BIOLOGICAL AND BIOCHEMICAL INVESTIGATIONS 0N THE NEMATODE, SYNGAMUS TRACHEA (MONTAGU, 1811) CHAPIN, 1925 The“: for ”1.: Degree of DI1. D. MICHIGAN STATE UNIVERSITY Russell Francis Krueger 1958 THESIS 6.3. This is to certify that the thesis entitled BIOLOGICAL AND BIOCHEMICAL INVESTIGATIONS ON THE NEMATODE, SYNGAMUS TRACHEA (MONTAGU', 18mm, T921;— presented by Russell Francis Krueger has been accepted towards fulfillment of the requirements for I Ph D. . Microbiology and ______degree In_ Public Health A} ;_ i -, in ,x‘. ("z ‘ / David T. Clark Major professor Datemcuw— 0-169 L I B R A R Y Michigan State University BIOLOGICAL AND BIOCHEMICAL.INVESTIGATIONS ON THE NEMATODE, SYNGAMUS TRACHEA (MONTAGU, 1811) CHAPIN, 1925 By Russell Francis Krueger A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1958 '1 Q‘\ II V A I. I ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. D. T. Clark, of the Department of Microbiology and Public Health, for his helpful advice and constant encouragement throughout this investigation. The author would also like to thank Dr. R. U. Byerrum, of the Department of Chemistry, and Dr. W. D. Lindquist, of the Department of Microbiology and Public Health, for their advice and criticisms. Appreciation is also extended to Dr. L. C. Ferguson, of the Department of Microbiology and Public Health, and to Dr. R. D. Barner, of the Department of Veterinary Pathology, for their suggestions and criticisms of this thesis. The author is indebted to Mrs. Betsey J. Krueger for her untiring aid in the preparation of the manuscript and constant encouragement during this investigation. Russell Francis Krueger candidate for the degree of Doctor of Philosophy Final examination: August h, 1958, 1:00 P. M., Room 101 Giltner Hall. Dissertation: Biological and Biochemical Investigations on the Nematode, Syngamus trachea (Montagu, 1811), Chapin 1925 Outline of Studies Major subject: Parasitology Minor subjects: Biochemistry, Veterinary Pathology Biographical Items Born, September 1, 1926, Milwaukee, Wisconsin Undergraduate Studies, Marquette University, lghh and Isue-SO Graduate Studies, Bowling Green State University, 1950-51, Michigan State University, 1955-58 Experience: Member United States Navy, lghh—hé, Special Graduate Assistant, Bowling Green State University, 1950-51, Researcher, Abbott Laboratories, 1951-55, Graduate Assistant, Michigan State University, 1955-56, Special Graduate Research Assistant, Michigan State University, 1957, Temporary Instructor, Michigan State University, 1957-58 Member of American Society of Parasitologists, American Association for the Advancement of Science, Midwest Conference of Parasitologists, Illinois Society of Bacteriologists, Illinois Society for Medical Research, National Society for Medical Research, Society of the Sigma Xi, Michigan Society of Bacteriologists BIOLOGICAL AND BIOCHEMICAL INVESTIGATIONS ON THE NEMATODE, SYNGAMUS TRACHEA (MONTAGU, 1811) CHAPIN, 1925' By Russell Francis Krueger AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1958 Approved Russell Francis Krueger ABSTRACT The purpose of this investigation was to study para- sitism as it is represented in the life cycle of the nema- tode, Syngamus trachea, and concurrently to establish bio— chemical Characteristics of this species with special emphasis on the role of respiration and carbohydrate metabolism. Oral, intravenous, intraperitoneai, and subcutaneous routes of inoculation of chickens and turkeys were tried. No infection was obtained with intraperitoneal and subcuta- neous inoculation. A previously untried route of infection, intravenous inoculation of suspensions of larvae, proved to be superior to established methods because of the ease of controlling the degree and intensity of infection. Syngamus was recovered from the trachea as early as eight days fol- lowing intravenous inoculation and had a lhfi day prepatent period. Oral inoculation resulted in gapeworms in the trachea on the ninth day and a prepatent period of 15 days. After approximately two weeks of age chickens became more difficult to infect and after three to four weeks of age became refractory to gapeworm infection. Turkeys as old as 186 days could easily be infected. The intensity Of gapeworm infections in turkeys began to decline after 2h days and by 37 days infections were negligible. Gapeworms were recovered from turkeys as long as 82 days after inocu- lation. Russell Francis Krueger The red pigmented pseudocoelomic fluid of Syngamus appears to be an iron porphyrin compound which is probably a hemoglobin. This hemoglobin is not identical with that of the host's hemoglobin. These hemoglobins are compared and differences noted. Wet and dried weight values covering the life span of Syngamus in the trachea of turkeys are given. The percent dried weight of paired Syngamus was 26.2% i 2.9%. The metabolic activities of Syngamus -— aerobic oxygen consumption and carbon dioxide liberation, endogenous and exogenous carbohydrate utilization, carbohydrate content, butyric acid utilization, and anaerobic carbon dioxide liberation -— were most pronounced in the youngest and smallest gapeworms. These metabolic activities all gradual- ly decreased as the weight of the gapeworms increased. There appeared to be a relationship of metabolic activity tO egg production. Respiration studies were conducted by standard mano- metric techniques. The rate of oxygen utilization of paired Syngamus decreased from 18.5h to h.08 microliter per mg dried weight per hour as the worms aged and increased in weight. The rate of oxygen consumption was decreased by such inhibi- tors as azide, cyanide, 2,h-dinitrophenol, fluoride, iodo- acetate, and malonate. Inhibition by fluoride and malonate took place only in a calcium free substrate. Oxygen utili- zation by Syngamus was also decreased following gassing with carbon monoxide and was further decreased when gapeworms Russell Francis Krueger (I) were maintained in darknes . The respiratory quotient (R0) in a phosphate buffered substrate, with or without 0.005 M glucose, was approximately 0.87. In a substrate containing 0.065 M butyric acid the R0 for Syngamus was 0.708. A R0 of approximately 1.0 was obtained with an acetone powder prepared from Syngamus in a substrate containing 0.005 M glucose. The range of the polysaccharide content in Syngamus was 0.33% to 0.76% and the rate of endogenous polysaccharide utilization was about 0.35% wet weight of Syngamus per 2h hours. Exogenous glucose utilization decreased with in- creased size of gapeworms from 3.89 to 0.23 mg per gram wet weight per hour. There was a pronounced increase in the rate of exogenous glucose utilization when gapeworms were maintained in vitro for ten days. TABLE OF CONTENTS Page INTRODUCTION - ----- - - - — — - - - - - l HISTORICAL REVIEW 1. Biological - - - — - _ - - - - - _ - h 11. Biochemical - - - ------ - - - 8 MATERIALS AND METHODS I. Biological A. Studies on the life cycle - — - 12 B Infection of turkeys and chickens ----------- 13 C. Acquisition, care and manage— ment of turkeys and chickens - -lh D Care and maintenance of earth— worms - - — - ~ — ----- - - 15 II. Biochemical A. Recovery of Syngamus trachea —- 16 B. Determination of differences in body substance Of Syngamus trachea --------- - — — 17 C. Identification and Characteri- zation of a red pigment in the pseudocoelomic fluid of Syngamus trachea ------------ 17 D. Quantitative investigation of endogenous and exogenous carbo— hydrate utilization by Syngamus trachea ------------ 21 E. Characterization of metabolism Of Syngamus trachea ------ 23 RESULTS 1. Biological ---------- - - - 26 II. Biochemical ------------ 38 DISCUSSIO - - - - - - - - - - ------- 69 SUMMARY - ----- — - — - - ------ - 85 REFERENCES CITED - - - - - - — - - - - - — — 89 INTRODUCTION Studies in parasitology are no longer confined to areas of morphology, taxonomy, and ecology, but rather have been extended to include biochemical and physiological aspects of parasitism. One