Abstract
Diplomonad flagellates of Spironucleus can cause intestinal and systemic infection, with significant morbidity and mortality in aquaculture.4 Despite their economic importance, many aspects of host-diplomonad interactions, including microhabitat preference, are not well understood. In young farmed rainbow trout Oncorhynchus mykiss, S. salmonis are often found in the pyloric region of the intestine.3 This localisation may be predominantly due to chemical factors, rather than morphology, since the intestinal-rectal valve is indistinct.2 It has been suggested that pH may play a decisive role in determining microhabitat preference of S. salmonis, and limited in vitro studies have reported a pH tolerance of 5-10, and an optimum of 7.5-8.0.1 However there are no in vivo studies that test this hypothesis.
Knowledge of diplomonad microhabitat preference and the role of pH as a determining factor has important applications in fish health, such as 1) indicating possible preferred hosts based on their intestinal pH, 2) determining the optimum pH from in vivo studies, which may in turn lead to improved in vitro culture protocols, and 3) predicting the impact of changes in pH associated with changes in diet (such as those with increased plant protein) on density of diplomonad infections.
To test the hypothesis, we approached our investigations by posing three main questions: (i) what is the relationship between occurrence and density of infection, and intestinal regions in trout, (ii) what is the pH profile along the intestinal tract of uninfected and infected trout, and finally (iii) what is the relationship between density of infection and pH in different intestinal regions.
We investigated 698 juvenile rainbow trout and recorded occurrence, density of infection with S. salmonis (semi-quantitative: light, moderate, and heavy), and pH in four intestinal regions; pyloric, anterior, middle, and posterior intestine, with an accuracy of 0.2 by using pH indicator strips. The Chi-squared test, ANOVA and t-test (for normally distributed data), and the Mann-Whitney and Kruskal-Wallis test (for non-normally distributed data) were used to test for significant differences among groups of data; results were considered significant if p < 0.05.
Diplomonads showed decreasing gradients of occurrence and density from pyloric to posterior regions, with a significant preference for the pyloric region. The pH profile was characterized by more neutral conditions in the pyloric and posterior intestine (mean pH of 7.3 and 7.1 respectively), which were significantly different from more alkaline conditions in the anterior and middle intestine (mean pH of 7.7 in both regions). Uninfected fish had similar pH profile. In the pyloric region, the mean pH in light, moderate, and heavy infections (7.4, 7.3, and 7.3 respectively) was not significantly different.
The present study demonstrates that for S. salmonis in trout, pH does not appear to be the dominant factor in microhabitat preference, and that the optimum pH may be close to 7.3, this being the mean in the pyloric region. We suggest that further factors including bile, bacterial fauna, and nutrient content of the digesta, determine the decreasing gradient of diplomonads from anterior to posterior intestine, and needed further investigation.
Acknowledgments
We thank Prof. Werner Kloas, and Prof. Frank Kirschbaum from IGB for their financial and logistical support. We gratefully acknowledge the Nachwuchsförderung des Landes Berlin (NaFöG) for the award of a Ph.D. scholarship to Mr. Reza Fard, the Amt für Ausbildungsförderung for Bafög to Ms. Claudia Weisheit, and the Deutsche Forschungsgemeinschaft for the award of a Mercator Visiting Professorship to Dr. Sarah Poynton.
References
1. Buchmann K, A Uldal. 1996. Temperature, pH and bile dependant in vitro cultivation of Hexamita salmonis from rainbow trout Oncorhynchus mykiss intestine. Dis. Aquat. Org. 24:169-172.
2. Ezeasor PN, WM Stokoe. 1980. Scanning electron microscopic study of the gut mucosa of the rainbow trout Salmo gairdneri Richardson. J. Fish. Biol. 17:29-539.
3. Fard MRS, Jørgensen A, Sterud E, Bleiss W, SL Poynton. 2007. Ultrastructure and molecular diagnosis of Spironucleus salmonis (Diplomonadida) from rainbow trout Oncorhynchus mykiss in Germany. Dis. Aquat. Org. (in press)
4. Woo PTK, SL Poynton. 1995. Diplomonadida, Kinetoplastida and Amoebida (Phylum Sarcomastigophora). In: P.T.K.Woo (ed.). Fish Diseases and Disorders, Vol. 1, Protozoan and Metazoan Disorders. CAB International, Wallingford, England. Pp.27-96.