Herpetomonas megaseliae sp. n. (Protozoa: Trypanosomatidae) from Megaselia scalaris (Loew, 1866) Schmitz, 1929 (Diptera: Phoridae)

Abstract

A new intestinal trypanosomatid was found parasitizing Megaselia scalaris. Forms with posterior kinetoplasts occurred in both natural infections and cloned cultures, but not in experimental infections. Average size and morphological variation were reduced in experimental infections with cloned stocks, compared to natural infections. A growth comparison was made between Herpetomonas muscarum and H. megaseliae. Differences in day of peak population, maximum numbers, and total time of survival were observed in culture. Attempts to infect Drosophila melanogaster with H. megaseliae were unsuccessful. The primary characteristic used to separate Herpetomonas from other genera of insect trypanosomatids is a posterior kinetoplast in up to 40% of culture forms (Rogers and Wallace, 1971). An intestinal trypanosomatid which exhibited a high percentage of posterior kinetoplasts in culture was discovered in Megaselia scalaris (Loew, 1866) Schmitz, 1929 (see Borgmeier, 1965, for synonyms). The organism was isolated and a study was made of its morphology in culture and in the insect host. In addition, a comparison with Herpetomonas muscarum was made of growth in culture. Morphological and host differences between the newly isolated trypanosomatid and published descriptions indicated that the parasite was an undescribed species of the genus Herpetomonas. The isolate was cloned and the description below is based on cloned material in culture and in experimental infections. MATERIALS AND METHODS Four culture media were used in the study: (1) Mansour's Medium (Dollahon and Janovy, 1971); (2) 1% proteose-peptone-glucose (PPG); (3) Wallace's Medium (Wallace and Clark, 1959); and (4) A medium consisting of 5 ml Locke's Solution (Tobie et al., 1950) into which one guinea pig fecal pellet was placed (LPG). The LPG was then autoclaved. The parasites were isolated from the larvae of M. scalaris into Mansour's Medium with 700 mg dihydrostreptomycin and 700 units procaine penicillin-G (Penstrep) per ml. The isolate was cloned 4 times, twice by serial dilution and twice Received for publication 4 January 1972. * This study was supported in part by a grant from the University of Nebraska Research Council. by isolation of a single organism with a capillary pipet. Each clone was made from a stock which had been previously cloned. Our present stock has thus been successively cloned 4 times. A combination of 4,000 units of procaine penicillin-G and 5 mg dihydrostreptomycin (Penstrep) per ml was used in the first transfer after the fourth cloning. A week-old culture in Wallace's Medium, a descendant of the final clone, was used to study the morphology and to describe the trypanosomatid in culture. Cultures were maintained at 25 C. H. muscarum, isolated from Phaenicia sericata, was obtained from Dr. F. G. Wallace, University of Minnesota. It was received in Wallace's Medium and subsequently maintained in our laboratory in Mansour's or Wallace's Medium. For growth studies, 20 ml of LPG, PPG, or Mansour's Medium was added aseptically to sidearm flasks. A flask of each medium was inoculated with 0.25 ml of a H. muscarum culture of the new trypanosomatid. Hemocytometer counts were made on the 3rd, 5th, 7th, and 10th days after inoculation. Zero time populations were calculated from hemocytometer counts of the inoculum. Colonies of uninfected host flies, M. scalaris (identification confirmed by Dr. William Robinson, Virginia Polytechnic Institute), were initiated with eggs gathered from guinea pig litter pans. Eggs were washed once in a 1:10,000 zephiran-chloride solution and once in distilled water. Washed eggs were placed in 8-oz cotton-gauze-stoppered Frenchquartered bottles containing 50 ml of a Drosophila medium (Strickburger, 1967) which had been modified by the substitution of 250 ml of autoclaved, ground Purina Guinea Pig Chow for 38 ml dried yeast. Uninfected fly colonies were periodically checked for infection. Colonies of infected flies were initiated with larvae from guinea pig litter pans. Drosophila melanogaster, Oregon-R strain, were obtained from Dr. D. D. Miller, University of Nebraska, and were maintained in the same fly medium. The Drosophila were not found to be naturally infected with trypanosomatid parasites. Insects were infected with culture forms con-