must recognize and appreciate the past con- tributions which serve as a foundation for the field of parasitology, and at the same time realize that a constant evolution is taking place. In this present day a perusal of the literature on bio- chemical studies of parasites will reveal a real dearth of information. The parasitic protozoa have been less neglected than the parasitic metazoa in this respect. In an attempt to gain insight into the physiology of the parasitic metazoa, the nematode, Syngamus trachea (Montagu, I811) Chapin, 1925, was chosen for biological and biochemical investigation. Syngamus trachea has as its habitat the trachea of birds. Frequently the bird experiences difficulty in breathing which results in gasping or gaping, thereby serving as basis for its common name, the gapeworm. It has a characteristic red color and presents a forked appearance by reason of the perpetual syngamy of the male and female nematodes. The very location and nature of Syngamus are reason enough for selecting this worm for investigation. In such an unusual habitat, the trachea, Syngamus trachea I is exposed to an abundance Of oxygen. Oxygen is also availa- ble to parasitic helminths which live in the blood, lungs, and swim bladder. Most helminths are found in an environment poor in oxygen, such as the lumen of the intestinal tract; others, although few in number, exist in body tissues where oxygen is available only by way of the host's Circulatory system. von Brand (l9h6) calls attention to this situation and states that there is practically nothing known about parasitic worms that live in environments rich in oxygen. von Brand (1952) also raised the question concerning the relationship of carbohydrate metabolism of such parasitic invertebrates having a free excess of oxygen and the free- living forms. This question and the status of these parasitic forms remains unanswered. Syngamus trachea is a member of the large superfamily of nematodes, Strongyloidea. Some of the representatives of this superfamily are of vast medical and veterinary importance. Most Of these Strongyloidea, as do most parasitic nematodes, inhabit the lumen of the intestinal tract. Here they attach themselves by strong, pronounced buccal capsules and suck blood while the teeth or cutting plates in the buccal capsules tear Off bits of mucosa. At the same time, the parasite is subject to a wide variety of intestinal contents. For such parasites it is difficult to ascertain the nutritional or environmental relationships in the presence of such a hetero— geneous assortment of nutrients, wastes, and metabolic products of assorted biota, as well as enzymes and secretions of the intestinal tract. Syngamus trachea possesses similar morpho- logical and physiological attributes as other Strongyloidea but inhabits a quite simple, constant, and easily defined environment. Its nutrition in the trachea of the bird is limited to constituents of the blood, lymph, and tracheal mucosa (Clapham, 1935, 1939; Rogers, l9h0; and Wehr, l9h0). With data obtained from an investigation of Syngamus one can feel more assured that it represents characteristics of the parasite. Such information might then be compared with data obtained on species from highly variable environments. The data obtained and techniques developed might be applicable to particular members of the Strongyloidea which are of medi- cal and economic importance. The aim of this investigation was to analyze parasitism as it is represented in the life cycle of Syngamus trachea, and concurrently to determine biochemical characteristics of this species, placing special emphasis on respiration and the role of carbohydrate metabolism. HISTORICAL REVIEW 1. Biological The earliest published account of Syngamus trachea was by Dr. Andrew Wiesenthal (1797), a physician from Baltimore, Maryland. The English naturalist, George Montagu (1811) first described the parasite on a scientific level. He named it Fasciola trachea. von Siebold (1836) gave a better descrip- tive account of the parasite and also changed the scientific name to Syngamus trachealis. Chapin (1925) pointed out that the species name was incorrect according to the International Code of Zoological Nomenclature and therefore he Changed the species name back to the original, resulting in the name it is known by today. Other early studies on the parasite were by Dujardin (l8hS) and Cobbold (1861). The first positive contribution on the life cycle of Syngamus trachea was by Ehlers (1872). He concluded that transmission to a new host occurred by ingestion of eggs containing mature larvae. Mégnin (1880, 1881a, 1881b, 1882) and Railliet (1901) arrived at the same conclusion unaware of Ehlers‘ earlier work. The findings of these early workers were confirmed by later investigators (Ortlepp, 1923; Leiper, 1926; Lerche, 1928; Szidat, 1928; Taylor, 1928; Rice, 1929; Morgan and Clapham, 193M; Wehr, 1937, 1939; and Olivier, l9hh). It was observed by Mégnin (1880) and Theobald (1899-1900) that h the eggs of Syngamus, if kept moist, remained viable for about one year. Mégnin (1880), Ortlepp (1923), and Lerche (1928) suggested that development in the egg to an infective larva occurs within one week. Walker (1886) reported that it took much longer. Years later Wehr (1937) redescribed the larval stages of Syngamus. He found that the larvae moult twice and are in- fective as third stage larvae. In addition to direct transmission by ingestion of Syngamus eggs containing infective larvae, a Franklinville, New York, physician, Dr. H. D. Walker (1886), demonstrated that the earthworm can become infected with a larval form of Syngamus and thus serve as a vehicle for transmission to the bird. The importance of Walker's findings were subverted by Salmon (1886, 1899) who cast doubt on certain aspects of his work. However, the role played by the earthworm as a trans- port host was confirmed by other investigators (Garman, 1897; Ranson, 1916; Waite, 1920; Clapham, 193M; Morgan and Clapham, 193M; Taylor, 1935; Ryzhlkov, l9hl). These investigators not only established the role of the earthworm in transmission, but they also incriminated other invertebrates such as slugs, snails, and insects. Taylor (1935, 1938) found that the larvae may remain viable in the earthworm up to four and one- half years. Madsen (1952) believes there is much evidence in favor of an invertebrate host acting as an intermediary in most natural infections. The infection is more difficult to establish without an intermediate host suggesting that the 1% larvae are subjected to some physiological stress which seems to be eliminated when an intermediate host is used (Clapham, 1938). Once larvae are ingested by the bird their fate from intestine to lungs is unknown. Ortlepp (I923), Wehr (1937), Clapham (1939), and Guilford and Herrick (l95h) postulate that the route of migration from the digestive tract to the lungs is through the blood stream. They base their conclu- sion (0 on finding third stage infective larvae approximately twenty-four hours after ingestion in various portions of the body such as the lungs, liver, air sac, and postcaval vein. Although their observations indicate that the blood stream is a likely route of migration this postulate has not been satisfactorily answered (Biester and Devries, l9hh; Morgan and Hawkins, 1953). Wehr (1937) states that the fourth stage larvae attaches to the female fourth stage larvae in the lungs sometime be- tween the third and seventh day after infection and then mi- grate as attached pairs to the trachea. Guilford and Herrick (195k) agree with Wehr that the paired gapeworms reach the trachea on the ninth day after infection, but Walker (1886) believed that only six to seven days were required. A wide range Of time has been given for the prepatent period of Syngamus. Fourteen to 25 days have been cited (Walker, 1886; Ortlepp, 1923; Lerche, 1928; Wetzel and Ouittek, l9h0). Syngamus has been reported as living in a chicken for