Q2. What was the primary fixation of the head?
Primary fixation was of whole heads (eyes punctured) in a solution of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M pH 7.4 cacodylate buffer for 3h.
Q3. What is the main reason why the present study is limited to pit vipers?
The present study is limited to pit vipers because IR sensing in vipers is much more strongly associated with the presence of a single type of anatomically complex pit, because of the previous evidence (now known to be incorrect) that pit vipers possess one fewer type of retinal photoreceptor than non-pit vipers, and because the precise taxonomic limits of IR sensing and marked variations in retinal anatomy are not known in pythons, boas and their closest relatives (pythons and boas are not each other’s closest relatives and may have, at least to some extent, evolved IR sensing independently themselves).
Q4. What is the definition of two-tiered retinas in snakes?
Walls (1942) and Underwood (1967, 1970: 62) considered two-tiered retinas to be a characteristic of nocturnal snakes (and vipers are generally nocturnal).
Q5. What was the phylogenetic tree used to estimate the substitution rates of sw?
Codeml from the PAML 4.7 package (Yang, 2007) was used to estimate non-synonymous(dN) and synonymous (dS) substitution rates and their respective ratio (dN/dS or ω) for the sws1, lws and rh1 genes in vipers (without outgroups) using a phylogenetic tree congruentwith that of Pyron et al.
Q6. What is the significance of the substitutions in the spectral sites?
positive selection has been detected at spectral sites not known to have substantial tuning effects, but these substitutions have occurred independently within vipers and suggest that fine-tuning of visual pigment λmax is of some importance in viper sensory ecology.
Q7. What software was used to analyze the flat mounts?
The immunolabelled flat mounts were analyzed with a laser scanning microscope (LSM) Olympus FluoView 1000 using the FV 1.7 software (Olympus).
Q8. How long did the incubation time in the secondary antiserum mixture be extended?
Here incubation time in the mixture of the two opsinantisera JH492 and sc-14363 was extended to three days at room temperature, incubation in the secondary antiserum mixture was for 1 h.Stained sections were analyzed with a Zeiss Axioplan 2 microscope equipped withepifluorescence.
Q9. What was the fit model for all three opsin genes?
jModelTest 2 (Darriba et al., 2012) was used to ascertain that GTR+G+I was the best-fit model of sequence evolution for all three opsin genes according to AIC and BIC scores.
Q10. What is the likely site for tuning in snakes?
If an additional tuning site, previously unknown for vertebrate LWS, might be operating in snakes, then the most likely candidate that can be identified from current data is site 174, which is phenylanaline (F; a neutral hydrophobic amino acid) in A. contortrix (which conforms to predictions) but leucine (L; an aliphatic non-polar amino acid) in B. arietans, P. regius and X. unicolor (which have surprisingly low λmax values).
Q11. What is the topic of substantial interest in sensory and evolutionary biology?
This is a topic of substantial interest in sensory and evolutionary biology, and among vertebrates there are several striking instances of new and/or elaborated senses occurring in taxa in which other senses are degenerate.