Goodheart JA et al. (2020), Laboratory culture of the California Sea Firefl...

ECB-ART-49761

Sci Rep 2020 Jun 26;101:10443. doi: 10.1038/s41598-020-67209-w.

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Laboratory culture of the California Sea Firefly Vargula tsujii (Ostracoda: Cypridinidae): Developing a model system for the evolution of marine bioluminescence.

Goodheart JA , Minsky G , Brynjegard-Bialik MN , Drummond MS , Munoz JD , Fallon TR , Schultz DT , Weng JK , Torres E , Oakley TH .

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Bioluminescence, or the production of light by living organisms via chemical reaction, is widespread across Metazoa. Laboratory culture of bioluminescent organisms from diverse taxonomic groups is important for determining the biosynthetic pathways of bioluminescent substrates, which may lead to new tools for biotechnology and biomedicine. Some bioluminescent groups may be cultured, including some cnidarians, ctenophores, and brittle stars, but those use luminescent substrates (luciferins) obtained from their diets, and therefore are not informative for determination of the biosynthetic pathways of the luciferins. Other groups, including terrestrial fireflies, do synthesize their own luciferin, but culturing them is difficult and the biosynthetic pathway for firefly luciferin remains unclear. An additional independent origin of endogenous bioluminescence is found within ostracods from the family Cypridinidae, which use their luminescence for defense and, in Caribbean species, for courtship displays. Here, we report the first complete life cycle of a luminous ostracod (Vargula tsujii Kornicker & Baker, 1977, the California Sea Firefly) in the laboratory. We also describe the late-stage embryogenesis of Vargula tsujii and discuss the size classes of instar development. We find embryogenesis in V. tsujii ranges from 25-38 days, and this species appears to have five instar stages, consistent with ontogeny in other cypridinid lineages. We estimate a complete life cycle at 3-4 months. We also present the first complete mitochondrial genome for Vargula tsujii. Bringing a luminous ostracod into laboratory culture sets the stage for many potential avenues of study, including learning the biosynthetic pathway of cypridinid luciferin and genomic manipulation of an autogenic bioluminescent system.

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	Figure 1. Map of localities where collections (and attempted collections) of Vargula tsujii were made. Black points represent geographic annotations for reference, purple points refer to the location where ostracods were collected that were ultimately cultured, blue where animals were found or collected, orange where Kornicker originally collected animals, and red where no V. tsujii were found. (A) Map of the California (USA) coastline where collections were attempted; (B) Map of the region near Two Harbors (Santa Catalina Island, California, USA) where collections were attempted. Figure generated using the R (version 3.6.2) package ggmap. Map tiles by Stamen Design, under CC BY 3.0. Data by OpenStreetMap, under ODbL.
	Figure 2. Diagrammatic representation of aquarium setup for Vargula tsujii. (A) Source water flows into (B) a reservoir tank, which overflows to keep the reservoir full and water level constant. (C) We use a siphon to carry water from a reservoir tank to main aquarium where ostracods live. Water level of the main tank drains through sand (stippled), through (E) an outflow that is raised to a level that would cause water to remain at the level of the dashed line, even if a siphon is interrupted to cause water to stop flowing into the main tank. (F) Plastic bottle and parts used to create small aquarium using the same logic as main aquarium. (G) Custom-built acrylic aquarium with drain in bottom and raised outflow with adjustable rate.
	Figure 3. Haplotype networks for (A) COI sequences, and (B) 16S sequences of Vargula tsujii animals from three localities sampled. Abbreviations: CI, Catalina Island; SD, San Diego; SP, San Pedro.
	Figure 4. Embryogenesis within brooding females of Vargula tsujii post-release into the marsupium. (A) Day 0, (B) Day 1–4, (C) Day 5–7, (D) Day 8–9, (E) Day 10–13, (F) Day 14–17, (G) Day 18–19, (H) Day 20–21, (I) Day 22–23, (J) Day 25–38. Scale bar = 1 mm.
	Figure 5. Summary of the timing of stages of embryogenesis in Vargula tsujii. This represents the complete range of first-appearance of each feature across multiple broods, including: yolk separation (range = 5–8 days, N = 7); eye-spot formation (range = 10–18 days, N = 12); upper lip formation (range = 15–19 days, N = 3); limb formation (range = 18–23 days, N = 11); hatching (range = 25–38 days, N = 11).
	Figure 6. Images of selected individuals within each inferred instar stage: (A) Newly hatched A-V instar beside its shed chorion and an embryo still within the chorion, (B) A-V, (C) A-IV, (D) A-III, (E) A-II, (F) A-I male, (G) A-I female, (H) Adult male, (IU) Adult female. Scale bar only for B-I, no scale for A. Abbreviations: A-V, instar 1, ch, chorion, em, embryo still within chorion.
	Figure 7. (A) KMeans clusters of Vargula tsujii developmental stages based on carapace height and width. Instar stages were assigned to each cluster via comparison to P. annecohenae and additional analysis of male and female size-dimorphism. (B) Points representing laboratory cultured animals overlayed on wild caught and unknown data. Different colors represent different clusters in the KMeans analysis, and are labeled with their inferred instar/adult stages. (C) Shows wild-caught animals dissected and identified as female in relation to the KMeans clusters, and (D) shows wild-caught animals dissected and identified as male in relation to the KMeans clusters.
	Figure 8. Plot of length versus eye-to-keel distance (EKD) in animals assigned to the A-I Instar/Adult Male cluster in the previous KMeans analysis (Fig. 7A). These points were then clustered via KMeans analysis into three clusters, which we assigned to A-I Males (red), A-I Females (blue), and Adult Males (yellow).
	Figure 9. Mitochondrial genome of Vargula tsujii. Note the duplicated control regions, CR1 and CR2 (in gray). Figure produced with Circos (version 0.69; http://circos.ca/)89.

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Bernt, MITOS: improved de novo metazoan mitochondrial genome annotation. 2013, Pubmed