Larry S. Roberts

Developmental physiology of cestodes. IV. In vitro development of Hymenolepis diminuta in presence and absence of oxygen.

Abstract After 6 days postinfection in vivo, Hymenolepis diminuta was cultured in vitro for 12 days under gas phases containing 0, 1, 5, and 20% oxygen. Worms from all groups increased significantly in mass and produced substantial numbers of proglottids and viable progeny. Some morphological and reproductive abnormalities were observed. Total carbohydrate concentration of worms cultured in vitro approximated normal, but lipid concentration was somewhat less than normal in in vivo grown worms. No effect of oxygen presence or concentration was detected.

Developmental physiology of cestodes. I. Host dietary carbohydrate and the “crowding effect” in Hymenolepis diminuta☆

Abstract The development of Hymenolepis diminuta (Cestoda: Cyclophyllidea) in hosts on diets containing suboptimal carbohydrate has been compared to that of worms in rats with crowded infections. Host diets varied in carbohydrate concentration and quality, i.e., high starch, low starch, and sucrose. Worms were collected for measurements and observations at 5, 10, and 15 days postinfection. Length, fresh weight, chemical composition, and numbers and development of proglottids were measured. The largest size was attained by worms from rats infected with 5 cysticercoids each and fed the high starch diet (5-HS control group); the smallest worms were from rats infected with 5 cysticercoids and fed the sucrose diet (5-Su). Tapeworms from hosts infected with 25 cysticercoids and fed the high starch diet (25-HS) and from hosts infected with 5 cysticercoids and fed the low starch diet (5-LS) were intermediate and approximately equal in size. Carbohydrate was highest and lipid lowest in the 5-HS group, both on a per unit dry weight basis and a per unit nitrogen basis. The opposite was true in the 5-Su worms. The 25-HS and 5-LS groups were intermediate between the other two. Numbers of mature and gravid proglottids were reduced in the 25-HS, 5-LS and 5-Su groups, the 5-Su being the lowest. Similarities in developmental characteristics of worms from crowded infections and those from hosts with suboptimal carbohydrate diets provided further evidence that competition for host dietary carbohydrate is involved in the crowding effect in cestodes. The implications of these findings in terms of the operational mechanism of the crowding effect are discussed.

THE CROWDING EFFECT REVISITED

Much has been learned of the biology of cestodes since Clark Read published his paper in 1951, yet in many respects our knowledge remains distressingly meager. Read observed that, in Hymenolepis diminuta and several other species, the size attained by the worms in their definitive host was roughly inversely proportional to the number of worms present, a phenomenon he called the ‘‘crowding effect.’’ The adaptive value of the crowding effect seems self-evident: the size of the individual worms is limited, by whatever means, to an aggregate mass that the host can tolerate without adverse consequences. The known list of cestodes that demonstrate a crowding effect has increased little in the past 40 yr, and a definitive explanation for the effect’s mechanism of operation remains elusive. Inasmuch as development and growth of the worms is being controlled by some means external to the individuals in the population, the mechanism of this control is of considerable biological interest. In their accompanying paper, Bush and Lotz (1999) point out that ecological consideration of crowding can relate to predator‐prey relationships or to competition. Because we can disregard predator‐prey relationships, I focus here on competition, which in turn can take the form of exploitative competition (required resources reduced in supply by the organisms) or interference competition (substances released by organisms having an adverse effect on others in the population). I also examine the possible role of the host immune response in the crowding effect. Exploitative competition? Investigations for the next 25‐30 yr after Read’s paper (Read, 1951) assumed that the mechanism was exploitative in form. In that paper he suggested that oxygen might be the required resource and that the resource was ‘‘probably not a food substance obtained from the foodstuffs ingested by the host.’’ Additional investigation by Read and his coworkers showed unequivocally that quantity and quality of host dietary carbohydrate did indeed affect worm growth (Read, 1959). He concluded that cestodes require carbohydrate for growth and reproduction, that the carbohydrate is obtained from host ingesta, and that ‘‘[t]he crowding effect in cestode infections may be interpreted in terms of competition for utilizable carbohydrate by the individual worms in the populations.’’

