Guide MALE GENITALIA: 20 Minute Digest

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Genome sequencing library statistics are available in Appendix 4—table 1. These results are consistent when considering the possible systematic error of kmer spectral analysis and flow cytometry genome size estimates. The heterozygosity is lower than that measured for P. Potential contaminants in Alat1. First, scaffolds were compared to known sequences by performing a blastn v2. This process removed scaffolds 1. Scaffolds were taxonomically annotated as described in Appendix 2. Live specimens were anesthetized on ice and dissected during the day.

The lantern tissue was dissected from the abdomen and contains the cuticle, photocyte layer and reflector layer. The fragmentation of mRNA was performed for 4 min. The enrichment PCR was done using six cycles. Sequence quality was inspected with FastQC Andrews, A non strand-specific de novo transcriptome assembly was produced with Trinity v2.

Peptides were predicted from the de novo transcripts via Transdecoder v5. De novo transcripts were then aligned to the A. A reference guided transcriptome was produced from all available A. Reads were first mapped to the A. Finally, the produced. GTF files were merged using StringTie --merge. A protein-coding gene reference set for A.

For transcripts, we combined reference guided and de novo transcriptome assembly approaches. Notably, these reference guided and de novo transcriptome assembly approaches differed from the current de novo Appendix 2. In the reference-guided approach applied here, RNA-Seq reads were mapped to the genome assembly with TopHat and assembled into transcripts with Cufflinks parameters: --min-intron-length 30 Trapnell et al.

The ORF sequences were mapped to the genome using Exonerate in est2genome mode for splice-aware alignment. We processed homology evidence at the protein level using the reference proteomes of D. These reference proteins were split-mapped to the A. These gene models derived from multiple evidence were merged by the EVM program to obtain the reference annotation for the genomes.

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Lastly, gene models for luciferase homologs, Ps, and de novo methyltransferases DNMTs which were fragmented or were incorrect e. The official gene set. A de novo species-specific repeat library for A. This process yielded a final library of interspersed repeats. We then used this library and RepeatMasker v4. This repeat library is dubbed the Aquatica lateralis Official Repeat Library 1. The Elateridae includes about 10, species Slipinski et al. These luminous species are recorded only from tropical and subtropical regions of Americas and some small Melanesian islands, such as Fiji and Vanuatu Costa, ; Costa et al.

All luminous species are closely related - luminous click beetles belong to the tribes Pyrophorini and Euplinthini Costa, ; Arias-Bohart, of the subfamily Agrypninae, with the single exception of Campyloxenus pyrothorax Chile in the related subfamily Campyloxeninae Stibick, This near-monophyly of bioluminescent elaterid taxa is supported by both morphological Douglas, and molecular phylogenetic analysis Sagegami-Oba et al. This suggests a single origin of bioluminescence in this family. The genus Ignelater was established by Costa in and I. The genus Ignelater is characterized by the presence of both dorsal and ventral photophores Costa, ; Rosa, An unreviewed report suggested that the adult I.

Phylogenetic analyses based on the morphological characters suggested that the genera Ignelater and Photophorus which contain only two species from Fiji and Vanuatu are the most closely related genera in the tribe Pyrophorini Rosa, The exact function of bioluminescence across different life stages remains unknown for many luminous elaterid species. Bioluminescent elaterid beetles typically have two paired lanterns on the dorsal surface of the prothorax, and a single lantern on the ventral abdomen, which is only exposed during flight. Several bioluminescent Elateridae produce different colored luminescence from their prothorax and abdominal lanterns Oba et al.

Harvey reported that there was not a marked difference in the luminescence color of the dorsal and ventral lanterns of Puerto Rican I. Like fireflies, elaterid larvae often produce light, with the glowing termite mounds of Brazil that contain the predatory larvae of Pyrearinus termitilluminans being a striking example Costa and Vanin, A description of the anatomy of the larval light organ of Pyrophorus is provided by Harvey, , and a more modern photograph of the larval light organ is provided by Bechara and Stevani, Like other bioluminescent elaterid larvae, I.

Adult I. Once a female is observed, the prothorax lanterns of the male go dark, the ventral lantern becomes illuminated, and the male approaches the female via a circular search pattern. Mating is brief, reportedly taking only 5 seconds. Unlike fireflies, bioluminescent elaterid species are not known to have potent chemical defenses. A defense role for I. Similarly, I. Individuals were captured at night on April 20th and April 28th during flight on the basis of light production.

Identification to species was performed by comparing antenna and dissected genitalia morphology to published keys Costa, ; Rosa, ; Rosa, Appendix 3—figure 1.

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Specimens collected at the same time, but not those used for genitalial dissection, were used for sequencing. Although the genitalia morphology of the sequenced specimens was not inspected to confirm their sex, sequenced specimens were inferred to be male, based on the fact that female bioluminescent elaterid beetles are rarely seen in flight Personal communication: S.

Velez and the dissected specimens collected in the same batch as the sequenced specimens were confirmed to be male. A Dorsal and B ventral view of an Ignelater luminosus aedeagus, dissected from the same batch of specimens used for linked-read sequencing and genome assembly. The species identity of this specimen was confirmed as I. The genome sizes of 5 male I. After a minimum of 30 min staining in the dark and cold, the average fluorescence channel number for the PI red fluorescence of the 2C diploid nuclei of the sample and standard were determined using a CytoFlex Flow Cytometer Beckman-Coulter.

The 1C amount of DNA in each sample was determined as the ratio of the 2C channel number of the sample and standard times Mbp. The genome size of these I.

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Genome size inference via Kmer spectral analysis of the I. The resulting library was then sequenced on one HiSeqX lane. A summary of the library statistics for the genomic sequencing is available in Appendix 4—table 1. The draft genome of I. The reported mean molecule size was A Supernova v2. Manual long-read based scaffolding was then applied to produce a final assembly Ilumi1. Before analysis, 10x Chromium barcodes were trimmed off Read1 using cutadapt v1.

The heterozygosity is higher than that measured for P. The read error rate for this library is also significantly higher than the P. We sought to systematically remove assembled non-elaterid contaminant sequence from Ilumi1. A tab delimited text file containing the results of this blobtools annotation is available on FigShare DOI: This approach removed scaffolds Kbp , representing 0.

While filtering the Ilumi1.

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Upon closer inspection, we found conflicting information as to the most likely taxonomic source of these scaffolds. Although Hymenolepis diminuta infects mammals, it also spends a period of its life cycle in intermediate insect hosts, including beetles, as cysticercoids Center for Disease Control and Prevention, ; Sheiman et al. For a beetle like I.

Scaffolds were taxonomically annotated as described in Appendix 3. Manual inspection of the initial gene-models for Ilumi1. In order to run Pilon efficiently, we split the taxonomically filtered Ilumi1. We determined via manual gene-model annotation of Ilumi1.

Reads were mapped to Ilumi1. Inspection of mapped reads with Integrative Genomics Viewer v2. Notably, this repeat unit was present the right edge of Ilumi1. Although our Nanopore sequencing did not unambiguously span this repetitive element and bridge the two scaffolds, we surmised that this information was sufficient to manually merge these scaffolds Appendix 3—figure 5.