as long as 1h? days and 22h days in a turkey (Wehr, 1939). There appears to be a distinct difference in susceptibil- ity of Chickens and turkeys to infection with Syngamus trachea. Olivier (l9hh) in summarizing his investigation and the ob- servations of others on species susceptibility suggested that turkeys are more susceptible than Chickens. He also concluded that young turkeys are more susceptible than young chickens of the same age. Wehr (1939) was in agreement with these observations and in addition thought that chickens lost their infections more readily and became refractive to infection at an earlier age. Madsen (1952) stated that this difference in susceptibility "in this respect seems to have been somewhat overstressed", inasmuch as he and others, Theobald (1899- 1900), Klee (1903), Waite (1920), Ranson (1921), Morgan (1931), Crawford (l9h0), and Olivier (l9h3) have encountered gapeworms in adult chickens. It was suggested by Ranson (1921) that age, stress, and debilitation might be the reasons for find- ing this infection in chickens. Clapham (1933, 193B) was able to demonstrate that Vitamin A and general mineral defi- ciencies produced sufficient physiological Changes in ten week-old chickens that they could be infected. The investigations of Olivier (l9h3, l9hh) using turkeys and chickens, and Guilford and Herrick (195h) using pheasants, showed that an acquired immunity develops in birds infected with Syngamus trachea. Immunity was demonstrated by challenge doses of infective larvae. Madsen (1952) concluded that a number of "natural hosts for gapeworm exists among the passerine birds and among gallinaxxnmabirds, the partridge and turkey". He discusses and summarizes the distribution and epidemiology of Syngamus trachea infections throughout the world. In conclusion he states,"the epidemiology of the gapeworm is thus very com- plicated and much work remains to be done". 11. Biochemical There have been no biochemical investigations of the gapeworm, Syngamus trachea. Inroads have been made into some few biochemical aspects of other nematodes. There is surprisingly little information on an essential basic biochemical characteristic, the dry weight of nematodes. Too frequently this information, which is essential for the determination of respiratory values, does not appear in publi— cations. Values for less than a dozen nematodes are availa- ble for consideration. Information on dry weight has been largely confined to the two large nematodes, Ascaris lumbri- coides (Weinland, 1901; Flury, I912; Gurtner, 1948) and Parascaris equorum (Schimmelpfennig, 1903; Flury, 1912). Only a few investigators have included such values for the smaller nematodes (von Brand, 1938, 1952; Bueding, 19h9; Lazarus, 1950). A second basic consideration of parasitic nematodes is that of glycogen content. It is the most frequent carbohy- drate determination made on nematodes. Unquestionably glyco- gen is the polysaccharide stored in the parasitic nematodes. Baldwin and King (l9h2) found the glycogen present in Ascaris lumbricoides to be approximately the same as that in mammals. The distribution and amount of glycogen found in several species of nematodes are summarized by von Brand (1952). Most of the glycogen present in nematodes in localized in the subcuticle, muscles, ovaries, and eggs. One must admit the existence Of simple sugars in para- sites because it is in these forms that assimulation and utilization occurs. Reducing sugars have been identified in Ascaris by Rogers (l9h5), in Litomosoides carinii by Bueding (l9h9), and in Parascaris by Fauré-Fremiet (1913). The investigations on various aspects of carbohydrate metabolism and aerobic and anaerobic gas exchange have been conducted on less than two dozen nematodes. Some of the. larval forms of nematodes which have been studied with re- gard to metabolic processes associated with carbohydrate utilization and respiration are: Eustrongylides ignotus (von Brand, 19h2, l9h5; von Brand and Simpson, 19hh), Neoaplectana glaseri (Rogers, l9h8; Massey and Rogers, l9h9, 1950), Nippostrongylus muris (Rogers, l9h8), Strongyloides papillosus (Costello, 1957), Trichinella spiralis (Stannard, McCoy, and Latchford, 1938; Goldberg, 1956). Some phases of intermediate carbohydrate metabolism of Strongyloides ratti have been studied by Jones Eh 31., (l955a, 1955b). The carbohydrate metabolism and utilization, and aerobic and anaerobic respiration of adult nematodes have been in- vestigated in the following species: Ascaridia galli, Nematodirus spp., Neoaplectana glaseri, Nippostrongylus muris (Rogers, l9h8; Massey and Rogers, l9h9, 1950); Ascaris lumbricoides (Adam, 1932; von Brand, l93h; Kruger, I936; Laser, 19hh); Dracunculus insignis (Beuding and Oliver- Gonzéles, 1950); Haemonchus contortus (Rogers, 19h8); Heterakis spumosa, Ostertagia Circumcincta, Strongylus equinus, Strongylus vulgaris, Syphacia obvelata (Lazarus, 1950); Litomosoides carinii (Bueding, 19h9); and Nematodirus spp. (Massey and Rogers, 19h9, 1950). A few other nematodes which are not included above have been investigated for various specific biochemical Characteristics. Of all the nematodes, Ascaris lumbricoides has received the major attention from numerous investigators. From knowledge that is available on carbohydrate meta— bolism of a few nematodes, one might assume that the well known processes of the Meyerhof—Embden system and Krebs cycle sequences take place in parasitic nematodes as it does in mammalian tissues. von Brand (1950) suggests that the problems which arise Concerning the metabolism of carbo- hydrates in nematodes are linked with the character of respiration for individual species. Hemoglobin in nematodes was first demonstrated and re- ported by Keilin (1925). He recovered it from the muscle and perivisceral fluid of Ascaris lumbricoides. At that time he raised the question of its physiologic significance. The functional nature Of such a respiratory accessory still has not been settled (Davey, 1938; Wharton, l9hl; Davenport, l9h9; Rogers, l9h9a). 11 Rogers (19h0) in a study on the hematological nature of the material present in the intestine of nematodes and trema- todes used Syngamus trachea as one of the representatives of nematodes which ingest hemoglobin. He found hematin present in the black~pigmented ingesta. However, he was unable to extract a hemoglobin. In his discussion on the nature Of the ingesta of the species studied he states that the body fluid Of S. trachea contains hemoglobin. There were no data or references cited for such a fact, and von Brand (1952) did not include S. trachea in the list of nema- todes for which hemoglobin has been identified. It is apparent from the foregoing review that further investigation is necessary in order to explain certain aspects of the life cycle of Syngamus trachea. This, coupled with a biochemical investigation, will not only elucidate the nature of Syngamus but may also be applicable to other parasitic nematodes. MATERIALS AND METHODS I. Biological A. Studies on the life cycle. The nematode was originally obtained in the larval form encysted in a collection of heterogeneous earthworms. These earthworms were secured from the pheasant rearing enclosures on the Michigan Department of Conservation Game Farm, Mason, Michigan. Approximately a dozen earthworms were fed to each of seven 21 day old White Leghorn Chickens. The birds were sac- rificed 17 days later. The tracheae were removed and exam- ined for the paired, adult Syngamus. Only one pair was re- covered from the seven chickens and it was identified as Syngamus trachea. The female worm was mature and passing typical eggs. The uteri were removed from the worm and ground in a small amount of tap water using a pestle and mortar. This suspension of eggs and debris was filtered through several layers Of cheesecloth and placed in a petri dish. Enough water was added to the suspension to obtain a depth of about one cm. The petri dish was left uncovered at room