LDLo does not differentiate from LD50 across studies.
65 out of 160 ophidian species had at least one LDLo value. These 65 ophidian species had 196 LDLo values, and 809 LD50 values. In the meta-dataset, 28 of these 65 ophidian species (~43%) had an LDmin that was, indeed, an LDLo. However, 25 of the 65 ophidian species (~38%) had LDLo values that were the meta-study LDmax, which was against expectations. (see Equation 1 in the Supplementary Files)
For figure 2 and 3, the range fraction is determined by equation 1. This way, relative toxicity between ophidian species is normalized, and comparison of variation can be done between ophidian species. In Figure 2, the mean of the LDLo fractions is 31.1%, and the mean of the LD50 fractions is 30.0%. The standard deviation of the LDLo fractions mean is 36.6%, and standard error is 2.9%. The standard deviation of the LD50 fractions mean is 33.0%, and standard error is 1.6%. Even by the less stringent method of standard error, the difference between them is insignificant. Thus, the mean of the LDLo and LD50 range fractions for the 65 snake species out of 160 are not meaningfully differentiated.
One might argue that significant difference might be seen in LDLo values for single test-animal-species, so this was examined in figure 3. Here, as above, the data does not show this. Instead it shows that for 6 out of 7 test-animal-species, mean LDLo is higher than LD50, and for 3 of them, this exceeds standard error. (Rat, mouse and dog.) Note that this also occurs for the highest N dataset (mouse). Only the sparse data for cat has mean LDLo below meanLD50, which is probably not significant. However, using median, 3 test animal species have higher LD50 than LDLo (dog, cat, and rabbit),
Strengthening this point, for 10 ophidian species, both LDmin and LDmax were LDLo’s. The mean number of DB entries for an ophidian species with one or more LDLo values was 2.9.
Consequently, because LDLo did not differentiate significantly from LD50 when examined across studies, LDLo designations were categorized together with LD50 for the rest of this meta-analysis.
Route of inoculation minimum and maximum lethal dose
In figure 4, there is good support for the concept that IC (intra-cerebral) < IV (intra-venous) < IP (intra-peritoneal) < IM (intra-muscular) < SC (subcutaneous). This is the only hypothesis not falsified in this analysis. However there are contradictory instances.
Out of 22 intracerebral injections (IC), 15 were LDmin values, which is as expected. So, approximately 2/3rds the time, an IC injection was the minimum, and the N should be meaningful at 22. Using a synthetic x-axis the LDmin 0.859 R2 coefficient of determination suggests that approximately 86% of the distribution fits the assumption that route of inoculation varies as IC < IV < IP < IM < SC. The curve fit for LDmax shows the opposite trend, with a good R2 suggesting approximately 86% of results can be attributed to route of inoculation distributed in this manner.
However, 18 out of 228 of the subcutaneous injections were LDmin values, which makes SC, unexpectedly the route of highest toxicity for 11% of the 160 ophidian species. Out of these 18, there were 2 venoms with strong hemotoxic or nephrotoxic effects, and 13 were neurotoxic.
Venom toxicity range fold change
The range of venom toxicity per ophidian species within the mouse test species has a mean average of 2.00 logs (98.97 fold change) within a single test-animal-species, as shown in figure 5. This is 2.47 times the 1.6 logs (40 fold change) documented in literature for toxicity difference between test-animal-species, as discussed above. For all test-animal-species together, the mean range is 2.97 logs (936.59 fold change), which is approximately 23 times what current literature indicates.
The largest range fold change seen for an ophidian species tested in mouse for one route of inoculation is Crotalus oreganus, 2.15 logs (141.3 fold change), N(studies) = 26. The largest range fold change for an ophidian species tested only in mouse for all routes of inoculation is Crotalus horridus, 3.6 logs (4,150 fold change), N(studies) = 16, routes of inoculation: SC/IM. For one ophidian species data for all test-animal-species, including all routes of inoculation, the largest range fold change is Naja naja, 4.76 logs (57,471 fold change), N (studies) = 27, N (test-animal-species) = 9, routes of inoculation: unknown/IC between rabbit (LDmax) and rat (LDmin). These are among the highest N studies counts for ophidian species. Note that a specific ophidian species mentioned does not mean this species has been determined to be the most venomous, or the widest range of all.
