Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae

The life cycles of many free-living nematodes contain developmental switches that allow individuals to either develop directly to adulthood, or to arrest development as a stress resistant and long-lived dauer larval stage. Here, in a panel of recombinant inbred lines derived from the Caenorhabditis briggsae isolates HK104 x AF16, we use methodologies developed for to map C.elegans quantitative trait loci (QTLs) affecting the number of dauer larvae present at the point of food patch exhaustion. These analyses provide strong support for three QTLs and are suggestive of a further two. Simon Harvey ( ) Corresponding author: simon.harvey@canterbury.ac.uk Green J and Harvey S. How to cite this article: Quantitative trait loci mapping of dauer larvae development in growing populations of 2015, :1447 (doi: [version 1; referees: 3 approved with reservations] Caenorhabditis briggsae F1000Research 4 ) 10.12688/f1000research.7546.1 © 2015 Green J and Harvey S. This is an open access article distributed under the terms of the , Copyright: Creative Commons Attribution Licence which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver The author(s) declared that no grants were involved in supporting this work. Grant information: Competing interests: No competing interests were disclosed. 15 Dec 2015, :1447 (doi: ) First published: 4 10.12688/f1000research.7546.1 Referee Status:


Introduction
Caenorhabditis elegans is an important model species, but there is a need to further understand its ecology and that of the other species with which it is associated [1][2][3] .Active, growing, populations of Caenorhabditis nematodes are normally associated with nutrient and bacteria-rich substrates such as rotten fruit or very fresh compost 1,3,4 .Growing populations can reach very large sizes, and worms would be expected to have optimized fitness (population growth and/or the production of dispersal stages) under such conditions.Appropriate development of growing worms as dauer larvae, the developmentally-arrested alternate third larval stage, is therefore likely to be central to genotype survival.
We have previously looked at dauer larvae development in growing populations of C. elegans [5][6][7] .These analyses identified both extensive variation between wild isolates in the number of dauer larvae present when a bacterial food patch is exhausted 6 and large numbers of QTLs effecting this trait in introgression lines (ILs) produced from the isolates N2 and CB4856 6,7 .Naturally, C. elegans has however been found to be associated with both C. remanei and C. briggsae 1,3,8 .Here, using the methodologies developed for C. elegans, we have investigated dauer larvae formation in growing populations in a panel of C. briggsae recombinant inbred lines (RILs) produced from the isolates AF16 and HK104 9 .

Methods
The C. briggsae RILs were generated from reciprocal crosses between males and sperm-depleted hermaphrodites of isolates HK104 and AF16 9 and were obtained from Asher Cutter (University of Toronto).Worms were maintained using standard methods 10 and all assays were conducted at 20°C.Assays were performed as previously described 6 , with populations initiated from single fourth larval stage worms (L4s) grown from synchronized, arrested, L1s with 100 μl of a 20% w/v suspension of Escherichia coli in water and monitored daily until food exhaustion.At this point, the population size and the number of dauer larvae were determined 5,6 .RILs were analyzed in three experimental blocks with AF16 and HK104 assayed in each block and five populations initiated per genotype.Treatments (genotypes) were randomized within blocks and plates were blind coded such that counts were performed without knowledge of worm genotype.Populations that failed to grow were discarded.
The RILs have previously been genotyped at 451 markers distributed across all six chromosome pairs 9 and these data were used for this analysis.QTL mapping was performed by composite interval mapping (CIM) using QTL Cartographer v2.5 11 .Genome-wide thresholds (0.05) were estimated based on 1000 permutations of the data and CIM analysis undertaken with a 1.0cM walk speed.In combination with the marker distances, these data were used for QTL mapping and the generation of the results shown in Figure 2 and Table 1.

Results
Comparing the AF16 and HK104 controls between the experimental blocks indicates that the number of dauer larvae at food exhaustion varies extensively between blocks (H = 11.06,p = 0.004 and H = 8.21, p = 0.007, for AF16 and HK104, respectively: Figure 1A).This variation is much greater than that seen for population size at food exhaustion (Figure 1B).Such variability between experimental blocks has been observed before with this assay, and is likely to be due to variation in either the actual or perceived food quality between batches of bacteria 6 .Given the variation in dauer larvae formation between the experimental blocks we considered it unlikely that analysis of raw data would identify any QTLs, and this is what was observed (Figure 2A).Two approaches were therefore taken to determine if we could control for this variation.Firstly, we scaled the number of dauer larvae observed for each RIL either to the AF16 or HL104 controls within that experimental block (Figure 2A), e.g. the AF16 scaled value for a RIL was determined as (RIL mean -AF16 mean)/AF16 mean and this was used for mapping.Secondly, we analyzed each of the experimental blocks independently (Figure 2B).
In combination, these approaches identify three QTLs on chromosomes IV, V and the X, that affect the number of dauer larvae present at food exhaustion (co-localizing QTLs present in both Figure 2A and B) (Table 1).The analysis is also suggestive of additional QTLs on chromosomes II and V (Figure 2A and B; Table 1).A similar analysis of the population size at food exhaustion did not detect any QTLs.