temperature for twelve days. Evaporated water was re- placed by adding tap water daily, and the suspension was agitated slightly by gentle rotation of the dish. On the twelfth day larvae and the associated debris were loosened 12 p—n (,3 from the bottom of the dish by means of a rubber-tipped rod. The embryonated eggs and freed larvae were washed several times in tap water. Much Of the debris was removed by this step. The embryonated eggs and larvae, suspended in a small amount of tap water, were added to a petri dish filled with soil which contained ten medium size (four to five cm) earth- worms, Allolobrophora caliginosus. Fourteen days later these earthworms were fed to a ten day old Leghorn chicken. The bird was sacrificed 17 days later. The trachea was removed and examined for the presence of Syngamus. The specimens recovered were identified as S. trachea. These gapeworms served as parent stock for the investigation. The initial procedure used for obtaining infective lar- vae was modified slightly. The changes consisted of in- creasing the incubation time of the eggs to ID days and the exposure time of the earthworms to larvae to 21 days. This schedule was maintained throughout the investigation when the earthworm was used. B. Infection Of chickens and turkeys. A number of routes of inoculation were used. The oral route Of inoculation was studied in infections established by passage of laboratory prepared suspensions of larvae directly into the crop by means of a plastic tube, and the forced feeding of earthworms which contained the infective larvae. Food was withheld from the birds six to eight hours prior to per os inoculation. Suspensions of larvae were also administered intraperitoneally, intravenously, and subcu- taneously. The medial wing vein (venae profunda humeri) was used for intravenous inoculation. Usually the number of larvae given were determined by counting aliquants of the suspensions containing larvae. Suitable dilutions were then made in order to place the inoculum in a volume of one ml. In order to determine the time required for the appear- ance of gapeworms in the trachea and the prepatent period of the worm, turkeys were inoculated intravenously with larvae and orally with earthworms. Beginning on the sixth day fol- lowing inoculation two birds were sacrificed each day for 19 days and Observations were made immediately following death. C. Acquisition, care, and management of turkeys and chickens. The chickens and turkeys used in this investigation were obtained as day old birds. The Poultry Husbandry Department of Michigan State University supplied the White Leghorn chickens and some Broad-breasted Bronze turkeys. Broad- breasted Bronze turkeys were also Obtained from Janssen Farm's Hatcheries, Zeeland, Michigan. A majority of the birds were hens although no attempt was made to select one sex or the other. New Hampshire cockerels were obtained from Delamarter‘s Hatchery, East Lansing, Michigan. All the birds were kept in electrically heated brooders at suitable temperatures until they were four weeks old. After this they were housed either in standard wire bottom poultry cages or in isolation rooms in which wood shavings t——‘ \Jl were used as litter. Infected birds were kept separate from uninfected birds. The feed used throughout these studies contained no antibiotics or other medication. The chick starter mash and turkey starter crumbles used were manufactured by A. E. Staley Manufacturing Co., Decature, Illinois and Valley City Milling Co., Portland, Michigan. Feed and water were sup— plied ad libitum. D. Care and maintenance of earthworms. The colony of earthworms, Allolobrophora caliginosus, used as transport hosts for the larvae of Syngamus trachea was established from a single identified specimen obtained in the vicinity of East Lansing, Michigan. This specimen was placed in soil which had been autoclaved for D5 minutes at 1210 C. To this soil scraps of organic material (vegeta- bles, fruit, oatmeal, coffee grounds, etc.) were added. Moisture as well as additional organic materials were added to this worm bed as the need demanded. y... 0‘ II. Biochemical A. Recovery of Syngamus trachea. The biochemical investigations were conducted on Syngamus trachea recovered from infected turkeys. Turkeys were infected intravenously with the third stage infective larvae, thereby insuring knowledge of the exact age of the gapeworms. Where at all possible every pair of gapeworms used for any one biochemical determination was obtained from the same turkey. This minimized possible variation which might occur in gapeworms obtained from different birds. In the trachea the male gapeworm is found permanently attached to the female. In separating the pair one member is usually destroyed; therefore, because of this inseparable nature, all investigations were conducted on paired Syngamus. The turkeys were killed by cutting the jugular veins and piercing the brain. The trachea was removed immediately. The entire length of the trachea was opened and the paired Syngamus removed with a moistened camel‘s hair brush. The anterior end of the male gapeworm usually was embedded in the mucosa, and was loosened with a fine probe. The gape- worms were placed in cold (30 C) 0.9% saline and left there until all were removed from the trachea. They were then \vashed in six to eight changes of saline (30 C) followed by immersion for 15 minutes in saline warmed to 370 C. Anti- biotics were added to the warmed saline to obtain a final concentration of h00 units penicillin and 0.h mg dihydro- streptomycin per ml. After five minutes in the solution 17 containing antibiotics the gapeworms were wahed in four changes of saline (30 C). The entire procedure required 30 to N5 minutes. The gapeworms were then held in saline at 30 C until used. B. Determination of differences in body substance of Syngamus trachea. To determine the wet (live) weight of Syngamus the gape- worms were removed from the cold saline solution with a camel's hair brush and placed on Whatman No. 1 filter paper to remove excess moisture. They were then transferred to tared containers and weighed to the fourth decimal place on an analytical balance. Dry weights were determined by drying to constant weight in a hot air oven (1000 C) for 10 to 12 hours. Total carbohydrate and polysaccharide determinations were performed on pairs of gapeworms. The procedure used was similar to that of Bueding (l9h9). Polysaccharides were determined according to the methods of Good, Kramer, and Somogyi (1933) and Somogyi (l9h5). Total carbohydrates were determined after deproteination (Nelson, l9hh) using the spectrophotometric method of Somogyi (19h5). Values obtained .by the Somogyi method are expressed in mg percent glucose. C2. Identification and characterization of a red pigment in ‘the pseudocoelomic fluid of Syngamus trachea. To Obtain the red fluid present in the pseudocoelom of fSyngamus ten large (approximately 3 mg wet weight each) 18 female gapeworms were removed from the final cold saline rinse with a camel's hair brush and blotted dry on Whatman No. 1 filter paper. They were then transferred to the in- side rim of a 12 ml conical, graduated centrifuge tube. The tube contained ten ml of dilute ammonium hydroxide (eight ml concentrated NHuOH in one liter distilled water) or 0.1 N hydrochloric acid for collection of the pseudocoelomic fluid. The female gapeworms were oriented on the wall of the tube in such a way that the posterior ends came within five mm of the fluid while the anterior ends were quite close to the rim of the tube. With a fine probe the tips of the posterior ends of the gapeworms were pierced. A cotton plug was in- serted into the tube in such a way as to secure the gape- worms. The tube was centrifuged at 750 rpm for ten minutes in a size 2 International Centrifuge. A second method for the collection of the red pseudo- coelomic fluid consisted of placing the pierced gapeworms directly into a conical centrifuge