Identification and Venom Characterization of Two Scorpions from the State of Chihuahua Mexico: Chihuahuanus coahuliae and Chihuahuanus crassimannus

Abstract

Chihuahua is the largest state in Mexico. The ecosystem of this region is composed of large area of bushes, forests, and grasslands, which allows for a specific diversity of fauna; among them are interesting species of non-lethal scorpions. Most of the Chihuahuan scorpions have been previously morphologically and molecularly described; however, this manuscript could be the first to describe the composition of those venoms. This work aimed at the collection of two scorpion species from the region of Jiménez (Southwest of the State of Chihuahua), which belong to the species Chihuahuanus cohauilae and Chihuahuanus crassimanus; the two species were taxonomically and molecularly identified using a 16S DNA marker. Reverse-phase high-performance liquid chromatography (RP-HPLC) of C. coahuilae and C. crassimanus venoms allowed the identification of three fractions lethal to mice. Additionally, three fractions of each scorpion displayed an effect on house crickets. In the end, three new fractions from the venom of C. coahuilae were positive for antimicrobial activity, although none from C. crassimanus venom displayed growth inhibition. Despite being a preliminary study, the venom biochemical analysis of these two uncharacterized scorpion species opens the opportunity to find new molecules with potential applications in the biomedical and biotechnological fields.

Keywords: C. coahuliae; C. crassimanus; Chihuahua; antimicrobial; insecticidal; scorpion venom.

Figure 1

Scorpion collection. Specimens were collected around Jiménez in the south of the State of Chihuahua, Mexico (27.114384, −104.879348), map generated by authors using QGIS Software v 2.18.12.

Figure 2

Taxonomic classification. (A). Chihuahuanus coahuilae. (A1) Carapace, dorsal aspect, showing one pair of central eyes and two pairs of lateral eyes. (A2) Ventral photo of a scorpion showing its pectins. (A3) Venom gland (telson, lateral aspect). (A4) Pedipalp of a scorpion covered with sensory hairs. (B) Chihuahuanus crassimanus. (B1) Carapace, dorsal aspect, showing one pair of central eyes and two pairs of lateral eyes. (B2) Ventral photo of a scorpion showing its pectins. (B3) Venom gland (telson, lateral aspect). (B4) Pedipalp of a scorpion covered with sensory hairs.

Figure 3

Molecular identification and phylogenetic analysis of the 16S gene. (A) PCR amplification products; 12S rRNA gene (450 bp), 16S rRNA gene (400 bp), COI gene (540 bp). The primers were reported by Borges et al. (2010) [17]. (B) Molecular phylogenetic analysis of the 16S rDNA mitochondrial gene using the maximum likelihood (ML) method generated with the Tamura 3-parameter Model with Gamma distribution (T92+G) with 100 bootstrap replicates. The tree with the highest logarithmic probability is displayed. The percentage of trees in which the associated taxa clustered is shown next to the branches. Each sequence is indicated by its GenBank accession number. The boxes indicate the sequences obtained in this project.

Figure 4

Venom separation analysis of Chihuahuanus coahuilae. The venom obtained from scorpions was separated by RP-HPLC, using a C18 column in a 0 to 60% acetonitrile linear gradient, in 0.1% TFA. Selected fractions marked with numbers and red arrows were further analyzed by mass spectrometry.

Figure 5

Venom separation analysis of Chihuahuanus crassimanus. The venom obtained from scorpions was separated by RP-HPLC, using a C18 column in a 0 to 60% acetonitrile linear gradient, in 0.1% TFA. Selected fractions marked with numbers and red arrows were further analyzed by mass spectrometry.

Figure 6

Surveillance of antimicrobial activity in venom fractions obtained from Chihuahuanus coahuilae. Selected HPLC fractions were assayed at 3 µg/µL in (A) Gram-positive Staphylococcus aureus ATCC 29213 and (B) Gram-negative E. coli ATCC 25922. Red circles display the inhibition halo of the selected fractions. Ampicillin and Kanamycin as a positive control, and H₂O negative control.