Cellular anatomy is essentially invariant between individuals of the same nematode species and the number of neurons is highly conserved between nematode species, allowing the identification of neurons across different species of nematodes 610173435.

Using the neuronal map of C. elegans (Figure 2A) 35, we identified the amphid cell bodies in other species. The amphidial neurons are present posterior to the nerve ring in all the species. In C. elegans, the ASH neuron is part of a five neuron group (ASJ, AUA, AWC, ASH, and ASE) arranged in the shape of a 'V' (Figures 2A and 2B). The position of these neurons is more lateral than all other neurons in the same focal plane. An important feature of this arrangement is the presence of two neurons anterior to the AWC neuron, RMD and SIBD (Figures 2A and 2B). These two neurons along with the five neurons form a 'Y'. We found that this arrangement is conserved in all species examined (Figures 2C–E). We observed that in all species, the position and size of the putative ASH neuron was similar to C. elegans (Figures 2C–E). However, in C. tripartitum and P. redivivus, the putative ASH neurons are larger than those in C. elegans, consistent with the larger size of these species (Figures 2F and 2G). The RMD and SIBD neurons, which are small in C. elegans, are also larger in these species (Figures 2F and 2G). The dorsal triplet amphid neurons ASI, ADL, and ASK are found in a different focal plane and these cells were also found in the other species in the same position (Figure 2A).

In C. elegans, amphid and phasmid neurons take up lipophilic dyes such as DiI, DiO, and DiD from the environment 363738. These dyes have been used to visualize the ASI, ADL, ASK, AWB, ASH, and ASJ amphid neurons, as well as the PHA and PHB phasmid neurons 39. We used DiI to visualize the amphid and phasmid neurons in all the species, and found that all species labeled most of the amphid neurons (Figures 3A–E). In addition, DiI staining showed that the morphology of the amphid neurons was very similar in most of the species as they label all parts of the neuron (Figures 3A–E). In C. elegans, all amphid neurons showed relatively strong dye uptake (Figure 3A). C. briggsae and Caenorhabditis sp. 3 also showed strong staining of all the amphid neurons (Figures 3B and 3C). In C. tripartitum, we could visualize only four of the six amphid neurons: ASI, ADL, ASK, and ASJ. We did not observe any staining of the ASH neuron in this species even though we were able to identify the neurons using differential interference contrast microscopy (Figures 3D and 2E). In P. pacificus, the ASH, ADL, and AWB neurons were stained relatively weakly compared with the other species (Figure 3E) and P. redivivus had the weakest staining for ASI, AWB, and ASJ neurons, respectively (Figure 3F). The differential dye uptake in the diverse species indicates that dye uptake is not sufficient to define equivalent neurons; however, it is consistent with general conserved properties of amphid neurons as having open sensory endings, and could have revealed major differences in morphology or position of neuronal cell bodies 711.

Based upon standard assays, avoidance of the volatile odorant 1-octanol is remarkably conserved across all species tested (Figure 4). C. elegans responds quickly to 100% octanol (~2–3 seconds) 4041 Additonal File 1. In C. elegans, in the presence of food, response to 100% octanol is primarily mediated by the ASH neuron, and in the absence of food ASH, ADL, and AWB neurons are responsible for sensing 100% octanol, suggesting that feeding status of the animal affects response to 1-octanol in C. elegans 40. All the species except P. redivivus responded after ~2–3 seconds of octanol exposure, indicating that these species are similar in sensitivity to C. elegans 42. P. redivivus showed significantly delayed sensitivity to 1-octanol, responding in an average of 5.9 seconds. The delayed response could be due to the requirement for a higher concentration of the chemical to generate a response. To confirm the identity of the putative ASH neuron identified by position and morphology (Figures 2B–G), we used a laser microbeam to kill the ASH neuron and assayed for octanol avoidance. Ablation of the ASH neuron in all the species is sufficient to abolish 1-octanol avoidance (Figure 4). As a control, we ablated the AWC neuron in all the species and found that ablation of this neuron did not result in loss of avoidance response to 1-octanol, suggesting that the ASH neuron is specifically required for avoidance to 1-octanol in all species (Figure S1 in Additional file 2).