Developmental Physiology of Cestodes. II. Effects of Changes in Host Dietary Carbohydrate and Roughage on Previously Established Hymenolepis diminuta

The effects of changes in host dietary carbohydrate and roughage on previously adult Hymenolepis diminuta were examined. In one experiment rat hosts were infected with five cysticercoids each, maintained on Purina Laboratory Chow (pellet ration) for from 21 to 28 days, then divided into treatment groups as follows: (1) continued feeding pellet ration, (2) fed a diet containing 30% starch as carbohydrate component, (3) fed a diet containing 30% sucrose, and (4) fed a carbohydrate-free diet. Worms from hosts on sucrose and carbohydrate-free diets rapidly decrease in weight, numbers of gravid and mature proglottids, and carbohydrate concentration. Lipid accumulates to high concentrations, and nitrogen rises slightly, but after a week nitrogen drops back to levels in the pellet and starch groups. Worms from rats on sucrose and carbohydrate-free diets have defective female reproductive systems, apparent from histopathology in late mature and gravid proglottids. In other experiments rats were infected and maintained as above, then changed from pellet ration to diets containing 30% cellulose, 5% cellulose, or no roughage. After 6 to 7 days worms from the high roughage diets are smaller, have lower lipid concentrations, and have slightly higher carbohydrate concentrations than worms from hosts on pellet rations, low roughage or roughage-free diets. Possible explanations for the observed effects are discussed in the light of pertinent knowledge of host intestinal physiology. Diet and feeding habits of the host may have a profound effect on its tapeworm parasites. Levine (1938) reported that limitation of host food decreased proglottisation in Davainea proglottina. Starvation for only 24 hr depressed proglottid production for a week following. Reid (1942) found that the scolex of Raillietina cesticillus shed its strobila after 24 hr of host starvation, a new strobila being formed only if normal feeding was resumed. The destrobilation of R. cesticillhs was correlated with a dramatic decline in the glycogen content of the worm. Evidence that tapeworms were affected by the carbohydrates in their hosts diet was reported by Chandler (1943), who found that absence or restriction of carbohydrate in host diet resulted in reduced establishment of Hymenolepis diminuta and stunting. Chandler et al. (1950) observed similar effects of carbohydrate lack whether the worms were previously established or the rats infected after they were already being fed low carbohydrate diets. Further, they found that neither sucrose nor glucose were adequate substitutes for starch in the host diet, but worms from rats on diets containing glucose were larger than those whose hosts were fed Received for publication 19 September 1966. * This investigation was supported by Grants AI06153 and 5 TI AI-226 from the NIH, U. S. Public Health Service. diets with sucrose as the only carbohydrate. They attributed the difference between glucose and starch diets to the fact that glucose is absorbed much more rapidly from the host intestine, while the gradual digestion of starch would make glucose available over a greater length of host gut. These results were confirmed and extended by Read and Rothman (1957b), who also stated that the effect of carbohydrate deficiency was the same whether the worms were previously established or the infection was initial. It is necessary to assume, however, that the worms must have arrived at their stunted condition by two different routes. If the host is being fed a diet inadequate in carbohydrate when it is infected, then normal worm growth does not occur, and maximum size cannot be attained. In contrast, if the worm is already at maximum size when the host is changed to a low carbohydrate diet, then worm size must be reduced. Such reduction might be the result of destrobilation, as found by Reid (1942), or proglottisation rate might be decreased, as apparently is the case in D. proglottina (Levine, 1938). Effects of changes in host diet on previously established worms may have some importance in nature, helping to account for seasonal fluctuations in egg production. Oliger (1950) reported that cestodes of tetraonid birds destrobilated when the birds changed to their

Intestinal digestion and absorption of starch in the intact rat: Effects of cestode (Hymenolepis diminuta) infection

Abstract 1. 1. Gastric emptying and intestinal transit were rapid (0·5 and 3·5 hr, respectively) after administration of a liquid test meal, and digestion of starch was incomplete. 2. 2. Absorption of starch from the liquid test meal was higher in the presence of Hymenolepis diminuta (Cestoda: Cyclophyllidea). 3. 3. Gastric emptying and intestinal transit were slower (4-0 hr and more than 5·0 hr, respectively) after a solid meal, and digestion was essentially complete. 4. 4. Effects of H. diminuta were minor after the solid meal. 5. 5. Tapeworms had no effect on gastric emptying or intestinal transit with either meal type.

In vivo effects of putative crowding factors on development of Hymenolepis diminuta

During in vitro incubation, Hymenolepsis diminuta secretes substances into the medium that inhibit DNA synthesis in the germinative region of freshly isolated, uncrowded worms. Of the many substances that are released by H. diminuta into the medium, earlier studies indicate that only succinate, acetate, glucosaminic acid, and cGMP are responsible for the inhibition. In the present report, effects of these putative crowding factors on worm development in vivo were examined. At 7 days postinfection the proximal end of the hosts intestine was catheterized and perfused with test solution. The test solution contained 28 nM cGMP, 250 microM glucosaminic acid, 120 mM succinate, and 40 mM acetate. The solution was perfused by a peristaltic pump at a rate of 50 ml/day. At 2 wk postinfection, worms were recovered for subsequent analysis. Worms developing in the presence of crowding factors were 53% less in wet weight than control worms. Carbohydrate concentrations in worms from experimental groups were not different from those in control groups; therefore, the inhibition in growth was probably not due to carbohydrate deprivation. Worms from experimental groups had fewer immature, mature, and gravid proglottids than did worms from control groups. The results are consistent with the hypothesis that the tested substances, which inhibit DNA synthesis in H. diminuta in vitro, are a part of the cause of the crowding effect in vivo.