Discussion
For a comparative analysis of dauer larvae development in growing populations, a panel of C. briggsae RILs were analyzed.These analyses indicate that the general properties of growing populations of C. briggsae are similar to those described for C. elegans 5,6 .This means these methodologies will be suitable for more direct comparisons between the species.The RIL analysis identifies a number of QTLs (Figure 2; Table 1) although these are not resolved to narrow genomic regions and therefore contain many hundreds of genes.They do however provide a starting point for attempts to identify causal loci.

Data availability
F1000Research: Dataset 1.The number of dauer larvae and the population size at food exhaustion for replicate populations of the RILs and the respective AF16 and HK104 controls from three experimental blocks, 10. 5256  Dauer larvae are dispersive forms of nearly all terrestrial nematodes, and are typically induced by the onset of food limitation and/or pheromone cues that are correlated with it.Given that the correctness of the decision to form a dauer (and thus abandon the direct path to reproductive adulthood) is expected to be highly contingent upon the exact ecological context in which it occurs, this trait should be both subject to strong selection and be potentially variable.This may be especially true for , which has a C. briggsae greater degree of geographic population structure than does its more famous congener, .

C. elegans
The authors take advantage of a set recombinant inbred lines between genetically distinct strains (lines that my own lab played a role in characterizing in 2011; see the Ross reference) to examine whether et al. there is indeed heritable variation for dauer formation, and if so whether there are any loci of variation with effect size big enough to show significant association with dauer formation.I have no doubts that this project is addressing a significant issue in biology, and the authors make some headway.
General enthusiasm aside, as Erik Andersen also notes in his review the main concern here is the noisiness of the assay that is mapped.In understanding how the dauer formation assays is done, I think I may see why it is so noisy.The authors are founding each plate with a single L4 larva and a fixed amount of food.This is easy to reproduce, so I see the appeal.However, to then reach starvation on the plate, we presumably are looking at the grandchildren of the founder worm.As an exponential process, tiny differences in the generation time or rate of egg laying in the first few hours would produce big variability in the total number of worms, and also in the increase of the signals that are stimulatory for dauer production.
One could try to model the nature of this variation, but I don't think this is author's goal.An alternative, though admittedly requiring a whole new set of experiments, would be to seed plates with 20 or 200 L4 larvae, and score dauer formation in the first generation.This ought to be far less variable, yet should also be assessing the same basic phenotype, which could be seen as the threshold signal required to induce dauer development.Rather than repeating the entire scan, perhaps the authors could take their most discordant RILs and repeat this assay on them?It may produce a stronger signal, or at least confirm the initial screen.
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.Competing Interests:

Dataset 1 .Dataset 2 .
The number of dauer larvae and the population size at food exhaustion for replicate populations of the RILs and the respective AF16 and HK104 controls from three experimental blocks http://dx.doi.org/10.5256/f1000research.7546.d109088The AF16 and HK104 data are presented in Figure 1.The RIL data are used to calculate the trait values used in QTL mapping.Marker distances (from Ross et al., 2011) for informative markers in the RILs analysed http://dx.doi.org/10.5256/f1000research.7546.d109089Dataset 3. RIL genotypes (from Ross et al., 2011) and analysed trait values for the RILs http://dx.doi.org/10.5256/f1000research.7546.d109090

Figure 1 .
Figure 1.Dauer larvae formation is variable across experimental blocks.The A) number of dauer larvae, and B) population size, at food exhaustion in AF16 and HK104 controls in the three experimental blocks.Values from each plate are shown, with red bars indicating the median values.

Figure 2 .
Figure 2. Quantitative trait mapping identifies regions affecting the number of dauer larvae at food exhaustion.A) CIM of the number of dauer larvae (solid line), and the number of dauer larvae scaled to the numbers in the AF16 (dashed line) or HK104 (dotted line) controls from that experimental block.B) CIM of the number of dauer larvae observed in each experimental block separately.Horizontal lines indicate thresholds.
of Biology, University of Maryland, College Park, MD, USA In this report, Green and Harvey address the genetic architecture governing variation in one of the best-studied examples of developmental plasticity in animas, the dauer larva Caneorhabditis nematodes.

Table 1 . QTLs affecting the number of dauer larvae at food exhaustion.
Position is the 1 LOD interval for the QTL.R 2 denotes the proportion of the inter-RIL variance explained by the QTL, and effect indicates how the QTL alters dauer larvae numbers, with a positive value indicating that the AF16 allele increases the number of dauer larvae compared to the HK104 allele.

dynamics and habitat sharing of natural populations of Caenorhabditis elegans and
Maintenance of C. elegans.(february 11, 2006), WormBook.ed. the C. elegans Research Community, WormBook, 2006.