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This may be due to arthropods expressing only a single variant of voltage gated sodium channel [ 48 , 49 ]. Alternatively, since the differences between Gallus, Danio, and Mus may be the result of the former two being tested in an early stage of development, the large difference in variability between the arthropod LD50 values and the Mus LD50 values could be indicative of a selective pressure on scorpion venom for high lethality to vertebrate predators.

Since the family Buthidae is considered monophyletic, this difference may be the result of a single evolutionary event. Materials and Methods 4. Species Selection We selected 10 scorpion species from three families in order to sample a broad range of venom potencies.

We selected five target species consisting of two arthropods the larvae of the wax moth, Galleria mellonella; and mealworms, Tenebrio molitor and three vertebrates zebrafish embryos, Danio rerio; chicken embryos, Gallus gallus; and mice, Mus musculus.

The contact points were wetted with saline to facilitate electrical conductance.

The telson was not part of the circuit, thereby avoiding any changes to the venom due to the applied voltage. The amount of dry venom content per specimen was obtained by dividing the total dry mass by the number of extractions required to obtain it.

Scorpions were given a resting period of at least two weeks between consecutive milkings. Venom solutions were applied to three days old chicken embryos, and mortality was ascertained after 24 h by candling the eggs. See [ 30 ] for details on this method. WT ABTL zebrafish embryos were injected with serially diluted venom solutions at three days post fertilization DPF in sample sizes of 20 embryos per venom concentration.

Survival was monitored at 24 h post injection by visual inspection of heartbeat.

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Embryonic bodyweight was estimated as the average drained weight of 30 embryos at three DPF, measured in triplicate on a high precision scale. Every mealworm was individually weighed on a high precision scale. Venom solutions were applied to mealworms of Venom was injected laterally on the ventral side, between the sixth and seventh abdominal segment, keeping the needle as close to the body wall as possible to avoid damaging the internal organs.

Different dosages of venoms were tested; 0. Higher concentrations for Babycurus jacksoni and Buthus ibericus where needed to calculate an accurate LD50 and therefore additional treatments of 4 and 2.

Mortality was assessed over a five-day period by looking at color change mealworms turn black quickly after death and by applying physical stimuli to elicit a response.

The waxworm assay was similar to the mealworm assay. Waxworms at the last instar before pupation were used. Different dosages of venoms were tested, 0.

Additional treatments for Centruroides gracilis and Androctonus australis were needed to accurately calculate LD50, 2 and 0. The LD50 test was carried out on male albino Swiss mice of approximately 19 g body weight. Different amounts of venom from G. Three mice were used in each dose and in the negative control.

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Injections were performed intraperitoneally using physiological saline solution as vehicle and negative control. We analyzed the intoxication level during the first 2 h after the injections, evaluating any recovery after 20 h after the injection as described by Valdez-Cruz et al. All further statistical analyses were performed in R 3. Our own LD50 dataset was augmented with LD50 data from mice available in the literature.

For some species, several LD50 values are available in the literature.

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When this occurred, we used the mean of the published values for further analysis. As the LD50 values span several orders of magnitude, they were log10 transformed prior to further statistical analysis. Conversely, to test if scorpion species were similar in their effects on the panel of target organisms, we performed correlations on the responses of the target organisms for the 10 scorpion species.

For the scorpion dataset, CO1 sequences were used to calculate the phylogeny and branch lengths See Table S1 in Supplementary Materials. The tree was calculated in MEGA 6.

We then used the mean path lengths method to estimate relative node age [ 56 ] and obtained an ultrametric phylogeny, setting the root age to one. This tree was also scaled to a root age of one. Differences between buthids and non-buthids were tested per target species using a t-test.

This work was funded by an NWO bezoekersbeurs None of the funding bodies were involved in the study or the preparation of the manuscript.

Supplementary Materials Click here for additional data file. Several milkings were made per sub adult specimen in some cases, Table S3: Toxicological analysis of the venom of Grosphus grandidieri. Note that Grosphus flavopiceus has been used to represent the position of G.

Numbers at branches indicate divergence times in millions of years. Author Contributions A. Conflicts of Interest The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Descargar Veneno Para La Corona 2ヲ (Narrativa) [pdf] Toti Martinez de Lezea - crinarwhorep

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