tube containing distilled water or 0.9% saline and centrifuging at 1600 rpm for ten minutes. The fluid containing the red pigment was then re- moved with a pipette. This method proved to be as reliable as the first for the collection of pseudocoelomic fluid. The identification and characterization of the red pig- Inent in pseudocoelomic fluid was by means of absorption curves obtained with a Bausch and Lomb Spectronic 20 colori— Ineter. Absorption curves were determined on pseudocoelomic fluid of Syngamus and on similarly treated samples of turkey blood. Treatments and procedures used on the paired samples were discussed or suggested by Hawk, Oser, and Summerson (19D?) and Hunter (1951). The absorption spectrum of oxyhemoglobin was demon- strated on samples taken in dilute ammonium hydroxide. The tube containing the sample was shaken vigorously to aerate the solution and absorption curves were then determined. Reduced hemoglobin absorption curves were secured under four different conditions. (1) Two to three drops of 10% sodium hydrosulfite were added to a dilute NHMOH solution containing the pigmented pseudocoelomic fluid and gently heated for 20 minutes to a temperature of not more than 550 C. Absorption curves were determined at pH 7 and pH 8. The pH of all solutions was determined with a Beckman pH meter and adjusted through the use of acetic acid. (2) The second condition was made by the addition of two to three drops of 10% sodium hydrosulfite to the samples, followed by evacua- tion and heating (not higher than 550 C) for 20 minutes. The colorimeter tube was sealed with a rubber stopper and a vacuum was drawn and maintained during heating. After an absorption curve was determined the tube was cooled, unstop- pered, shaken vigorously, and a second absorption curve was determined on the aerated sample. (3) A third sample was I>laced in a colorimeter tube and sealed with a rubber stop- Iber. The tube was evacuated and a vacuum was maintained Vvhile it was gently heated (not higher than 550 C) for one Tiour. Absorption curves were obtained on this sample while warm; and again after it was cooled to room temperature, un- stoppered, and shaken to permit aeration. (h) A final con- dition was established by heating the sample (not higher than 550 C) and paSsing a constant flow of nitrogen (95% N2 - 5% C02) into the solution. The excess gas was removed by maintaining a negative pressure above the sample. After one hour of such treatment an absorption curve was determined on the sample while warm, and another absorption curve was ob- tained after cooling, unstoppering, and shaking the sample. The methemoglobin absorption spectrum was determined on samples collected in ammonium hydroxide solution. Two to three drops of saturated potassium ferricyanide were added, and the solution was adjusted to the desired hydrogen ion concentration. Absorption curves were obtained at pH 6, 7, and 8. Acid hematin was determined by using dilute hydrochloric acid according to the method of Cohen and Smith (1919). Acid hematoporphyrin resulted when two to three drops of concen- trated sulfuric acid were added to a sample collected in dilute ammonium hydroxide (Hunter, 1951). Carboxyhemoglobin was demonstrated by gassing with car— bon monoxide (95% CO - 5% 02) for 30 minutes. Two methods Vvere used. Carbon monoxide was bubbled through a dilute Eimmonium hydroxide solution containing pseudocoelomic fluid 21nd then absorption curves were determined. In the second Tnethod carbon monoxide was bubbled through a saline solution <2ontaining whole gapeworms. The pseudocoelomic fluid was 21 then released into the saline by piercing the worms and cen- trifugation. Absorption curves were determined on this solu- l tion. D. Quantitative investigation of endogenous and exogenous carbohydrate utilization by Syngamus trachea. The endogenous rate of carbohydrate utilization was established by determining the decrease in total carbohy- drates and polysaccharide content which resulted when Syngamus was maintained in;vi££2 for 2h hours at 370 C. Gapeworms were divided into three groups. The first group served as the controls, and total carbohydrates and polysaccharides were determined immediately with this group using methods described above. A second group was placed in the substrate used for respiration studies. The composition of this substrate is described later. One hundred units peni- cillin and 0.1 mg dihydrostreptomycin were added to each ml Of the substrate. The substrate of the third group contained antibiotics as in the second group plus 0.005 M glucose. Groups II and III were placed in 25 ml of the proper substrate in a standard petri dish and incubated at 370 C for 2h hours. Following incubation the gapeworms were washed six times in saline as a preparatory step for the determina- tion of total carbohydrates and polysaccharides. These (determinations were made following the same procedure used JFOr the control group. The exogenous rate of carbohydrate utilization was ob- ‘Lained by determining the rate of glucose uptake from a known substrate. The amount of glucose was measured spectro- photometrically by the method of Somogyi (l9h5). The rate of glucose uptake was investigated under two conditions. Paired gapeworms were maintained in 20 x 150 mm screw-cap culture tubes in 2 m1 of the respiration substrate (described later) containing 0.005 M glucose. Antibiotics were added to the substrate to obtain 100 units penicillin, 0.1 mg di- hydrostreptomycin, and 100 units nystatin per ml. The tubes were placed in a horizontal position in a 370 C incubator. The substrate was Changed daily, and the amount of glucose in the substrate that was removed was determined. The gape- worms were maintained under these conditions for ten days. Sterility tests were performed on the substrates used for both endogenous and exogenous carbohydrate studies. Brain-heart infusion broth and N.I.H. thioglycolate were used for sterility tests. The second condition under which the exogenous carbo- hydrate utilization by Syngamus was examined occurred while the respiration of Syngamus trachea (in toto) was studied with the Warburg apparatus. Glucose concentrations of the substrate from reaction vessels containing parasites were compared with the glucose concentrations of the substrate in the thermobarometric flasks at the end of the determina- tion. The differences between glucose concentrations repre- Isented glucose uptake by the parasite. E. Characterization of metabolism Of Syngamus trachea. The nature of the metabolism of Syngamus trachea was determined by investigation of (l) aerobic respiration, (2) anaerobic respiration, (3) the relationship of carbohy- drates to respiration, and (A) the effects of various meta- bolic inhibitors. The studies on respiration were conducted in a Precision Warburg 20-unit respirometer at a shaking rate of 120 per minute and a temperature of 370 C. Standard manometric pro- cedures were followed (Umbreit et 31., 19h9). Oxygen uptake was determined by the direct method. A continuous estimate of carbon dioxide was obtained by subtracting oxygen uptake from the value Obtained in a paired vessel without potassium hydroxide. Thus the quantity of carbon dioxide liberated could be followed over a long period of time and comparative relationships noted. Anaerobic respiration values and esti- mates Of carbon dioxide used in respiratory quotients were obtained by making a correction for retension of CO2 in the buffered substrate. Using paired vessels, 0.2 ml 1 N sulfuric acid was emptied from the sidearm into one reaction vessel at the beginning and into the second vessel at the end of the determination. A total volume of three ml substrate was used in each reaction vessel. These vessels had an approxi- niate capacity of 15 ml. The common atmosphere for manometric Vvork on Syngamus was air. Gas atmospheres of nitrogen (95% DJ2 - 5% CO2) and carbon monoxide (95% CO - 5% 02) were se- csured through gassing