DEVELOPMENTAL PHYSIOLOGY OF CESTODES: CHARACTERIZATION OF PUTATIVE CROWDING FACTORS IN HYMENOLEPIS DIMINUTA

It was shown previously that worm-conditioned saline (WCS) prepared from crowded 10-day-old H. diminuta inhibited the incorporation of 3H-thymidine into DNA in the anterior regions of uncrowded worms and that the inhibition was partially accounted for by succinate and acetate excreted by the worms. The present study describes further characterization of the active components of WCS. An ultrafiltrate was fully as potent as untreated WCS, indicating that all detectable inhibitory components were less than about 500 daltons in molecular mass. Inhibitory factors in WCS were stable to heat (80 C for 30 min), cold (4 C for 48 hr), drying and reconstitution, alkaline pH (11 to 12 for 3 hr), and ethanolic extraction. Active compounds were probably not lipoidal in nature. Although the acidic ethanol extract of WCS was inhibitory, no activity was observed in fractions of WCS that contained basic, acidic and neutral amino acids. Amino compounds in the WCS were further investigated. Twenty-four amino acids were identified, 3 of which (phosphoserine, 1-methylhistidine, and 3-methylhistidine) have not been reported previously for H. diminuta. On a molar basis, alanine accounted for 40-50% of the amino acids released. The amino sugar, D-glucosaminic acid, was found in the WCS and also has not been heretofore reported from H. diminuta or any other cestode. In concentrations comparable to those in the WCS, D-glucosaminic acid inhibited incorporation of 3H-thymidine into the DNA of the tapeworms by 25-35%, suggesting that D-glucosaminic acid may be one of the crowding factors.

Developmental physiology of cestodes—X. The effect of crowding on carbohydrate levels and on RNA, DNA and protein synthesis in Hymenolepis diminuta☆

Abstract 1. 1. The carbohydrate concentration in the germinative and immature regions of Hymenolepis diminuta (Cestoda: Cyclophyllidea) was not affected by high population density. 2. 2. Crowding resulted in lower carbohydrate concentration in the mature and gravid regions. 3. 3. High population density partially inhibited RNA, DNA and protein synthesis along the strobila of 12-day-old H. diminuta . 4. 4. Uptake of precursors to macromolecular synthesis was also inhibited, but the inhibition of uptake was less than that of synthesis. 5. 5. RNA and DNA synthesis were more severely inhibited in the germinative, immature and mature regions than in the gravid area, protein synthesis was least affected in the germinative region of the strobila. 6. 6. The implications of the results in relation to the operational mechanism of the “crowding effect” in cestodes is discussed.

Hymenolepis diminuta: Lack of pathogenicity in the healthy rat host

Abstract Whether Hymenolepis diminuta (Cestoda: Cyclophyllidea) might affect adversely the growth of its host under normal conditions was studied. Rats were divided into four experimental groups: (1) rats infected with the tapeworm and fed ad libitum , (2) uninfected rats fed ad libitum , (3) and infected rats fed isocalorically with (4) uninfected rats. Growth rates of infected rats did not differ from infected animals. Infected, meal fed rats limited to 15 g synthetic diet/day grew as rapidly as their uninfected counterparts, and infected rats fed ad libitum did not consume more food than the comparable infected group. There were no significant differences in consumption or in excrement produced between groups (1) and (2) and groups (3) and (4). Weights attained by the worms were not affected by mode of host feeding ( ad libitum or meal fed), whether expressed as wet or dry weight. Since H. diminuta appears not to affect nutrient utilization or consumption in a healthy, unstressed host, at least on a gross level, it probably should be considered an endocommensal.

Amino acid metabolism in the rat tapeworm, Hymenolepis diminuta.

1. The amino acid metabolism of the rat tapeworm, Hymenolepis diminuta was investigated. 2. In addition to the characteristic end products of helminth metabolism, H. diminuta also forms substantial amounts of 14C-alanine during incubations in 14C-glucose. 3. Of 10 amino acids tested, only 14C-labelled asparate and, to a lesser extent alanine, generated substantial amounts of 14CO2 when incubated with H. diminuta. 4. 14C-aspartate was incorporated into both succinate and acetate, major products of the worms mitochondrial metabolism, but the rates were low when compared to the metabolism of exogenous glycogen. 5. These results suggest that amino acid metabolism in H. diminuta is very limited.