Review 1 (Joseph J Schall, University of Vermont, Department of Biology, Burlington VT, USA; nominated by Laura Landweber, Dept. of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ, USA)
The arrival of the avian malaria parasite Plasmodium relictum on the Hawaiian islands in the early 20th century (about century after a competent vector was introduced by accidental human action) led to an ecological catastrophe of almost biblical proportions. Many endemic bird species were reduced in density, altered in their distribution, or driven to extinction. The phenomenon of the bird community was destroyed. Apart from the heartbreaking esthetic loss, these events argue that pathogens may have always played a role in shaping biodiversity. Even rare such events would have long-term importance. And, the Hawaiian bird malaria story warns us the emerging infectious disease can have the broadest consequences.
The Hawaiian bird malaria team has produced a string of reports that rank among the most important in the history of parasitology. Here, the team members examine the genetic diversity of the TRAP gene. TRAP is an important surface protein involved in the weird gliding movement of apicomplexan cells.
The authors report three general findings:
1. The structure of the gene and protein is characterized in some detail.
I have only limited experience with such analysis, and so can offer no informed comments.
2. There is some relationship between virulence and allele diversity of TRAP per host. The manuscript brings up "host survivorship" [Abstract line 38] or "surviving birds" [first mention on line 136]. What this means is not provided until line 431/432, so this survivor issue drops out of nowhere. Also, there were only 18 birds under study, so with this small sample size, nothing can be inferred about the allelic diversity of TRAP and survival of experimental birds. Therefore, I will not pursue this issue here, and feel that the topic should not be included in an otherwise fine presentation.
Authors' response: This is quite right. This manuscript is intended as a genetic analysis of several individual hosts to begin to characterize diversity and develop additional methods for future population-level analysis based on trap. It not at all intended as an analysis of survivors and non-survivors of avian malaria. However, many of the individuals under study were involved in experimental infection studies. The author's feel compelled to include this data here, but we have reworked it so as to more accurately reflect its somewhat minimal significance.
3. There is a substantial diversity of TRAP alleles even within single infections. At least 88 alleles are identified, and this appears to underestimate the true diversity. Of these, 28 appear in more than one bird examined, and are thus given the closest attention. This allelic diversity could be a result of (i) a large number of genotypes of the parasite arriving over the past decades; (ii) mutation of the TRAP gene leading to diversity after the parasite became established; (iii) a combination of (i) and (ii); (iv) rapid mutation of TRAP within individual infections leading to new variants even if an infection began with a single allele; (v) PCR artifacts leading to an apparently high diversity. My reading of the results comes to a different outlook on the story than the authors', but this different conclusion would still would be a very interesting and important.
Malaria researchers for many years have suspected that the surface proteins of Plasmodium may be under selection to mutate rapidly. Diversity may be favored in response to the attack of the immune system. Previous studies on the malaria parasites of humans have found a very large number of alleles for surface protein genes cycling in a single population (84 alleles detected in one study, similar to the results for TRAP in P. relictum). A very rapid mutation rate may even lead to new alleles appearing during the course of a single infection.
The "Conclusions" begin with the statement: "This study shows that the genetic diversity of Hawaiian isolates of P. relictum is much higher than previously recognized. These results indicate the widespread presence of multiple-genotype infections in the native birds of Hawaii." But, another conclusion could be reached. If there is very rapid mutation of TRAP (at least the variable portions of the gene), we might imagine a clonal genetic structure, with no variation in the genome except for the genes coding for surface proteins. That is, case (iv) and even case (v) above may be the situation.
Authors' response: These are valid points. We have now included a phylogenetic network analysis (at the suggestion of another reviewer) from which we were able to provide a clearer presentation of the relationships of (sequenced) haplotypes. These data appear consistent with (iv) above, rapid mutation, and have now been included.
The number of infections studied was 2 from lab birds (from two infections collected in nature, I assume (isolates KV115 and K1), and 7 more wild-caught birds. So, we have 9 infections under study. From those 9 infections, there are 80+ alleles. Also reported is that more alleles are detected if the PCR is run for 80 cycles vs. 35 cycles. This last result suggests that mutations are taking place during the PCR. After all, PCR replicates what happens in cells, so this seems to indicate that the gene is very prone to copy errors.
Authors' response: While one can never totally eliminate the possibility of random PCR error, we took several steps to minimize inclusion of clones with higher potential for error. We only included clones produced independently in two or more birds under the assumption that random error is unlikely to occur independently twice at the same restriction sites. We used the Promega enzyme system for two reasons. One was that it allowed for the A- overhang needed for cloning, and also it was the common system in use at the time of data collection (prior to the common availability of the Roche enzyme system). We also included PCR+1 methods into our protocol to insure sequencing of only homoduplex clones, which eliminates issue of chimeric sequences due to heteroduplex formation in PCR. Even the clones produced primarily using the Roche enzyme system had at least one cycle using Promega (PCR+1 cycle, which also added the overhang). While there is some degree of randomness to the polymorphisms observed among sequences, the high degree of conservation of sequence in regions known to be critical to proper function (and are quite highly conserved across species) doesn't support the proposition of high PCR error, which one would expect to be random.
