Janis Weeks, Ph.D.

NemaMetrix logo - C. elegans research and phenomics


Purpose

The NemaMetrix ScreenChip platform was developed for C. elegans, which is a free-living (non-parasitic) species that feeds on bacteria and has both hermaphroditic and male sexes. However, C. elegans represents only one of the estimated 1 million members of the highly diverse phylum Nematoda, of which only ~25,000 species have been described. In addition to free-living species, other nematodes parasitize humans, animals or plants, with significant medical, veterinary and economic consequences. To demonstrate the utility of microfluidic EPG recordings from species beyond C. elegans, we adapted the platform to record from human and animal parasites, male and female members of a dioecious species, and a carnivorous nematode.

Results

Table 1 shows the species and life stages that we used for EPG recordings from parasitic (Figure 1) and free-living (Figure 2) species. Recordings were obtained using the ScreenChip system1, an 8-channel EPG chip2,3, or both platforms. Chips with varying channel sizes were used to accommodate differences in worm diameter3,4,5,6.

Species Habitat Stage(s) with EPG recordings obtained Reference
Hookworm, Ancylostoma ceylanicum Human intestinal parasite Infective L3 activated in vitro; host-stage L4 from hamsters 3
Hookworm, Ancylostoma caninum Canine intestinal parasite Infective L3 activated in vitro 3
Roundworm, Ascaris suum Swine intestinal parasite; may be the same as human parasite A. lumbricoides7 Host-stage L3 from pigs (lung stage) 3
Haemonchus contortus (“barberpole worm”) Ungulate (sheep, goats) gastrointestinal parasite L2 reared from eggs; L4 cultured from infective L3 in vitro 4
Panagrellus redivivus (“beer mat nematode”) Free-living; eats yeast Adult males & females 5
Pristionchus pacificus Free-living carnivore of other nematodes, or bacterivorous Adult 6

 

Pharyngeal Recordings (EPGs) taken from different species of parasitic nematodes

Fig. 1. EPG recordings from intestinal nematodes of humans (A); swine (B); dogs (C); and small ruminants (goats, sheep) (D). Images8 of the species at right.

Pharyngeal Recordings (EPGs) taken from different species of free-living nematodes

Fig. 2. EPG recordings from free-living nematodes. Ai,ii.P. redivivus adult male and female. B.P. pacificus adult. Images9 of the species at right; in B, P. pacificus has captured a C. elegans for ingestion.

The most extensive experiments were performed in A. ceylanicum and P. redivivus. For example, in A. ceylanicum we determined the concentration-dependence of 5HT (5-hydroxytryptamine; serotonin) treatment on pump frequency and demonstrated the inhibition of pumping by an anthelmintic drug3 and a natural product used traditionally as a vermifuge in Haiti10. In P. redivivus, we investigated whether the effect of 5HT or anthelmintic drugs on pharyngeal pumping differed between males and females5. Microfluidic EPG recordings have also been obtained in a plant parasitic nematode, Globodera pallida11.

Conclusions

The ability to use microfluidic EPG chips with species other than C. elegans broadens the utility of this technology. Suggested applications include assaying EPG phenotypes induced by RNAi or other manipulations, assaying drug resistance in human or animal parasites, screening drug candidates, comparative studies of nematode feeding behavior, and other instances in which an electrophysiological readout can provide unique insights into nematode biology.

The six species tested here are a small sample of those used in research laboratories. NemaMetrix staff are available to work with investigators who wish to adapt the ScreenChip system to additional species. The upcoming release of a NemaMetrix ScreenChip for worms as small as C. elegans first-stage larvae (L1; ~12 µm diameter) will further enable the use of microfluidic EPG recordings as a routine tool in nematode research.

 Methods

Species were reared as described elsewhere3,12. EPG recordings were made in M9 buffer13 or culture medium containing 5HT and/or serum and other additives. Parasite recordings were obtained at 35-38 oC using a heated chip dock. Data were acquired in Spike2 software (Cambridge Electronic Design) for 8-channel chips or NemAcquire software14 for the ScreenChip system. Detailed ScreenChip methods are available elsewhere15. Recordings were analyzed using custom software in IGOR Pro3, soon to be superseded by the expected release of NemaMetrix’s NemAnalysis software later this year.

References

  1. http://nemametrix.com/product/starter-kit/
  2. Lockery SR et al. 2012. A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans. Lab Chip 12:2211-2220.
  3. Weeks JC, WM Roberts, KJ Robinson, M Keaney, JJ Vermeire, JF Urban, Jr., SR Lockery & JM Hawdon. 2016. Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: a new tool for anthelmintic research. Int J Parasitol: Drugs Drug Res, in press.
  4. Weeks JC, KJ Robinson, B Storey & A Wolstenholme, unpublished data.
  5. Karanga-Senge W, KJ Robinson, WM Roberts & Weeks JC, unpublished data.
  6. NemaMetrix Inc., unpublished data.
  7. Leles D et al. 2012. Are Ascaris lumbricoides and Ascaris suum a single species? Parasit Vectors 5:42.
  8. http://www.cliniciansbrief.com/article/reconsidering-ancylostoma-ceylanicum
    http://ascarislumbricoides.org/facts-you-didnt-know-about-ascaris-lumbricoides
    https://en.wikipedia.org/wiki/Ancylostoma_caninum#/media/File:Hookworms.jpg
    http://www.ucalgary.ca/jsgilleard/research
  9. http://faculty.ucr.edu/~pdeley/vce/Panagrellus/redivivus/thumbs.html
    https://tuebingen.mpg.de/en/news-press/press-releases/detail/the-neurobiological-consequence-of-predating-or-grazing.html
  10. Keaney M, MacIntyre A, KJ Robinson, WM Roberts, JC Weeks & JM Hawdon, unpublished data; Wolpert BJ et al. 2008. Plant vermicides of Haitian Vodou show in vitro activity against larval hookworm. J Parasitol 94(5):1155-60.
  11. Hu et al. 2014. StyletChip: a microfluidic device for recording host invasion behaviour and feeding of plant parasitic nematodes. Lab Chip 14:2447-55.
  12. http://www.cflas.org/microworm-care-sheet/4159; modified from Conder GA & Johnson SS. 1996. Viability of infective larvae of Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis following exsheathment by various techniques. J Parasitol 82:100-2.
  13. Stiernagle T. 2006. Maintenance of elegans. WormBook, http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
  14. http://nemametrix.com/downloads/
  15. http://nemametrix.com/wpcontent/uploads/2016/08/ScreenChip_System_QuickStart_Guide.pdf
    http://nemametrix.com/tech-notes

Acknowledgements

Funding from the Bill & Melinda Gates Foundation, American Center for Veterinary Parasitology and UO Summer Program for Undergraduate Research.