The manuscript describes the identification of several novel conotoxins from a relatively understudied worm-hunting cone snail species using transcriptome sequencing on the Illumina platform. The authors have used specimens of small, medium and big sizes and sequenced the transcriptomes of each, in addition to sequencing a normalised dataset and the venom bulb. Overall their methods are sound and while most of the manuscript is of good quality, there are some issues that ought to be rectified prior to publication. If the authors address these comments, I think it will make it a well-rounded paper.

Method/work concerns:

1) The discrepancies between the text in lines 156-160 and table 1. For example, the text
suggests the middle-sized specimen's transcriptome produced 4.58 Gb of raw data, while
Table 1. Suggests it is 8.39. This needs to be resolved so that the results are reliable and
to ensure that the authors have not misinterpreted their data.

2) The authors have used a signal sequence cut-off of 75% to designate sequences into
superfamilies. While this was used in some of the earliest work in this area (E.g.: Dutertre
et al, 2013, Mol. Cell. Prot, and Jin A.H. et al, 2013, Mol. Cell. Prot.), more recent work
particularly in the light of the comparison of signal sequences in various superfamilies by
Lavergne et al, 2014, BMC Genomics suggests that 53.30 % is a more suitable cut-off.
Lavergne et al, 2015, PNAS, have used this cut-off and I have also incorporated it into a
paper on transcriptome analysis in Prashanth and Lewis, 2015, Toxicon. Alternatively,
Barghi et al, 2015, Mar. Biotechnol. have used different cut-offs for different
superfamilies. The authors should reclassify their sequences using the signal peptide
identity cut-offs used currently.

3) The authors report that they discovered the expression of toxins within the venom bulb in
the conclusions section (Line 393 - 395) but admit that the expression levels are rather
low. This contradicts a previous study (Safavi Hemami et al, 2010, J. Prot. Res) that
found no evidence of venom expression in the venom bulb. Figure 7. Compares the
expression of venom peptides in the venom bulb and duct, but since peptides exclusively
expressed in the venom bulb haven't been PCRed, this part of the study is inconclusive.
The bands from venom bulb sample are also extremely faint in keeping with the
extremely low levels of expression reported. The simplest explanation for what the
authors see is that the peptides likely arise from the proximal end of the venom duct,
which culminates within the venom bulb and I think the authors maybe over-interpreting
their data in this particular instance.

4) Please add a section in the methods to indicate how the RPKM calculations were
performed.

5) Figure 5. - This figure is a bit confusing and requires constant referral to the legend to
make any sense of it. To make it easier for readers and to tease out a better comparison of
the three transcriptomes, I suggest taking the top 20 peptides by RPKM for one of the
transcriptomes, say middle and comparing those values with the corresponding RPKM
values for the same peptides from the different specimens.

6) The authors need to add some discussion of Lavergne et al, 2015, PNAS that found that
despite using a variety of assembly tools and parameters on Illumina data, only an
exceedingly small fraction of putative conotoxin transcripts were found within the
assembled reads with upwards of two thousand putative transcripts found among the
unassembled data of which, many were also validated through proteomics. In this study,
the authors have compared various specimens using only assembled data, which runs the
risk of missing many of these low-level transcripts that have often been shown to contain
various novel conotoxins.

Other comments/suggested corrections:

Line 64: Remove the 'The' before 'venomous cone snails'.

Line 67: Cite Dutertre et al, 2014, Nat. Commun. for defensive role of cone snail venoms.

Line 72: Replace 'had' with 'have'. The age of the genus is widely accepted as being 55 million
years ago - See numerous reviews, studies etc.

Lines 75-78: Please rephrasing the sentence starting with 'The modified radular' as it is unclear.
Also, poison refers to substances that are ingested and venom is used to refer to substances that
are injected.

Line 79: Conotoxins cause paralysis (loss of movement and sensation) and not just numbness
(loss of sensation).

Line 84: Requires a reference. I suggest Lewis et al, Pharmacol. Rev., 2012.

Line 113 - 116: The authors suggest that the venom bulb has been mistakenly referred to as the
venom gland, but provide no reference for the claim. The venom gland has been used
interchangeably with venom duct. Eg: Terrat et al, 2012, Toxicon.

Line 138: Needs a reference. There are some good reviews/papers comparing venomics to
traditional approaches.

Line 140: Also add references to Barghi et al, 2015, Genome Biol. Evol., Dutertre et al, 2014,
Nat. Commun., SWA Himaya et al, 2015, J. Prot. Res., Lavergne et al, 2015, PNAS. All these
papers also present transcriptome-sequencing results of the venom ducts of cone snails.

Line 156-160: See point 1. Above.

Line 161: Should be assemblers rather than assembler.

Line 163: Replace 'were' with 'was'

Line 197: Please see point 2. Above.

Line 207: The number of novel superfamilies etc. should be revised based on the more current
signal sequence cut-off score of 53.3%

Line 249: Should be 'about the same number'.

Line 299: Should be 'As expected'.

Line 384: Should be 'As expected'.

Line 393-395: See point 3 above.

Line 414: Should be 'The second approach'.

Line 431: Shouldn't it be 'reads with more than 10% of unknown nucleotides were removed'?

Line 443: Should be 'homology searches'.

Line 459: See point 2 about classification.

Line 475: Should be 'cDNAs were reverse transcribed'

Level of interest
Please indicate how interesting you found the manuscript:
An article whose findings are important to those with closely related research interests

Quality of written English
Please indicate the quality of language in the manuscript:
Acceptable

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