The study has available the data to test the "real genetic variation" vs. "rapid generation of variants" hypotheses. Infection K1 was passed to 6 birds, and KV115 to 5 birds. Thus, we would expect to find the same alleles in all the recipient birds as in the donor bird. Are the same alleles found? Over the course of the infection, are the same alleles found? Or, are different alleles found in the recipients, and do new alleles show up during the infection? There are no such bird-by-bird results presented in the manuscript.
Authors' response: We wish we had the samples from the original infections with which to compare. Unfortunately, we don't. We are in the process of trying to complete this type of analysis at this time with more recently derived samples. Thank you for your suggestions.
Researchers working with human malaria parasites would read this paper with great interest, and would conclude that this is a very nice example of the rapid mutation of a surface protein gene, and another example of how these proteins interact with the immune system and are selected to generate variation (analogous to the VAT's in Trypanosoma).
Review 2: (Daniel Jeffares Wellcome Trust Sanger Institute, Population and Comparative Genomics Team, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK; nominated by Anthony Poole, Arrhenius Laboratories for Natural Sciences, Department of Molecular Biology & Functional Genomics, Stockholm University, SE-106 91 Stockholm Sweden)
In this work Dr. Susan Jarvi and colleagues examine the genetic diversity of the TRAP gene in Hawaiian isolates of the avian malaria parasite Plasmodium relictum. They PCR amplify the TRAP gene (which is known to be highly polymorphic in other Plasmodium species) from DNA extracts from the blood of 18 birds of various Hawaiian species, clone the products to allow detection of multiple parasite isolates within each bird, and examine the clones by restriction fragment length polymorphism and sequencing.
The study examined TRAP diversity from 7 birds that had been naturally infected with P. relictum, 5 birds that had been experimentally infected with one P. relictum isolate that had been maintained in laboratory contained mosquitoes, and 6 birds that had been experimentally infected with another laboratory maintained isolate.
The study found multiple TRAP haplotypes in most birds. Since Plasmodium parasites are haploid in blood stages this indicates that there is significant genetic diversity in Hawaiian P. relictum and that most infections contain multiple parasite clones.
Jarvi et al. clearly show there is significant genetic diversity in Hawaiian P. relictum and that most infections contain multiple parasite clones. However, greater understanding of the TRAP gene, or of the population genetics of Hawaiian P. relictum will require more data. However the reliable data produced are rather limited, and some of the data analysis is handled inappropriately, in my view. I detail these problems below.
First, the authors own controls indicated that their methods (PCR amplification with a non proof-reading Taq for 80 PCR cycles) produced some PCR-induced polymorphism artefacts. Although PCR-induced polymorphism is not expected to produce patterns such as geographic or phenotypic associations (rather just to add noise), it will increase measures of genetic diversity. The majority of data was produced with the non proof-reading Taq using 80 PCR cycles, however there is no consideration of this in results/discussion sections.
Authors' response: We completed this small trial to evaluate these variables in our system. We don't believe that PCR error is significantly impacting estimates of genetic diversity in this study as we have included only clones found independently in two or more separate birds. Please also see previous response to similar concerns of Reviewer 1.
Also, RFLP haplotype data from two birds were effectively removed from the study because they contained unique haplotypes. This will also have the effect of increasing estimates of diversity.
Finally there was a non-random selection of which clones to sequence. Proportionally more clones were chosen from the rarer RFLP haplotypes. This choice will also have the effect of increasing measures of genetic diversity from the sequence data. The result is that while estimates of genetic diversity are high from sequence (genetic diversity =~ 0.06) it is difficult to know how much of this value is artefact, and hence how to compare these values directly to other studies.
Authors' response: We removed the birds from the estimates of genetic diversity because of the minimal data collected (only 2 clones and 1 clone were obtained from each of these birds). We did not try to produce more clones from these birds, but kept them in the overall study as we had obtained sequence data from one of them. To include them in the estimates would bias them in the other direction. We sequenced 18 clones from the most common RFLP haplotype (77) and 15 clones representing 12 other haplotypes. I don't believe we disproportionately selected the more rare haplotypes. Since this is not a population-level study, these results may not be directly comparable to other studies.