One might ask whether the range fold change increasing holds up when a single species and single route of inoculation is examined. We see this in figure 6, compared to all data, where the range fold change is plotted against the number of studies. By inspection, one can see that the range fold change does appear to increase as the number of different studies rises, and the curve fit appears to be likely an exponential.
Figure 7, which looks at single test species for multiple routes of inoculation, shows a linear regression trend that reaches significance for the range fold change increasing as the N for number of studies gets larger. This graph appears to signal the same thing that a set of ecological diversity transects continuing to increase would. It indicates that to fully characterize ophidian venom lethal doses, probably requires more than 50 different studies.
Regressions of LDmin
Null hypotheses for minimum lethal dose: A.) Minimum lethal dose within each ophidian species varies by less than a factor of 2. B.) The minimum lethal dose does not correlate with the number of times the lethal dose has been tested (e.g. the number of LD DB entries).
Alternate hypothesis: Minimum lethal dose varies by more than a factor of 2, and minimum lethal dose correlates with the number of times dose has been tested.
Table 2: Regressions curve fit summary for LDmin, rounded.
Correlation examined
|
R2
|
Number of routes of inoculation
|
0.21
|
Number of test species tested
|
0.31
|
Number of LD DB entries (Number of studies)
|
0.396
|
Number of routes of inoculation correlation to LDmin
In figure 8, as the number of routes of inoculation increases, the likelihood of having more test-animal-species for the ophidian species also increases. The fitted curve is probably determined by the probability of inclusion of a lower lethal dose value rising as the number of inoculation routes goes to 5, because, as was seen above, IC < IV < IP < IM < SC does tend to hold true.
Here in figure 9 the apparent drop visible in the fitted curve is 1.5 logs, a fold change of 32X. Similarly to the above, it should be expected that lethal dose would drop some with larger numbers of test-animal-species, because literature indicates that some animals are up to 1.6 logs (fold change of 40X) more susceptible to certain venoms than others, and there is some frog data in the dataset.
Additionally, the more test-animal-species there are for one ophidian species, the more likely it is that there will be more routes of inoculation. However, in the dataset, there are multiple instances of the same test-animal-species occupying LDmin and LDmax, and quite a few are very close to this state, which suggests that, indeed, the number of times an ophidian species is tested is a major factor.
Number of DB entries (studies reported) per ophidian species correlation to LDmin
In figure 10 the “All data LDmin” fitted curve does not control for different test species. To address this criticism, “Mouse LDmin” shows the same graph filtered for inoculation of mice only. The R2 value of 0.245 is not as good as the 0.39 R2 value is for all data, however, the N is lower, and by inspection, there is a fairly good match for the curves. If there were no correlate by number of studies per species, then the fitted curves should be flat, whereas, both fitted curves span over 1.75 logs, and have fairly close exponents and constants. Consequently, these data suggest that the primary correlate for lethality is the number of studies that have been performed.
Regressions on range fold change
The range fold change is Rmax ÷ Rmin. The R2 values for these regressions are larger than what we see above. These data indicate that there is a correlation for all measures with number of DB entries for lethal dose (number of studies reported). The number of routes of inoculation has a meaningful correlation for all data, and for single test species. Fit equations in this section are of the form y = C∙emx .
Table 3: Regressions on all data for range fold changes of highest over lowest dose. All data (LD-ADrf), single species (LD-SSrf), single species, single route (LD-SRrf). Number of species tested is not applicable for LS-SSrf, of LDSRrf. Number of routes is not applicable forLD-SRrf.
R2 below 0.20 not shown. N = 160 ophidian species.
Data, by species
|
LD-ADrf R2
|
LD-SSrf R2
|
LD-SRrf R2
|
N
|
N = 160
|
N = 160
|
N = 113
|
Number of routes of inoculation
|
0.43
|
0.41
|
NA
|
Number of species tested
|
0.46
|
NA
|
NA
|
Number of LD DB entries
|
0.56
|
0.41
|
0.26
|
Table 3 confirms that the number of studies done is probably the primary correlate of toxicity for whole venom, and just above the correlation of number of test-animal-species used. The number of routes of inoculation roughly ties with the number of species tested.