by means of a lO—phase gassing manifold while the vessels where in the bath. Twenty minutes of con- tinuous nitrogen flow and NO to 60 minutes of carbon monoxide flow were the gassing periods. The substrate used throughout the investigation was selected following preliminary trial and error. It consisted of the basic components and proportions used for metabolism studies of schistosomes by Bueding (1950). The only modifi- cation was a lowering of the phosphate concentration. As used in this investigation it consisted of: 0.137 M sodium chloride, 0.0085 M potassium chloride, 0.005 M magnesium chloride, 0.0003 M calcium Chloride, and 0.03 M sodium phosphate. A pH of 7.5h for the solution was obtained through the use of appropriate amounts of monobasic and di- basic sodium phosphate and unless noted otherwise contained 0.005 M glucose. The pH of 7.5h is that Of turkey blood and the amount of glucose used is within the range of glucose concentration in birds (Spector, 1956). Twenty to 60 mg wet weight of gapeworms per flask was found most suitable for manometric investigation. Only paired gapeworms were used. A brei of the paired Syngamus was prepared as outlined in Umbreit__tl_l. (l9h9). Gas exchange (02 consumption and CO2 liberation) was calculated and expressed in terms of micro- liters per mg dry weight of parasite per hour, except when brei or an acetone powder (Green .2.313’ 1937) of the para- site were used. These calculations were based on a minimum period Of two hours. The compounds used as inhibitors were introduced from I\) \H the sidearm of the reaction vessel, or were placed directly into the substrate. Comparisons were made between paired flasks with and without the inhibitors. The method of Robbie (l9h6) was used to control cyanide concentrations. In the investigation of malonate and fluoride as inhibitors calci- um was omitted from the substrate because Massey and Rogers (1950) found greater inhibition when a calcium free sub— strate was used. Studies using carbon monoxide were con- ducted both in artificial light and in the dark. Darkness was obtained by wrapping the reaction vessels in aluminum foil. Other inhibitors used were azide, iodoacetate, and dinitrophenol. To remove endogenous nutrients an acetone powder of Syngamus was prepared according to the method of Green et .31. (1937). The activity of the incompletely defined enzyme system which occurs in the acetone powder on a 0.005 M glu- cose substrate was studied by standard manometric methods. Butyric acid was substituted for glucose in the sub- strate to demonstrate utilization of fatty acids. The con— centration of butyric acid used necessitated neutralization of the substrate with sodium hydroxide. The chemicals used in this investigation were of U.S.P. or A.C.S. quality. Specific organic reagents were products of Eastman Organic Chemical Division of Eastman Kodak Co., Rochester, New York. Antibiotics were purchased from E. R. Squibb and Sons, New York, N. Y. RESULTS 1. Biological A comparison of the routes of inoculation of chickens revealed that infection could be established through intra- venous inoculation Of infective larvae and forced feeding of earthworms containing encysted infective larvae (Table 1). Per 22 and subcutaneous inoculation of infective larvae pro- duced no Syngamus infection. It appeared to be more diffi- cult to infect Chickens after ten to fourteen days of age. Chickens older than three weeks of age could not be infected by inoculation of earthworms containing larvae andchickens more than 30 days of age could not be infected by intrave- nous inoculation of larvae. Earthworms infected under natu- ral conditions, in the pheasant rearing areas of the Mason Game Farm, were no better source of inoculum for older Chickens. There was great variation in the degree of Syngamus in- fections in chickens inoculated with the same number of earthworms. This can be noted in Table 1. Within a group or between groups of earthworm which were similarly infected and maintained, the number of pairs of Syngamus recovered per chicken fluctuated widely. A more uniform degree of in— fection of Chickens was Obtained by intravenous inoculation of infective larvae. The earliest appearance of Syngamus in chickens was in a single bird which died eight and one-half days after 26 ' Amman oxoc co pmscHHCOOH o\o o NH o AH om>th He H mbomzumH HE H m\0 o NH m m: mEcOSHHCmo m m\0 o :H o m: *mECOBHHcmm m\o o :H m om mEHO3cHumm m N\H H NH w Hm *wEuozzoumm H\H m SH H mH s OH mm\mH A.m.:.m . .m.m.m.m.H.H.H.H.H mH mm AH s H H\H my.” \IH H OH : OH H\H m AH H oH = o mxm A.:.: on m H s H m\o o m m H s H o\d m:.m: .H:.mm.wm.bm.bH.:H NH OH mm o H EuOBHHHmo H Hm QMZHm IHHB mzmeHIU LO ZOHHUMLZH LO mmmoma MIR LO ZOmHmcmH msEmmczm m>HuOchH HO cmaEsc CBOCxC: Ho COHm:onsm s '.‘ \‘l 0“ .cmchon sCOmmZ Hm Scam oewo oompm EOHH cochHno wEuochumm Q" "\ mxo O NH m com s He o.H H\o O NH H omH = He o.H H\o 0 OH H NA = HE m.o m\o O NH m m: = H5 o.H mxo O NH m o: z He o.H o\H H NH 0 on s Hs o.H m\m om.om.mm mH m :H s H5 o.H m\m om.om mH m 0H om>th Hs o.H moozm>th He o.H Hmxmae co coHHdtso m>mxmae ZOHHm QMZHm IHHS m>mmeH LO ZOHHOMLZH LO mmmomm NIB LO ZOmeumH o>HoOmHGH m .om>cmH mafiamcaw o>HuooHcH Lo Congas czocxc: HO mconcOQmsm * HxH mN OH H NA s H8 m.o m\o o NH m cm z OHS o.H m\o 0 NH m cm z NHE o.H o\m oH.wH.AH NH b on s H5 o.H N\N m.N m N cm = H5 o.H Nxo o A N on = He o.H NxH HH NH N AH z HE m.o. HAH ON HH H A = HE mN.o HAH mH :N H m = He m.o H\H m :N H m om>cmH He N.o wbozm>52 He o.H mDOmzmxm3A AUOSCHHCOOV m mgm<fi after inoculation. When turkeys were inoculated intravenously with suspen- sions containing known numbers of larvae the ratio of paired Syngamus recovered to larvae given was obtained. These ratios appear in Table 3 along with the number of larvae given, num- ber of paired Syngamus recovered, and duration of infection. In noting the age of the turkeys infected and number of worms recovered it again appears that turkeys of a wide range of ages can be infected equally well. The highest ratio of pairs of Syngamus recovered to lar- vae given intravenously was 0.1875. A total (sum of days 13 and lb) of 10h8 paired Syngamus were recovered when 5610 lar- vae were given; and this, when reduced to smaller numbers represents three Syngamus recovered when eight larvae were given, or three paired gapeworms for 16 larvae. When the duration of infection is considered with the ratio of Syngamus recovered it can be noted that after 2h days there is a consistent and rapid decrease in number of gapeworms. This consistent decrease in gapeworms continued until after 37 days only negligible infections remained. It is apparent from inspection of the data (Table 3) on the number of larvae given turkeys compared to the number of gapeworms recovered that a direct relationship exists. If the numbers of larvae given intravenously are grouped for infections of 2h days duration or less, the relationship shown in Figure 1 exists for paired Syngamus per larvae given. Such a linear relationship was better demonstrated under more 74 H3. Homwa onc co oochHcOOH A: o ooA .QOA .omo A .m .H omoo. :m mo H so: oN ooze. No uo H :o: oN come. on ooH .OA N oooH .oomH AHH .AN Nomo. oN AA H Hmoo 03H ooqo. AN mo.Ho.oo.HA m omNN.omNN.mo:.moo.ooN Ho.Hm.oH.Ho.ON A030. oN ooH .:A N oooH .oomH mom .oo oomH. :N oo.mo.oo.oo m oooH.Aoo.Aoo.mNs.mom NoN.omH.:oH.oA.o: :HmH. AN oHH .Ho .AH o omNN .ooA .ooo ooH .ooH .HA OAoo. ON NoH H on m: ONmo. oH oHH H ooo 0A OAHH. oH oHH .AA .:A o Hmom .ommH .ooo :om .oA .H: Nsoo. AH QNH .oHH N 00A .ooo oo .oo oomH. oH ooH .oHH N oooH .ooo NNN .Ho momH. mH :oH .ooH .A: H ooA .ooo .ooo .oom mAH .oHH .oo .NA onH. :H NoH .HoH .A: : ooA .on .on .ooo ooN .o:H .Ao .Ao onH. oH NoH .oHH N on .ooo ooH .OA moNH. NH oo H Hmoo 0o: oomH. HH om .o: o HWoooH -- :N mNmH -- H ommH. oH Ho H oooH oo mNoo. o mzmo CH pmHmHsOOcH AHmsocN>mHHcH Scions Hum muHmL mhma CH omcmm om< confisz Co>Hm om>cwH > *oHHNm GOHHOOHCH m>mxm3A o>HHooHdH .oz monomuo -osmmdzm Ho