the genetic, developmental, and evolutionary body size divergence of C. inopinata and add to the discussion of body size differences between these two closely related species.