Nose touch avoidance behavior also showed a high degree of conservation among five of six species tested (Figure 5A). In C. tripartitum, after the first few positive trials, the animals failed to respond to nose touch, showing an overall avoidance of < 10%, suggesting that C. tripartitum adapts to the nose touch stimulus faster than C. elegans and the other species tested (Figure 5A). However, during the first three trials, C. tripartitum showed a quantitatively similar response to that of C. elegans (Figure S2 in Additional file 2). Ablation of the ASH neurons in five nose-touch responsive species resulted in loss of nose touch avoidance (Figure 5A). Four of five species showed nose touch responses in 20–30% of the trials (Figure 5A). However, in Caenorhabditis sp. 3, ASH-ablated animals completely lacked the ability to respond to nose touch (Figure 5A). Ablation of the AWC neuron did not affect response to nose touch in the different species (Figure S3 in Additional file 2).

In C. elegans, nose touch response is mediated by two non-amphid neurons, FLP and OLQ, in addition to the ASH neurons 25. To test whether nose touch response might be mediated by a single sensory neuron (ASH) and to check the role of the non-amphid neurons, we assayed nose touch response in FLP-ablated, OLQ-ablated, FLP/OLQ-ablated and FLP/OLQ/ASH-ablated animals in both C. elegans and Caenorhabditis sp. 3 (Figure 5B). In C. elegans, FLP-ablated animals were significantly less responsive to nose touch compared with unablated animals. OLQ-ablated C. elegans showed no effect on nose touch compared with unablated animals (Figure 5B). However, ablation of both the non-amphid neurons FLP and OLQ in C. elegans resulted in significantly decreased nose touch avoidance compared with either of the single neuron ablations, suggesting that among the non-amphid neurons, FLP plays a major role in mediating nose touch whereas OLQ plays a minor role 25 (Figure 5B). In C. elegans, FLP/ASH-ablated animals were significantly different from ASH-ablated animals (Figure 5B). Animals lacking FLP, ASH, and OLQ neurons were significantly different from ASH-ablated animals but not different from ASH/FLP-ablated animals (Figure 5B).

In Caenorhabditis sp. 3, the FLP and OLQ neurons have no effect on nose touch response (Figure 5B). Moreover, ablation of both the two non-amphid neurons (FLP, OLQ) did not show any significant difference from either of the single ablated animals (Figure 5B). In Caenorhabditis sp. 3, ablation of all three neurons (ASH, FLP, and OLQ) did not significantly differ from ASH-ablated animals (Figure 5B). These results suggest that the ASH neuron completely mediates nose touch avoidance behavior in Caenorhabditis sp. 3, as compared with three neurons in C. elegans. It is conceivable that in Caenorhabditis sp. 3, other neurons could be involved in mediating nose touch behavior. However, ASH-ablated animals in Caenorhabditis sp. 3 show less of a nose touch response compared with ASH/FLP/OLQ-ablated animals in C. elegans, suggesting that ASH mediates most of the nose touch response in Caenorhabditis sp. 3 (Figure 5B). The sensory response network for mechanical stimuli in Caenorhabditis sp. 3 thus shows a reduction in the number of neurons involved compared with C. elegans.

Osmotic avoidance behavior was assayed using the standard drop assay 23 (Figure 6A). C. elegans responds within a second of drop delivery by backing away from the drop of osmotic solution 23. The avoidance index (a.i.) was calculated by dividing the number of positive responses to the total number of trials 23 (Additional file 1). Within the Caenorhabditis clade, all three species tested responded similarly to C. elegans with respect to response times to 2 M glycerol. However, the phylogenetically distant species, P. pacificus, C. tripartitum, and P. redivivus, had longer response times compared with C. elegans (Table 1). The average response times for P. pacificus, C. tripartitum, and P. redivivus were 5.2, 7.4, and 8.1 seconds, respectively (Table 1). P. redivivus, the most distant species tested, showed significantly decreased sensitivity to 2 M glycerol compared with the other species (Figure 6A). We speculate that P. redivivus could be adapted to high osmotic conditions because it was initially isolated from high sugar concentration environments.

