Comparative genomic analysis of crustacean hyperglycemic hormone (CHH) neuropeptide genes across diverse crustacean species

The manuscript presented by Chang and Lai describes a collection of CHH neuropeptide genes in over 100 crustacean species.

I read with interest this short report and could appreciate that a majority of neuropeptide studies have been limited to decapod crustacean species previously. In addition the presence of neuropeptide transcripts in other divergent crustaceans including those from basal lineages (copepods and branchiopods) are presented.

Like in their previous work (Chang and Lai, 2018, BMC Genomics¹), a broad sampling strategy was followed. Here the approach was across multiple crustacean lineages, which allows for drawing conclusions on the evolutionary trajectory of CHH genes and their potential functional role during life histories.

Although the paper describes a single finding, it is a valuable asset, as it provides an inventory of a high-quality list of carefully curated CHH neuropeptide transcripts for future functional studies.

The paper is well-written and concise, and definitely fits with the scope and eligibility of the F1000Research guidelines.  

As a minor comment: it would be good to describe how the tree in Figure 1 was made in the legend.

The authors collected and analyzed transcriptome datasets for 112 crustacean species (representing three Classes - Malacostraca, Branchiopoda, and Copepoda), as well as those for 5 non-crustacean (arthropod) species (Insecta, Arachnida and Chilopoda) (Supplementary Table 1; Fig. 1), retrieved from public repository.
 
Collectively, 413 genes are annotated based on sequence and motif similarity as encoding peptides belonging to the so-called CHH/MIH/GIH family. The number of CHH/MIH/GIH genes identified from each crustacean species is shown as heat maps (Fig. 2). Protein sequence, annotation information, etc. are listed (Dataset 1 and 2). Between-species % identity is reported (Supplementary Table 2). Multiple sequence alignment on representative CHH/MIH/GIH sequences is presented in Fig. 3 to show the 6 conserved cysteine residues (Fig. 3). A phylogenetic tree is built based on the multiple sequence alignment data (Fig. 4).   
 
The main drawback of this work is it only results in a 413-sequence inventory of CHH/MIH/GIH family peptides, but with little information (nucleotide length, amino acid sequence).

The authors did not describe how the tree presented in Fig. 1 was built with what sorts of data. If they took a constructed tree from other source, this should be cited. There are obviously other current interpretations of a phylogenetic relationship of Crustacea, in addition to the one shown in the manuscript.

For annotation information (Dataset 2), it should be more discerning in that peptide should be assigned as CHH, MIH/GIH, MOIH (mandibular organ-inhibiting hormone) or ITP (ion transport peptide), instead of only giving an inclusive description - Crustacean CHH/MIH/GIH neurohormone family.

Results derived from further analysis of the sequence data (Figs, 2, 3, and 4) are not adequately discussed or lead to conclusions that have been previously established.
For example, the only conclusion made with Fig. 3 is the 6 highly cysteine residues, a signature of the family already extensively described.  Figure 4 is left without discussion. Moreover, it is not mentioned which type of peptide (CHH, MIH/HIV, or ITP) the different clusters highlighted in the tree belong to.

Part of the discussion (left column, p. 4) starting with “It was previously reported that multiple gene duplications of CHH family peptides…..” is not particularly relevant to the data being discussed and is again making a point already extensively described and discussed. It should also be mentioned that genomic data are more adequate than transcriptomic data when discussing gene duplication and gene copy number (Fig. 2). 
Figure 2 is also left without discussion. In addition, data presented in this figure might be suffering from the criticism that the number of CHH/MIH/GIH genes assigned to each species is likely biased as the number will be absolutely influenced by the type of tissue used for sequencing (eyestalk ganglia, other tissue, or whole animal) and sequencing depth. Accordingly, the relevant information should be given in Supplementary Table 1 and comparison should only be made among species with the same sequenced tissue type (e.g., eyestalk ganglia) and comparable sequencing depth. 

Overall, while the methods employed are largely (but not entirely) appropriate for the intended analysis, the goal of the study is not clearly set and this work only produces a sequence inventory without novel finding or solid discussion. Additional analyses expected to yield novel finding should be added to the manuscript. For example, with a vast amount of sequence information that could be extracted from a set of 413 peptides from animals encompassing 3 Classes, would it not be possible to uncover some function-defining sequence characteristics or motif? This piece of information would be useful for functional analysis, which the authors thought should be an important aspect of the next stage of research.