doHHmuoa >HmDOZM>HO m<§mmmeH EOLL Qmmm>OOmm m QmmHcNH o>HHOOHcH .pmuo>oomu one: mEc03ome oon paw mmH OH oco EOHH 6cm UOHONLCH ocm3 mH UOHNHSOOCH mzmxudu bH Nap LON .:o>Hm om>cmH o>HHOmLcH LO HonEdc ozu OH Umuo>oooc monomuu mssmmczm LO wchQ on» mH OHHmc one * moH o OOAo.oomo.oomo oH .H .o QAoo. Nm AHH H com o 0006. A no H Aoo o 0000. :H ooH N ONoH .oomH m 6Noo. o AHH .oo N Hmoo .00A o 0000. om ooH .om N oomH .ooo oH .m oooo. Am AHH H 00A No ooze. mo wkmo CH oonHsoocH kasocO>mchH zoxcsb com mchL mzmo CH omdmfllbm< HomEmz, Co>Hm om>ANH AOHHNL COHHOOLCH m>oxm3A o>HHooHcH .oz monomto mosmmczm No coHHmooo AUOSCHHCOOV O r\ MHm5L. .Cm OL‘ .QU i-«U (Om um >3 v—lF-I (GO +JL O [—4 0.5 Average mg wet weight of paired Syngamus ‘W 2.8 6:1 111.2 19‘.7 total carbohydrate olysaccharide 10 20 30 Days after intravenous inoculation Figure B. Comparison of percent total carbohydrate and polysaccharide content of paired Syngamus trachea to age and average wet weight 81 compared with samples of turkey blood used as reference standards. I The absorption curve of Syngamus pseudocoelomic fluid was quite similar to that of oxyhemoglobin of turkey blood as is shown in Figure 5. The d absorption maxima of turkey blood was 575 mu while for worm pseudocoelomic fluid it was 570 mu. The 0 absorption maxima for both samples was 5h0 mu. Similarly treated samples of turkey blood and the pig— mented body fluid of Syngamus were deoxygenated by physical and chemical methods. Reduced hemoglobin absorption curves were obtained for each sample. When the samples were deoxy- genated by heat, evacuation, and maintained in a vacuum fol- lowing heating there was a difference of five mu between Syngamus and the turkey hemoglobin maxima (Figure 6A). Syngamus pseudocoelomic fluid, when deoxygenated by gassing with nitrogen and heated in a vacuum resulted in a broad ab— sorption maximum with a peak at 535 mu (Figure 7A). When samples were deoxygenated by sodium hydrosulfite and heat in a vacuum similar flat absorption patterns as presented in Figure 8A resulted. In each case when the deoxygenated sam- ple was cooled and aerated, a typical oxyhemoglobin absorp— tion curve with3 4.) H m C Q) 'C To O 00-! # Turkey 0. O Syngamus LL 1 _l_ 520 5N0 560 Wave length (mu) 580 Figure BB. Absorption curves of above samples following cooling and aeration. reduced hemoglobin. Similar treatment of Syngamus pseudo— coelomic fluid resulted in an absorption pattern of deoxy- genated hemoglobin (Figure 9). This absorption pattern at pH 8 was identical to that of hemoglobin which was similarly reduced but maintained in a vaCuum, however, at pH 7 there was a slight absorption maximum at 535 mu. Absorption curves quite like that of carboxyhemoglobin of turkey blood were obtained with the pseudocoelomic fluid from Syngamus. These absorption curves are presented in Figure 10. Similar absorption maxima were evident at 570 mu and 5N0 mu. Treatment of turkey hemoglobin with potassium cyanide resulted in oxidation of the ferrous form of hemoglobin to the ferric form, methemoglobin. The absorption curves of turkey methemoglobin were similar at pH 6, 7, and 8. The absorption patterns of Syngamus pseudocoelomic fluid which was treated with KCN were dissimilar at each pH and also dif- ferent from those of turkey methemoglobin. These differences in absorption patterns can be noted in Figure 11. The absorption curves of acid hematin Of turkey blood and the acid preparation Of the body fluid of Syngamus were essentially the same. This can be seen in Figure 12. Treatment of hemoglobin with concentrated hydrochloric acid results in a hemoglobin artifact, hematoporphyrin. The absorption patterns of the hematOporphyrin of turkey blood and a sample of the pigmented body fluid of Syngamus treated in the same manner are moderately different. Such a differ- ence is apparent in Figure 13 in the wave length range between Optical density N7 \¢_‘\\T\ Syngamus pH 7 LSyngamus pH 8 _A A 500 550 ' 600 Wave length (mu) Figure 9. Hemoglobin absorption curves of turkey blood and Syngamus trachea pseudocoelomic fluid in dilute ammonium Chydroxide, pH 7 and 8, reduced by NaZSZON and heat. Optical density 500 Figure 10. SAC 570 ’“\ // \ / /°\. \ / \ '\_\ \\ . \\ /, \\ / /‘ )‘\ //. .1 II/. \. \\ ‘ \ , \ \\ Syngamus I\. ‘Syngamusfi A 580 5110 Wave length (mu) *pseudocoelomic fluid taken from Syngamus after exposure of the intact gapeworm to CO. Carboxyhemoglobin absorption curves of turkey blood and Syngamus trachea pseudo- coelomic fluid in dilutE'ammonium hydroxide, pH 8, after exposure to carbon monoxide. N8 N9 .o ocm . . :d Hm ooH Ixocpzc EdHcerm ousHHo CH oHsHL OHEOHOOOOpzomQ monomuo w35mmcwm cam UOOHH zoxusb HO mm>uso coHumcownm chonoEonuoE .HH musmHL .UHSHH OHEOHNOO lowdown mocomub m3Emmczm .< Aaev cameoH o>m3 .oooHo HoxHoA .m HnEV HHmCNH o>m3 ooo ooo o:m on: coo ooo 03m 6o: .qumnw11urxvs/ \ .\. .I . / I .0wa I ‘- A Hi > 6 HH m H 3 w o mm A mo .17 m o mm m 1. FA Kitsuap [eorqdo ity 4‘ \ V Optical den \\ xSyngamus \ //’ _——___— \\\ /,/ \H“’/ N80 520 560 600 6N0 680 Wave length (mu) Figure 12. Acid hematin absorption curves of turkey blood and Syngamus trachea pseudo- coelomic fluid. \ 11 C) .oHsHH OHEOHOOOOuDOmQ monomcu m:Emmczm 6cm UOOHQ onusu mo mo>cso COHHQuOmnm CHHAHQHOQOHmEoc vHO< .mH ousmHL Anev cameoH o>m3 ooA ooo ONo oom osm com 6o: 1|1 wsEmmCNm K1Isuap 19911d0 N70 mu and 560 mu. The endogenous carbohydrate utilization of Syngamus was determined for a 2N hour period. The total carbohydrates and polysaccharides were determined on gapeworms maintained in a phosphate buffered substrate without glucose and on gapeworms kept in the same substrate with 0.005 M glucose. The difference between these carbohydrate values after 2N hours and carbohydrate determinations at zero hours represent the carbohydrate utilization which appears in Table 5. The zero hour carbohydrate values represent the controls. Total carbohydrate was found to be 0.009 mg per mg wet weight of Syngamus, or 0.9% of the wet weight. The polysaccharides contributed 0.51% of the total carbohydrates. The endogenous total carbohydrate utilization of gapeworms maintained in a phosphate buffered substrate was 0.29% of wet body weight 'while gapeworms from a 0.005 M glucose substrate utilized 0.15%. The endogenous utilization Of polysaccharides by ESyngamus in a glucose free substrate was 0.35% of wet body Vveight. In a substrate containing glucose the utilization Vvas 0.38% of wet body weight. .The differences between the i;ota1 carbohydrates and polysaccharides per mg wet weight Eslngamus was 0.0039 mg for the cOntrols, 0.0062 mg for Group I I, and 0.00N5 mg for Group III. Exogenous carbohydrate utilization by Syngamus trachea Vvas Obtained by determining the amount Of glucose removed f‘rom a phosphate buffered substrate which contained 0.005 M sglucose during aerobic respiration studies. When the .HcmHNS Ho: LO Hcoocoa mm UNmmOHQXO on Ome >NEN .08 mm.0 wm3 Noammcxm UOHHNQ Com H£0H03 H03 mmwuo>mH omoosHo Oz mm.o oHoo.o oN.o Hooo.o Houuom oomnomoao HHH anouo umwmsm oumzamozn om.o oHoo.o mH.o ono.o dH omoooHo z moo.o HH QBOHO 1111 Hm00.0 :11: 0000.0 mHOHHCOO H asouo panoz umz m5€dchm muzmHNB 003 msswmcxm New ooH\so HHmHoz so ooH\so HHoHoz coHHmNHHHH: Ho: mz\mz doHHmNHHHHp, Ho: mz\mz one $238338 mNASHoEOmES HEB O 50 H< mew > ZOHHIOmmHZm3H m 3mg \fl If gapeworms were young and concurrently smallest in size the glucose utilization was greatest. With increased body size there was a decline in exogenous glucose utilization. This .can be seen in Table 6. Exogenous glucose utilization was also determined on gapeworms maintained in'vitro for ten days at 370 C in a phosphate buffered substrate containing 0.005 M glucose. The daily rate of glucose utilization in terms of mg glucose utilized per gram of wet weight of Syngamus per hour for each of the ten days is shown in Figure IN. The rate of exogenous glucose utilization was lowest on the first day of the ten day period —- 0.56 mg glucose utilized per gm wet weight