Rehydration methods to recover cysticercoids of the rat tapeworm Hymenolepis diminuta from dry flour beetle carcasses

Abstract

Terrestrial arthropods host a variety of helminth parasites, yet quantifying the intensity of infection in these hosts post-mortem is challenging because carcasses may desiccate quickly. We recovered cysticercoids of Hymenolepis diminuta from desiccated flour beetle (Tribolium confusum) carcasses by modifying a published insect rehydration procedure. Without rehydration, carcasses dissected more than 1 day post-mortem had noticeable degradation of cysticercoids. Mild rehydration (soaking in water only for 2 days, or 0.5-10% KOH for 1 h followed by 1 day in water, or 0.5% KOH for 1 day) left carcasses tough and time-consuming to dissect, but all parasites could be recovered and were similar in body size to fresh cysticercoids. Moderate rehydration (5-10% KOH for 1 day) allowed all parasites to be recovered and facilitated dissection by partially dissolving internal organs of the beetle while causing little degradation of the cysticercoids. Harsh rehydration (5-10% KOH for 1 day followed by 5 days in water) not only dissolved internal beetle tissues but also severely damaged cysticercoids, such that parasite counts were unreliable. The degree of initial carcass desiccation had little effect on results following rehydration. However, regardless of treatment used, intact cercomers were rarely retained on rehydrated cysticercoids. Rehydration was less successful on early developmental stages of the parasite, which were recovered reliably only as they neared the cysticercoid stage. This method has utility for studies of parasite-induced mortality by permitting accurate and reliable parasite counts from dead, desiccated hosts.

Comparison of the effects of multiple variables on the levels of infection of the rat tapeworm,Hymenolepis diminuta, in its intermediate host, the confused flour beetleTribolium confusum

“…Frozen beetles were subjected to dissection and the presence (prevalence) and number of cysticercoids per infected beetle (intensity) was recorded. It should be noted that beetles that did not survived the process were eliminated from the analysis, although current research has now provided a way to include these carcasses in future studies (Chin et al ., 2017).…”

Longitudinal study of parasite-induced mortality of a long-lived host: the importance of exposure to non-parasitic stressors

“…Details of sources and maintenance of beetles ( T. confusum ) and parasites ( H. diminuta ), and methods for recovery of parasite eggs and infection of adult beetles (exposure to oatmeal flakes containing an aqueous suspension of eggs), are described in detail in Shostak (2009), and beetle necropsy procedures in Chin et al (2017). All procedures complied with the ethical standards set by the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and the Canadian Council on Animal Care (Ottawa, Canada) on the care and use of laboratory animals, using the minimum number of animals judged necessary to achieve statistically significant results, and were conducted under University of Alberta Animal Use Protocol AUP00000078.…”

“…The contents of each arena were passed through a 250 µ m mesh sieve, carcasses of dead hosts removed and stored in micro-centrifuge tubes, the dishes cleaned with 70% ethanol, fresh medium and/or DE reapplied, and surviving adults returned. Any hosts surviving to 6 weeks were killed by chloroform (Chin et al 2017) and stored in micro-centrifuge tubes for later necropsy.…”

“…All chemicals unless otherwise noted were from Fisher Scientific (Ottawa, Ontario, Canada). Briefly, each beetle was dissected in insect saline (Chin et al 2017), and its cysticercoids were counted and then placed in a 1·5 mL micro-centrifuge tube with 1 mL insect saline. The saline was replaced with 1 mL acid–pepsin solution [1 g pepsin (Ward's, Rochester, New York, USA), 0·5 mL concentrated HCl, 100 mL 0·85% NaCl] for 10 min, washed 3× with Tyrode's saline, stored in 1 mL Tyrode's bile–trypsin solution [0·5 g trypsin, 0·5 g porcine bile extract (Sigma, St. Louis, Missouri, USA), 100 mL Tyrode's saline] for 20 min, and then examined for the presence of liberated scolices.…”

“…Morphological examination was done on cysticercoids dissected in insect saline from freshly killed beetles and photographed as in Chin et al (2017). We used beetles from the baseline group in experiment 3 to represent 0-week-old cysticercoids (i.e.…”