Scorpions

All specimens figured and discussed here are held by the University of Wisconsin-Madison Geology Museum, Madison, Wisconsin, USA. Specimens photographed using a Canon EOS Rebel T3i Digital SLR with a Canon MP-E 65 mm macro lens and full spectrum lighting. Some images were made using low-angle lighting. Images were z-stacked and stitched using Adobe Photoshop CC. A corrosion cast of the pericardium and pulmo-pericardial sinuses of a present-day scorpion (Centruroides exilicauda) was used for anatomical comparison. Another present-day scorpion (Hadogenes troglodytes) was used for morphological comparison of medial structures. Explanatory diagrams and the reconstruction of Parioscorpio venator were created using Microsoft Surface Pro 3 with a stylus using Photoshop CC.

Patients with stage IV lung adenocarcinoma diagnosed and treated between May 2014 and August 2016 in two Kaohsiung Medical University-affiliated hospitals (Kaohsiung Medical University Hospital and Kaohsiung Municipal Ta-Tung Hospital) in Taiwan were identified and followed until December 2016. The diagnosis of lung cancer was confirmed pathologically according to World Health Organization pathology classification, and tumor staging was made by a special committee including clinical pulmonologists, medical oncologists, chest surgeons, radiologists, pathologists and radiation oncologists, according to the seventh American Joint Committee on Cancer staging system. Patients were included if they: (1) had adequate tumor specimens for EGFR mutation examinations and had susceptible EGFR mutations including exon 19 deletions and exon 21 L858R point mutations; (2) were chemotherapy-naïve and treated with 30 mg or 40 mg daily of afatinib as the first-line treatment.
Baseline clinical characteristics were determined by retrospective chart review, including age at diagnosis, sex, Eastern Cooperative Oncology Group (ECOG) performance status at the beginning of first-line afatinib treatment, smoking history, and tumor histology. Glomerular filtration rate was estimated using the Modification of Diet in Renal Disease formula (eGFR-MDRD). Mutations in the EGFR gene were analyzed using an EGFR RGQ kit (Qiagen, UK) which utilized amplification refractory mutation specific (ARMS) polymerase chain reactions and Scorpion technology for detection and/or direct sequencing as in our previous report [12 (link)–16 (link)]. The initial treatment response was classified based on serial imaging studies using the revised Response Evaluation Criteria in Solid Tumors (RECIST 1.1) criteria. The PFS and OS with first-line treatment were defined as the duration from the start of the first treatment to the date of disease progression on imaging studies and the date of death, respectively. ADRs were recorded by physicians and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.0.

Aligning ICKs, or any cysteine rich peptide, is difficult due to nonhomologous cysteines mistakenly being aligned. Thus, the finalized consensus disulfide connectivities were used to inform the alignment of ICKs using a similar approach to Pineda et al. 2020 [6 (link)]. Rather than align everything at once and then manually adjusting misaligned cysteines followed by realignment of regions between the two adjusted cysteines, only amino acids between cysteines participating in disulfide bonds common to all ICKs in the dataset were used for the alignment. Additionally, the regions between homologous cysteines were aligned separately while using the barcode “WWYHWYYHMM” to replace flanking cysteines to prevent inner cysteines from misaligning with flanking cysteines similar to the approach by Shafee et al. 2016 [68 (link)]. The alignment was then provided as input to IQTREE v1.5.5 [58 (link)] to test the amino acid composition using a Chi-squared test. Any sequences that failed the test were removed from the alignment, and the sub-regions were realigned following the previously described procedure. The resulting alignment was reverse translated to form a coding sequence alignment using PRANK [69 ].
The same outgroup as used by Pineda et al. 2020 [6 (link)] (disulfide-directed $\beta$ -hairpin from the whip scorpion Mastigoproctus giganteus) was added to the protein alignment using MAFFT v7.455 [56 (link)]. The phylogenetic relationships of the ICKs in this alignment were reconstructed using IQTREE and the default settings.
Adaptive molecular evolution is typically inferred in coding sequences by comparing ratios of the rates of nonsynonymous substitution and synonymous substitution ( $d_N/d_S$ or $\omega$ ), where $d_N$ exceeding $d_S$ indicates positive selection, $d_S$ exceeding $d_N$ indicates negative selection, and $d_N/d_S$ approaching unity indicates neutral evolution. The HYPHY [70 ] implementation of Branch-Site Unrestricted Statistical Test (BUSTED) for Episodic Diversification was used to assess whether a gene has experienced positive selection at at least one site on at least one branch. To determine if ICKs have experienced positive selection, the codon multiple sequence alignment and phylogeny were provided as input to BUSTED using default parameters.
In ICKs, specific amino acid sites may play an important role in the structure-function (e.g., binding specificity) and adaptive evolution. To identify specific amino acid sites that have undergone pervasive positive selection, the HYPHY implementation of a Fast, Unconstrained Bayesian AppRoximation (FUBAR) was used with the codon multiple sequence alignment and phylogeny provided as input and default parameters.
There may only be specific episodes where certain amino acids receive strong bouts of positive selection. To determine if amino acid sites have undergone positive selection, the HYPHY implementation of a Mixed Effects Model of Evolution (MEME) [71 (link)] was used to determine if certain amino acid sites have undergone episodic positive selection. The codon multiple sequence alignment and phylogeny were provided as input to MEME with default parameters and the phylogeny set as the background.
To evaluate specific instances on a phylogeny where positive selection has occurred, branch-site models are typically implemented. Much like how MEME is unable to statistically specify the exact branches within a site undergoing episodic positive selection, branch-site models are only able to identify specific branches where a certain portion of sites have undergone positive selection. To accomplish this, the HYPHY implementation of adaptive Branch-Site Random Effects Likelihood (aBSREL) [72 (link)] was used with default parameters, and the codon alignments and phylogeny were provided as input.
Aside from evaluating signatures of positive selection through calculations of codon substitution rates, we also investigated the co-occurrence between amino acid positions in ICKs, which may provide useful inferences into the evolution of their structure/function. This can be achieved using the HYPHY implementation of the Bayesian Graphical Model (BGM) [73 (link)], which maps amino acid substitutions to a phylogeny and reconstructs ancestral states for a given model of codon substitution rates that is then followed up by a series of 2 × 2 contingency table analyses.

L. rufozonatus was collected from the Beicheng snake and scorpion-breeding farm in Weifang, China, in November 2019. Fifteen healthy adult snakes of the same body length raised under the same environmental conditions were screened. No antibiotics or other drugs that can affect the intestinal microbes were administered to the snakes for 2 months before collection. After 30 days of fasting, a dose of lidocaine hydrochloride was injected into the brain for euthanasia, immediately after which an abdominal incision was made and the digestive tract was removed. The digestive tract contents were collected via sterile swabs, transferred into sterile vials, immediately frozen in liquid nitrogen, and stored at −80 °C until DNA extraction.