Another limitation of this study is that apart from the RFLP analysis, the sequence data produced in this study is relatively small. Only 14 TRAP sequences were produced from one of the cultured isolates (10 of which from a single bird infection), 16 from another (again, 10 of which from a single bird infection) and 5 from wild isolates (none of which are derived from high fidelity Taq). The result is that few of the more interesting conjectures in the discussion have significant support. For example there was no significant difference between diversity estimates of the two mosquito-derived isolates, or the wild isolates, nor between those birds that survived experimental infections vs. those that did not. Collection of more sequences from more isolates would allow analysis of the frequency distributions of polymorphisms, and be much more informative. To be fair the authors state that population level genotyping is underway. I would caution readers from drawing too many conclusions until this analysis is available.
For example, I would regard any clustering of TRAP isolates with respect to mortality with strong suspicion. While the TRAP gene may contribute to mortality, an association between mortality was not shown (recall that most sequence data are from two isolates from experimentally-infected birds from cultured parasites), and even if it were shown we would need evidence that this association was not due to population subdivision or to linkage with another causal variant.
In summary I think that the data has been extended beyond what it is capable of describing, and may also be limited by PCR-artefacts. Certainly there is no subterfuge about this in the manuscript (the authors for the most part describe the limitations), but the results could well have been limited to a simpler story. Further work on this system, particularly with the improvements that have been shown to be necessary with the data in this work, should provide a much more informative picture.
Authors' response: I think it is important to realize that this manuscript is only intended as an initial attempt to begin to genetically characterize diversity in a previously uncharacterized species of Plasmodium. It not intended as an analysis of survivors and non-survivors of avian malaria. However, as noted above, many of the individuals under study were involved in experimental infection studies and we feel compelled to include this data here. We have reworked the discussion to reflect this concern. We also included a more detailed description of the isolates. Thank you for your suggestions.
Review 3: (Susan Perkins, American Museum of Natural History, Assistant Curator, Division of Invertebrate Zoology, Central Park West at 79th Street, New York, NY 10024: nominated by Eugene Koonin, Senior Investigator NCBI, NLM, NIH Bethesda, MD 20894)
I found this manuscript, by Jarvi, Farias, and Atkinson to be very interesting and thorough. It also represents an important addition to our knowledge of the nonmammalian and/or non-model system malaria parasites with respect to the genetic diversity that is naturally present in these systems. When I first began reading this manuscript, I immediately worried that some of the high amount of genetic variability might have been caused by heteroduplex formation and bacterial repair during the cloning process, so was very relieved when the authors said that they also incorporated PCR+1 methodology into this study. That said, though, I found it slightly unusual that instead of bumping up the reaction to 80 cycles, they did not also choose to try a scenario of using a very small number of cycles and then pooling these products – a method that is commonly used among microbiologists who are trying to assess diversity in an environment in an attempt to recover rarer organisms (sequences) that might be swamped out by more common ones through the PCR amplification process. My second suggestion is that I think that their sequence data would be far better represented by a haplotype network rather than a traditional, bifurcating tree. As the authors admit, the bootstrap support values on most nodes are very low and this is no doubt a result of a more network-like scenario of evolution that resulted from one or a few haplotypes founding these populations and then accumulating point mutations as the populations expanded and dispersed. A bifurcating, distance-based tree such as the one in Figure , will be complicated and distorted by this pattern of evolutionary change. Representing the data as such, along with perhaps shading to depict those sequences found in survivors vs. non-survivors might reveal more interesting and clear-cut patterns than this NJ tree could ever do.
Authors' response: Yes, I wish I had thought of that at the time (i.e., pooling reactions using smaller numbers of cycles). Thank you for your suggestion of a haplotype network. We completed this analysis and it is much more informative than a NJ tree. We have incorporated these data into the manuscript.
At times, this paper also seemed like a mixture of what would be two or perhaps even three different publications. There was the central issue of how much genetic diversity of the TRAP gene – and ergo, number of parasite clones, exists within infected birds and whether or not this diversity is related to host or geography. There was also the attempt to look at the correlation of the presence of certain TRAP sequences to survival in infected birds and this was discussed, in part, with particular changes within the TRAP molecule. Finally, there was the embedded study of enzyme, cycle number, and methodology (PCR+1 or not) and their relationships to the number of clones recovered. Perhaps the authors should consider if these three things might not be better split as such to show that first, the optimum method for detecting clonal diversity was found and used and then that the resulting data on the diversity of genotypes was then analyzed in a context to allow for the most powerful ability to correlate virulence with genotype (ideally by sequencing all genotypes present in these infections).
Authors' response: Yes, we are packing a lot in a single paper, but individually, the pieces do not seem substantial enough to warrant a publication on their own.
You may want to explain the origin of the K1 and KV115 isolates – these are not "cloned isolates" in the sense that human and rodent malaria researchers are used to thinking about them.
I would advocate for including the primer sequences here, even if they are published elsewhere. This will facilitate the ability of others to use them, if desired, without having to track down multiple other publications, which they may or may not have easy access to.
Authors' response: We have included a section describing the origin of the isolates, as well as the sequences of all primers with their references. Thank you for your suggestions.