Ablation of the ASH neuron almost completely abolishes osmotic avoidance response in C. elegans 23. In the parasitic nematode S. stercoralis, avoidance response to high salt concentrations is completely mediated by the ASH neuron 20. Ablation of the ASH neuron in the other species resulted in reduced a.i. (Figure 6A). ASH ablation does not completely abolish osmotic avoidance in P. pacificus, suggesting the possibility of ASH-independent mechanisms of osmotic avoidance (C. elegans a.i. = 0.15 vs. P. pacificus a.i. = 0.36; Figure 6A). In C. elegans, ASH, ADL, ASK, and ASE neurons are known to mediate avoidance of chemical repellents 23. The residual osmotic avoidance seen in ASH ablated animals of P. pacificus could be mediated by these neurons. In C. elegans, ablation of the ADL neuron had no effect on osmotic avoidance (Figure 6B). Ablation of both ASH and ADL neurons in C. elegans did not show a significant change in osmotic avoidance index compared with ASH-ablated animals (Figure 6B). In P. pacificus, ablation of ADL neuron results in a significantly lower avoidance index compared with unablated animals (Figure 6B). Ablation of both ASH and ADL neurons in P. pacificus had a significantly lower avoidance index compared with ASH-ablated or ADL-ablated animals, suggesting an additive effect of both the neurons in osmotic avoidance behavior (ASH/ADL-ablated a.i. = 0.17 vs. ASH-ablated a.i. = 0.36; ASH/ADL-ablated a.i. = 0.17 vs. ADL-ablated a.i. = 0.62) (Figure 6B). Ablation of the ASK and ASE neurons did not change the osmotic sensitivity of P. pacificus, indicating that these neurons do not play any role in osmotic avoidance (data not shown). Hence, we believe that the ASH neuron is the primary sensory neuron for mediating osmotic avoidance in all nematode species, but additional sensory neurons, such as ADL neuron in P. pacificus, may contribute to mediating osmotic avoidance.

In C. elegans, the ADL neuron, along with ASH and ASE neurons, mediates avoidance response to heavy metal ions, but does not play a role on its own in mediating this avoidance 43. In the dog hookworm A. caninum, both ASH and ADL neurons are required to mediate avoidance of the detergent sodium dodecyl sulphate 21. Ablation of only the ADL neurons in the hookworm results in an assortment of responses, from avoidance to non-responsive behavior, suggesting that the animal is not able to decide whether to respond to the chemical 21. In P. pacificus, ablation of ADL neuron resulted in a significant reduction of avoidance response, indicating that this neuron is necessary for mediating avoidance to high osmotic conditions. The role of the ADL neuron during avoidance behaviors in different species thus remains unclear. Perhaps analysis of the genes expressed in the ADL neuron would provide insight into the role of this neuron in the different species.

We cannot rule out the possibility that ablation of specific neurons results in functional replacements by other neurons. However, if this occurs, then this aspect of nematode neurobiology varies among cells, behaviors, and species.

We hypothesized that nematodes would display habitat-specific sensitivity to the same stimuli rather than phylogeny based sensitivity. Therefore, we tested several different concentrations of the different noxious stimuli (chemosensation and osmosensation), and used a cluster algorithm to generate a behavioral dendrogram for the different avoidance behaviors (see Methods).

For octanol avoidance, we tested concentrations ranging from 0.1% to 100% octanol. The data for the different species was then normalized for each concentration with the maximal value for that concentration giving a normalized avoidance index for the different species (see Methods). P. redivivus displayed the least sensitivity to octanol compared with the other species at the different concentrations (Figure 7A). C. elegans and C. tripartitum show similar sensitivity to 1-octanol (Figure 7A). Also, in C. briggsae, Caenorhabditis sp. 3, and P. pacificus, sensitivity to octanol avoidance was more similar to each others' than to the other species' (Figure 7A).

