Assessing morphological preservation of gastrointestinal parasites from fecal samples of wild capuchin monkeys (Cebus imitator) stored in ethanol versus formalin

Abstract

The copromicroscopic identification of gastrointestinal parasites is a common, cost-effective method vital to understanding host-parasite interactions. However, its efficacy depends on effective preservation of the samples. In this study, we compare the preservation of ethanol and formalin preserved gastrointestinal parasites collected from a wild population of Costa Rican capuchin monkeys (Cebus imitator). Fecal samples were collected, halved, and stored in either 10% formalin or 96% ethanol at ambient temperature, then microscopically screened for the presence of parasites. Parasites were morphologically identified and rated based on their preservation using a newly developed rubric. We identified more parasitic morphotypes in formalin-preserved samples but found no difference in the number of parasites per fecal gram (PFG) between mediums. There was no difference in the PFG of two most prevalent parasite morphotypes, Filariopsis barretoi larvae and Strongyle-type eggs, and while Filariopsis larvae were better preserved in formalin, strongyle eggs showed no preservation difference between mediums. Our results support the suitability of both ethanol and formalin for morphological parasite identification in samples stored over 1 year, describe the morphological changes and challenges associated with parasite degradation, and highlight the potential for future studies to use both morphological and molecular methods in non-invasively collected samples.

Figure 1

Examples of parasitic larvae (A–F) and eggs (G–J) identified in capuchin monkey (Cebus imitator) fecal samples and their state of preservation (preservation score) in either 96% ethanol or 10% buffered formalin. Larvae with a preservation score of 3 (A,D) show very little damage to their cuticle and have no or relatively minor alterations to their internal and external structures. Larvae with a preservation score of 2 (B,E) show moderate damage to the cuticle, as well as the internal and external organs. Larvae with a preservation score of 1 (C,F) show significant damage to the cuticle and internal/external organs and are often difficult to morphologically identify due to changes in and degradation of internal structures and overall form (i.e., size, shape, characteristic organs and appendages, etc.). Eggs with a rating of 3 (G,I) are clear, intact, and show minimal or no signs of damage, while eggs with a rating of 2 (H,J) show minor to moderate damage of the eggshell and may exhibit uncharacteristic changes in size or shape. We did not identify any poorly preserved eggs warranting a score under a 2 in these samples, and so the scale for eggs preserved in either ethanol or formalin is 3–2, instead of 3–1 for larvae. However, an example of a poorly preserved egg is available in Supple Fig. S2.

Figure 2

Gastrointestinal parasites collected from a wild population of capuchin monkeys (Cebus imitator) living from Costa Rica. (A) Strongyle-type egg (~ 57 × 36 μm), (B) Acanthocephalan egg (possibly Prosthenorchis sp.) (~ 62 × 37 μm), (C) Coccidian oocyst (possibly Eimeria sp.) (~ 30 × 30 μm), (D) unidentified egg (38 × 24 μm), (E) unidentified strongyle-type egg (large) (~ 93 × 61 μm), (F) Filariopsis sp. larva (381 μm), (G) unidentified larva morphotype 1 (~ 380 × 27 μm), (H) unidentified larva morphotype 2 (~ 235 μm) (I) unidentified larva morphotype 3 (~ 308 μm), (J) unidentified larva morphotype 4(possibly filariform Strongyloides sp.) 551 μm.

Figure 3

Average preservation rating, morphotype diversity, and parasite per fecal gram for gastrointestinal parasites in fecal samples collected from a wild population of capuchin monkeys (Cebus imitator) and stored in either 10% buffered formalin or 96% ethanol. Average parasite preservation ratings and morphotype diversity were significantly higher in formalin preserved samples when compared using Wilcoxon signed-rank tests.