Description

Entomopathogenic nematodes (EPNs) form species-specific mutualistic symbiotic relationships with their gut bacteria; on the other hand, they are parasitic to insects, which they infect and rapidly kill with the assistance of their symbiotic bacteria (Dillman and Sternberg, 2012). Studies of EPNs can therefore provide useful insight into parasitism and mutualistic symbiosis. Research in this field has been so far limited by the lack of a genetically tractable system on the host side (Cao et al., 2021). S. hermaphroditum was first isolated in Indonesia (Griffin et al., 2001); that isolate was subsequently lost, but the species was recently rediscovered in India (Bhat et al., 2019). It is consistently hermaphroditic, and techniques and methods developed to study hermaphroditic Caenorhabditis elegans are largely applicable (Cao et al., 2021). Together with its bacterial symbiote Xenorhabdus griffiniae it provides a genetically tractable system for the study of symbiosis.

Nematode development consists of four larval/juvenile stages, known as either L1, L2, L3, and L4 (as in C. elegans), or as J1, J2, J3, and J4 (as in S. hermaphroditum). As an alternative to the third larval stage, nematodes can become a stress-resistant form known as dauers in C. elegans and infective juveniles (IJs) in EPNs in response to environmental conditions including limited food. As a crucial part of their life cycle, IJs search, invade, colonize, and subsequently feed on their insect host. Chemosensation is thought to play a major role in EPNs’ host-seeking behavior, and molecules including CO₂ have been identified as attractants for EPNs (Dillman et al., 2012; Gang and Hallem, 2016; Hallem et al., 2011).

The nervous system of the nematodes is highly conserved across species. For example, the tiny (~1mm) free-living nematode C. elegans has a neuroanatomy highly similar to that of the much larger (up to 40cm), evolutionary distant, and parasitic Ascaris (Schafer, 2016; Stretton et al., 1992; White et al., 1986). The functionality of nematode neurons are at least partially conserved. For example, ablation of the BAG neurons of both Steinernema and Heterorhabditis EPNs, identified by their conserved anatomy as corresponding to the C. elegans neuron responsible for sensing CO₂ eliminated the EPNs’ CO₂‑dependent attraction by insect hosts and the jumping response of Steinernema carpocapsae to CO₂ (Hallem et al., 2011). In C. elegans, amphid neurons are known to be important for chemosensation (Bargmann and Horvitz, 1991a), which is required for the decision-making process of entering and exiting the dauer larval state (Bargmann and Horvitz, 1991b). In C. elegans and other nematodes, including Steinernema carpocapsae, a dye-filling assay has been shown to be an efficient assay to label some of the amphid neurons (Han et al., 2015; Hedgecock et al., 1985; Perkins et al., 1986; Srinivasan et al., 2008; Tong and Burglin, 2010). In this study, we have developed a protocol for the efficient DiI (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate, Invitrogen) staining of S. hermaphroditum head sensory neurons.

We adapted the protocol described by Tong and Burglin (2010) for DiI staining, which has been utilized for the staining of the closely related nematode S. carpocapsae (Han et al., 2015). We optimized the staining protocol by altering the concentration of the DiI dye (Fig. 1A, top), buffer solution, (Fig. 1A middle) and staining duration (Fig. 1A bottom). We found that a 20µg/ml DiI concentration with 0.5% Triton X-100 in M9 for 5 hours gave us the best results when considering both the staining effectiveness (Fig. 1A) and the health of the worm. In C. elegans, head sensory neurons, including amphids- ASI, ADL, ASK, AWB, ASH, ASJ, and inner labials- IL1 and IL2 are known to fill with DiI (Burket et al., 2006; Shaham, 2006; Starich et al., 1995; Tong and Burglin, 2010) (Fig. 1B, C). In S. hermaphroditum, we found that neurons identifiable by position as analogs of the amphid neurons ASI, ADL, ASK, ASJ and the inner labial neurons IL1, and IL2 could be labeled using DiI filling (Fig. 1D-G). While amphid neurons are generally labeled in DiI dye-filling assays in C. elegans (Tong and Burglin, 2010), they were only intermittently stained in S. hermaphroditum (at least 1 putative amphid neuron is stained in roughly 20% of the treated animals). On the other hand, the inner labial neurons are much more accessible (approximately 90% of the treated animals have at least 2 putative IL neurons stained), including the IL1 neuron that is not commonly filled in C. elegans (IL1 staining in C. elegans is possible as shown in https://www.wormatlas.org/). Our observations are consistent with what has been observed in S. carpocapsae, in which putative IL2 neuron is dye accessible, and amphid neurons are stained only occasionally (Han et al., 2015). While our observation focused on the head neurons, it is known that in the tail phasmid neurons, PHAs and PHBs can also be labeled using dye-filling in C. elegans (Hedgecock et al., 1985; Tong and Burglin, 2010). However, we did not look for phasmid neurons in S. hermaphroditum hermaphrodites and it is unclear whether they were stained. As in S. carpocapsae (Han et al., 2015), no dye-filling was observed in the stress-resistant IJ animals (Fig. 1J). By contrast, staining is observed in C. elegans dauers (Peckol et al., 2001). The mechanism of dye-filling is not well understood, but the difference among species in uptake is likely caused by structural divergences in the cilia opening. Similarly, Heterorhabditis bacteriophora, another EPN, despite that it is evolutionarily much closer to C. elegans than that of S. hermaphroditum, is reported to have a similar staining pattern: their putative amphid neurons are not stained consistently, and their IJs are not stained (Han et al., 2015). Besides dye filling in the head neurons of non-IJ animals, we also observed dye uptake by the excretory pore on the ventral surface of the animal near the head and in the gut of nearly every worm observed (Fig. 1H, I). Overall, we showed that dye-filling assays could be applied in S. hermaphroditum, and the anatomic characterization based on the staining suggests that it has a neuroanatomy similar to that of other characterized nematodes.