As with osmotic avoidance behavior, we found that P. redivivus was the least sensitive to glycerol compared with the other species (Figure 7B). This observation correlates with the fact that P. redivivus has been isolated from high osmotic strength environments and hence could be adapted to high osmolarity 3344. However, at the highest concentration tested (4 M glycerol), P. redivivus also exhibited a very high avoidance, as did the other species (Figure 7B). C. briggsae, Caenorhabditis sp. 3, C. tripartitum, and P. pacificus exhibited similar sensitivity to osmotic avoidance (Figure 7B). C. elegans exhibited slightly different osmotic sensitivity different from all these species with the exception of P. redivivus (Figure 7B). Hence, other than P. redivivus, all other species exhibited similar sensitivity to different osmolarity conditions.

Finally, we combined the data of octanol and osmotic avoidance behaviors including the ASH neuron ablation data for all the species and found that P. redivivus exhibited the most different behavior in our analyses (Figure 7C). Based on relative distances computed by the algorithm, C. tripartitum is the next closely related species to P. redivivus. C. elegans, along with the other species, forms a whole branch on the behavioral dendrogram, with P. pacificus being closely related to it. C. briggsae and Caenorhabditis sp. 3 form a sub-branch, suggesting that these two exhibit similar behaviors. Comparing our behavioral dendrogram for octanol and osmotic avoidance behaviors with the phylogenetic tree, we see some interesting features (Figures 7C and 7D). We observe that for these two behaviors, the relative positions of P. redivivus and C. tripartitum resemble that of the phylogenetic tree (Figures 7C and 7D). P. pacificus seems to be behaviorally similar to the branch of Caenorhabditis sp. 3 and C. briggsae. Given the association of Caenorhabditis sp. 3 with rice weevils 28 and P. pacificus with beetles 45, this correlation makes sense. Unexpectedly, C. elegans does not exhibit similar behavioral properties like its close siblings C. briggsae and Caenorhabditis sp. 3. These data suggest that sensitivity to different stimuli varies among species and that the differential sensitivity could be linked to the functional sensory receptor repertoire of these species 46.

All strains were raised at 20°C unless indicated otherwise, using standard methods 51. Strains used in this study were C. elegans (N2 Bristol isolate) 51, C. elegans eat-4(ky5) MT6308 52, C. elegans glr-1(n2461) KP4 41, C. briggsae (AF16)53, Caenorhabditis sp. 3 (PS1010), P. pacificus (PS312) 54, C. tripartitum (SB202), and P. redivivus (PS2298/MT8872) 55.

A stock solution of DiI (2 mg/ml) was prepared in dimethyl formamide and stored at -20°C 38. Worms from the different species were washed from plates and resuspended in a small eppendorf tube to a 100 μl volume. The worm pellet was washed thrice in M9 buffer and worms allowed to settle down using gravity. To this pellet we added a 1:200 dilution of DiI solution and incubated in the dark for 2–3 hours. After the incubation, the worms were washed three times in M9 buffer and then mounted on a slide to visualize the amphid neurons. For some species, we used a longer incubation time (5–7 hours) to visualize neurons.

For all species tested, we used the L1 larva stage for our ablations as described previously 26. All behavioral assays were performed as described previously 232542. A detailed description of the ablations and behavioral assays is given in the Supplementary Materials and Methods in Additional file 1. Ablation of one of the two bilaterally symmetrical neurons did not affect the avoidance response to different stimuli.

For octanol avoidance sensitivity, we made different dilutions of 100% octanol (0.1%–100%) and each species was tested with each concentration at least on three different days. Data was represented as average avoidance time for each concentration.

Similarly, for sensitivity to osmotic avoidance, we tested concentrations ranging from 0.1 M to 4 M glycerol. Data was represented as mean a.i. (see Supplementary Materials and Methods in Additional file 1 for details).

For each of the behavioral assays, the normalized a.i. was computed for each species. This was done by normalizing the value for each species at each concentration to the maximum value for that concentration. The normalized values for the different concentrations were then plotted for each species in Matlab. For obtaining the dendrogram, data was then clustered using a Matlab hierarchical clustering algorithm (Euclidean distances and average linkage). The x-axis of the dendrogram indicates relative distance between the different species.

The statistical tests employed are described in Additional file 1.