of Syngamus per hour. A gradual increase in glucose utilization occurred until on the tenth day the rate was 1.36 mg glficose litilized per gm wet weight Syngamus per hour. Over the period c>f ten days the gapeworms decreased in average wet weight f‘rom 5.03 mg to 2.61 mg per pair of Syngamus. The aerobic respiration of paired Syngamus was investi- SJated by standard manometric procedures based on the Warburg rnethod. The oxygen consumption, carbon dioxide liberation 21nd respiratory quotient were Obtained on a size range of ESyngamus in order to determine the rate of the aerobic respi- r‘ation with increased age and size. All values of gas ex- C2hange were calculated in microliters per mg dried weight IDer hour. The general pattern of aerobic respiration can be seen (in Figure 15. No attempt was made to correct for absorption #3 HH/ .mcHxN:w usoszB musoz SHCOBH * *0m + : mm.0 NA. om.m 0.A 00.0 0A m©.m o.m oo.H o: _ N:~: 6.0 oo.H NA om.H m.o oo.m o: NA.o swam... smegmafimg Wows . Amman; OHNHQD omOOBHO 02 HO .Oz 08 omwcm>< mmOODHO E moo.o OZHZHm >m ZOHH000 HHHB OOHNHOONN mm omousHm Z m00.0 HHH3 NHNCHNOSN omuonsn obmaamoca m CH mssmmczm UNHHNQ LO sz>Hbom AHOHNHHQNOH OHOOHO< .mH ocsmHL HHdes ooHuo 6: 63H ONH 66H oo oo 61 ON 1 mHI NOOOIII O H I m I O In .1. 121110131w ‘\ 3 nwe u g qqfifam pallp Bw/Jnoq/ .as D 58 of some of the CO in the phosphate buffer, consequently, 2 the values for the carbon dioxide liberation and the respir— atory quotients are slightly lower. Nevertheless, these re— spiratory values were useful in determining the comparative relationship of body size to rate of respiration of Syngamus. The rate of respiration may be described as the quantitative Oxygen utilization and carbon dioxide liberation per mg dried weight of Syngamus per hour. The highest rate of respiration was obtained in the smallest and youngest gapeworms and a gradual decrease in rate with increased body size was noted. I t appears that the gradual decrease in respiratory rate be- comes negligible after the paired Syngamus obtain a dried weight size of about 2 mg. The hourly values for oxygen utilization and carbon dioxide liberation for some of the longer aerobic respiration S tudies are presented in Figure 16. Examination of the hourly rates of aerobic respiration suggest that as length Of time of the study increased the respiration rate of gape- Worms in the substrate with glucose increased. This increase 1 r1 respiration rate appeared to be more pronounced in gape- worms of lighter average weight. The pattern of respiration i 8 rather constant over the hourly period for gapeworms Of heavier weight and for gapeworms in a glucose free sub- Strate. Only the pattern of aerobic respiration for the various Vveights of Syngamus was represented in Figure 15. The aerobic respiratory activity of’Syngamus as measured by the Microliters/hour/mg dried weight +10 L Time in hours CJlEBday old Syngamus, 0.19 mg dry weight H H O 13 " " O.h5 mg . 23 H H n l. 65 mg H 9' ------- in phosphate buffered substrate in phosphate buffered substrate containing 0.005 M glucose Figure 16. Hourly rate of aerobic respiration of paired Syngamus trachea of different average weights in‘phosphate‘buffered substrate, pH 7.5M, glucose free or containing 0.005 M glucose. \fl \0 O\ Q respiratory quotient (RQ) appears in Table 7. Regardless of the size of the gapeworms used for the determinations there was a rather consistent respiratory quotient (average = 0.866) when the substrate contained 0.005 M glucose. Even when the substrate was free of glucose or butyric acid the respiratory quotient of Syngamus approximated that obtained when gapeworms were present in a substrate containing 0.005 M glucose (average = 0.887). However, when 0.065 M butyric acid was present in the substrate a respiratory quotient of 0.708 was obtained. The pronounced effect of butyric acid in the substrate on the respiratory quotient of Syngamus was not reflected by a change in oxygen utilization. The rate of oxygen utilization in the presence of 0.065 M butyric acid was the same as when 0.005 M glucose was present in the substrate (Table 8). The effect of butyric acid was due to decreased carbon dioxide liberation, thus causing a lower respiratory quotient. The rate of aerobic respiration of a brei of Syngamus was greater than an equal weight of intact gapeworms (Table 8). The respiratory quotient of the brei was 0.900 as compared to 0.u3u for the control group. An acetone powder prepared from Syngamus, when in the phosphate buffered substrate containing glucose, produced complete oxidation of the glucose molecule. This is shown by a respiratory quotient of nearly 1.000 (Table 9). The microliters of oxygen uptake and carbon dioxide liberation .oomuumoom may dfi cofipcopou m 00 uom noooouuou* .omNHHHu: 0o UmoMumoHH NOD mo owumu wag mH Aomv “Gowposv zqumuHQwomH mmoosfio Z moo. m®®.o Hw.m wo.qn $3.: mmousoo z moo. omo.o oo.o oo.:- oo.o mmousoo z moo. moo.o oo.m mo.o- om.H .............. mmo.o mo.m mo.o- om.~ mmoosuo z moo. onw.o HH.: oo.:- moH.H ----- ......... ooo.o No.: mm.m- mmH.H ooo< outsosm z moo. oos.o mo.m oo.m- No.H omoosoo z moo. moo.o om.o mo.m- -.o “no“: ) *mooo moo coma coo :m.m zo .momcouoam Mom ogoomz omummmsm wowzamoza uz\mE\muopHH0uoHE zoo m2 m QmmHmOHw omm~HH>HHU< >mOh compomou Qua“ EummUHm EOLm UwfiuQEo umo3oa mcoumo Hmcfim .momuomosm oopmmmso oomcamocm CH om>~0mmfiv mm: u6630q ocOpmow 20mm QmmHH>HHU< >mOH 2 sence of various substrates, appears in Table 10. The rate as reflected by carbon dioxide liberation (Q in the pre— TABLE 10 ANAEROBIC% RESPIRATORY ACTIVITY OF PAIRED SYNGAMUS TRACHEA IN PHOSPHATE BUFFERED SUBSTRATE Mg Dry Weight Q N Substrate with: Per Paired C02 Syngamus Microliters 0.201 +2.81; _ 0.005 M Glucose 1.190 +2.00 u.u60 +1.76 0.893 +3.26 0.065 M Butyric Acid l.u00 +2.22 0.825 +3.59 No Glucose 1.320 +2-h8 % Nitrogen atmosphere (95% N2 - 5% CO of anaerobic activity may be considered to be the quantita- tive rate of carbon dioxide liberated in manometric investi- gations. Regardless of the substrate used in the anaerobic investigations the rate of anaerobic respiration as expressed by carbon dioxide liberation was decidedly less than the QCOE of aerobic respiration (Table 7). It appeared that the an- aerobic respiration of Syngamus in a phosphate buffered substrate and in one with 0.065 M butyric acid was about the same while it was slightly less in the buffered substrate containing 0.005 M glucose. As was previously noted for aerobic respiration the rate of anaerobic respiration de— creased with increased weight of the gapeworms. The effect of various recognized inhibitors of metabo- lism on the aerobic respiration of Syngamus is summarized in Table 11. A decrease in oxygen utilization from the normal was used as a criterion of metabolic inhibition. The rate of oxygen utilization was markedly inhibited by azide, cyanide, and 2,u-dinitrophenol. In the presence of fluoride, iodoacetate, and malonate there was decreased oxygen uptake; however, this inhibition was less pronounced. Fluoride and malonate were inhibitory only in a calcium free phosphate buffered substrate. When Syngamus was exposed to carbon monoxide (95% CO - 5% 02) the pseudocoelomic fluid changed from a dark to a bright red color and a decrease in oxygen utilization occur- red. The difference between oxygen uptake in the presence and absence of light appears in Table-12. In the presence TABLE 11 EFFECT OF VARIOUS INHIBITORS ON THE OXYGEN UTILIZATION OF PAIRED SYNGAMUS TRACHEA IN PHOSPHATE BUFFERED SUBSTRATE OF pH 7.5K CONTAINING 0.005 M GLUCOSE O \ os . Concentration Mg Dry Wt. . Inhibitor (Molarity) Per Pair Rat1ol AZIDE 10:3 0. 33 0.288 10_6 8. 0 0.812 10 0.73 0.895 CYANIDE 10:E 0.273 death 10 0.239 0.088 _6 1.83 0.077 10 0.237 0.718 2.80 0.891 2,u-DIN1TRO- 10:E 0.708 0.387 PHENOL 10 ' 0.80 0.68 * “'6 6.50 0.852% 10 0.60 0.79 x 0.811 0.812 -8 1.38 0.89 % 10 0.76 0.89 % TTLUORIDE 10‘2 0.818 1.00 0.75 0.83 * 1.38 0.86 + brei 1.00 Im QmmHXO ZO MQHXOZOE ZOmm