Academic journal article Genetics

Potential Nematode Alarm Pheromone Induces Acute Avoidance in Caenorhabditis Elegans

Academic journal article Genetics

Potential Nematode Alarm Pheromone Induces Acute Avoidance in Caenorhabditis Elegans

Article excerpt

DETECTING danger is crucial for animal survival. Alarm pheromones are used to communicate danger by many animal species such as sea anemones, insects, fishes, and mammals (Wyatt 2003). Even humans have alarm pheromones (Mujica-Parodi et al. 2009). In these animals, chemical cues are released from injured or stressed animals and detected by conspecifics or closely related species to invoke innate alarm responses such as fleeing. Chemical compositions of alarm pheromones are often species specific, e.g., anthopleurine in sea anemone (Howe and Sheikh 1975), CO2 in fruit flies (Suh et al. 2004), chondroitin fragments in zebrafish (Mathuru et al. 2012), and 2-sec-butyl-4,5-dihydrothiazole in mice (Brechbühl et al. 2013b). The olfactory pathways that detect alarm pheromones largely consist of odorant receptors, G proteins (e.g., Gaq in flies, Gai in fish, and Gao and Gai in mice), and a second messenger (e.g., cAMP in fish and cGMP in mice) (Enjin and Suh 2013).

Surprisingly, it remains unclear whether there is an alarm pheromone in nematodes, considering that alarm pheromones exist in a wide variety of animals (Wyatt 2003) and that nematodes are the most abundant animals on earth (Lorenzen 1994). Nematodes are known to use a class of small molecules called ascarosides as pheromones to regulate behaviors such as mate-finding and aggregation (Ludewig and Schroeder 2013). However, there is no published report of an alarm pheromone in the nematodes.

Here we present evidence of a potential nematode alarm pheromone in the internal fluid released from injured worms. The fluid induces an acute avoidance without inflicting physical harm. This avoidance signal appears ascaroside independent and conserved among multiple nematode species. In Caenorhabditis elegans, detection of this signal requires a cGMP signaling pathway. Together, these data suggest the existence of a nematode alarm signal.

Methods

Animal maintenance

C. elegans strains were cultured on nematode growth medium (NGM) with OP50 Escherichia coli at 20° as previously described (Stiernagle 2006). N2 (Bristol) was used as the wild-type strain. All worm strains were obtained from the Caenorhabditis Genetics Center (CGC) except daf-37(ttTi3058) from the Centre National de la Recherche Scientifique, goa-1(sy192) from the Sternberg laboratory, srbc-64(tm1946) and srbc-66(tm2943) from the Sengupta laboratory, and Steinernema carpocapsae from the Hallem laboratory. Unoutcrossed strains that showed as a hit in chemoavoidance assays were outcrossed six times and tested again. Detailed information of all mutant strains is listed in Supplemental Material, Table S1 in File S4.

Unless otherwise specified, day-1 adult hermaphrodites were used in our behavioral assays. Synchronized L1s were collected by bleaching gravid adults as described (Stiernagle 2006) and cultured on OP50 plates until they reached adulthood.

To obtain starved worms, we washed well-fed young adult worms off the plates into M9 buffer. The worms were washed three additional times in M9 and placed in M9 at a concentration of one worm/ml. Control worms were placed in M9 with 1% OP50. Both groups were incubated at 20° and tested after 1, 3, and 5 hr of starvation.

To collect dauers, C. elegans plates were starved for 5 additional days after the worms cleared the bacterial lawn. Five holes were made on the wall of each plate above the agar level using a flamed needle. Five 100-ml drops of sterile water were placed on each lid where the plate wall touched. The plates were placed upside down sitting on the lids overnight. The water drops on the lids were then collected and examined for dauers.

S. carpocapsae were cultured as described (Ehlers and Shapiro-Ilan 2005). Five waxworms (PetSmart) were placed in a 60-mm Petri dish lined with filter paper (55 mm, Whatman, Maidstone, UK). A total of 200 ml of water containing ~100 infective juveniles (IJs) was dropped on top and around each waxworm. …

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