Strongyloides stercoralis: Amphidial neuron pair ASJ triggers significant resumption of development by infective larvae under host-mimicking in vitro conditions

Abstract

Resumption of development by infective larvae (L3i) of parasitic nematodes upon entering a host is a critical first step in establishing a parasitic relationship with a definitive host. It is also considered equivalent to exit from the dauer stage by the free-living nematode Caenorhabditis elegans. Initiation of feeding, an early event in this process, is induced in vitro in L3i of Strongyloides stercoralis, a parasite of humans, other primates and dogs, by culturing the larvae in DMEM with 10% canine serum and 5 mM glutathione at 37 °C with 5% CO₂. Based on the developmental neurobiology of C. elegans, resumption of development by S. stercoralis L3i should be mediated, in part at least, by neurons homologous to the ASJ pair of C. elegans. To test this hypothesis, the ASJ neurons in S. stercoralis first-stage larvae (L1) were ablated with a laser microbeam. This resulted in a statistically significant (33%) reduction in the number of L3i that resumed feeding in culture. In a second expanded investigation, the thermosensitive ALD neurons, along with the ASJ neurons, were ablated, but there was no further decrease in the initiation of feeding by these worms compared to those in which only the ASJ pair was ablated.

Introduction

Although once considered a very close relative of the much-studied free-living nematode Caenorhabditis elegans, the parasitic threadworm Strongyloides stercoralis is no longer thus classified (Blaxter et al., 1998). The cell bodies of its amphidial neurons, located in the lateral ganglia, are, nevertheless, located in positions similar to those of the amphidial neurons in C. elegans (White et al., 1986, Ashton et al., 1995), allowing positional homologs to be identified, and, consequently, like functions to be hypothesized (Ashton et al., 1995, Ashton et al., 1998, Ashton et al., 1999). For example, in laser-microbeam ablation studies with hatchling L1 of S. stercoralis, the amphidial neuron pairs ASF and ASI were killed and thus found to control the decision whether to develop via the homogonic (direct) or heterogonic (indirect) pathway (Ashton et al., 1998). These neurons are the positional homologs of those in C. elegans (ADF and ASI) that control a similar developmental decision, whether or not to enter into the dauer stage (Bargmann and Horvitz, 1991). Likewise, the finger cell neurons (AFD) are the thermosensitive neurons in C. elegans and ablation studies reveal a similar function, namely control of thermotaxis and of thermosensitive aspects of development, in their putative counterparts (ALD) in S. stercoralis larvae (Lopez et al., 2000, Nolan et al., 2004). Thus, it appears that neuronal identity and function is largely, although not necessarily completely, conserved between these somewhat distantly related nematode species, C. elegans and S. stercoralis.

In the C. elegans dauer larva, an environmentally resistant, developmentally arrested stage, the ASJ neuron pair is of primary importance in detecting environmental change, and, if this is favorable, the worm will exit from this stage and resume development (Bargmann and Horvitz, 1991). This function is supported by the action of other neurons, ADF, ASG and ASI. Resumption of pharyngeal pumping in this free-living nematode, resulting in the oral intake of nutrients, is one of the first steps in this process. This feeding response has been proposed also as a critical early physiological step in the initiation of parasitic development by infective nematode larvae (Hawdon and Schad, 1990). The transition between free-living and parasitic life is triggered by host-given chemical and/or physical signals that are similar for related nematodes with the same portal of entry into the host, but species-specific differences exist (Hawdon et al., 1993). Thus, in some species the initiation of feeding is delayed until after the second parasitic ecdysis (Gamble and Mansfield, 1996).

If the environmentally resistant infective stage (L3i) of S. stercoralis is the life-stage equivalent of the C. elegans dauer larva, as has been proposed (Hotez et al., 1993) and is increasingly accepted, then resumption of development on finding and entering an appropriate host—the equivalent of exit from the dauer stage—should be controlled, at least in part, by neurons functionally homologous with the ASJ pair of C. elegans. Furthermore, if such neurons are ablated, then significant numbers of infective larvae should fail to resume development, either upon entering a host, or on being placed in host-mimicking culture conditions. To test this hypothesis, we ablated neuron pair ASJ (Ashton et al., 1995) in hatchling first-stage larvae (L1) of S. stercoralis harvested from coprocultures, raised the operated larvae to the L3i stage, and examined their ability to initiate feeding in a host-mimicking in vitro system (see below).

On entering a homeothermic host from the external environment, an infective larva encounters an entirely new, very different and complex milieu. There is a change in temperature, as well as in osmotic, ionic, and other physico-chemical conditions. Thus, it is probable that additional neurons may detect this complex of changes and, consequently, have a role along with ASJ-class neurons in mediating resumption of development. Therefore, in addition to studies in which only the ASJ neuron pair was ablated, we also ablated the ALD thermosensory neuron pair, the so-called lamellar cells, along with the ASJ neurons. These neurons had been shown to influence developmental switching in the free-living larvae of S. stercoralis (Nolan et al., 2004). Laser-operated larvae, and appropriate controls, were then raised to the L3i stage and tested in the in vitro host-mimicking system.

In the research reported here, resumption of feeding was used as an indicator of reactivated larval development, i.e., in vitro-simulated parasitic development. In vivo, infective larvae of S. stercoralis are stimulated to feed after entering into mammalian skin. Host-specificity is not marked, the potential to feed being triggered in vivo during a brief period of adaptation in either gerbil or canine skin (Schad et al., unpublished). Feeding can be demonstrated by incubating larvae recovered from skin in standard tissue culture media, containing fluorescein isothiocyanate (FITC) as an ingestible marker, under an atmosphere of 5% CO₂ in air at 37 °C. Additionally, tissue culture media and supplements to these media (canine serum, glutathione, and both of these supplements combined) were found to activate feeding in vitro in infective larvae taken directly from coprocultures. In a system similar to that used previously to stimulate feeding in hookworm L3i (Hawdon and Schad, 1990), DMEM supplemented with 10% dog serum and 5 mM glutathione was found to be the optimal in vitro system for triggering and sustaining feeding by S. stercoralis L3i. Ingestion of FITC-labeled culture medium indicates resumption of development in L3i. The lumen of both the pharynx and the intestine fluoresce (Fig. 1). Unstimulated control larvae (not shown) do not, there being no autofluorescence in S. stercoralis L3i.