EXPERIMENTAL

Glucose

PARASITOLOGY

Utilization

70,25-34(1990)

Rates Are Linked to the Internal Gradient in the Rat Tapeworm

Free Glucose

EAIN M. CORNFORD Southwest Regional Veterans Administration Epilepsy Administration West Los Angeles Medical Center, Los of Neurology, University of California Los Angeles, California

Center, Neurology and Research Services, Veterans Angeles, California 90073, U.S.A., and Department at Los Angeles School of Medicine, 90024-1769, U.S.A.

CORNFORD, E. M. 1990. Glucose utilization rates are linked to the internal free glucose gradient in the rat tapeworm. Experimental Parasitology 70, 25-34. Hymenolepis diminuta is able to acquire plasma-borne glucose 3-O-[‘4C]methylglucose) in vivo. Free glucose concentrations estimated for this helminth in vivo are comparable to that of the host intestine. Both in vivo and in vitro examinations indicate that the scolex-neck regions (first quartile) of this tapeworm have the highest glucose content, and an anterior-posterior gradient along the second, third, and fourth quartiles was observed. Substrate concentration was rate affecting for glucose utilization rates (measured as substrate depletion from the medium in vifro). Glucose utilization per minute exceeds glucose content by a factor of more than 5. The half-life of glucose was about IO sec. emphasizing that sugar metabolism is a very rapid process. In addition, utilization was highest in the first quartile and decreased in succession in the second, third, and fourth quartiles. It is concluded that while the exogenous glucose concentration remains stable, regional differences in glucose utilization rates are linked (R = 0.98; P < 0.01) to free glucose content in H. diminuta. 6 1990Academic FWSS. IIIC. INDEX DESCRIPTORS: Hymenolepis diminuta, the rat tapeworm; Metabolic gradient; 30-Methylglucose; Free glucose content; Glucose utilization rate; Coupling of transport and glucose utilization.

INTRODUCTION

1959; Roberts 1966); egg production ceased (Read and Rothman 1957) and destrobilization was observed concomitantly with the initiation of intravenous feeding (Castro et al. 1976). The ability of this tapeworm to compete successfully for dietary carbohydrate is exemplified by the suggestion that up to 50% of the ingested carbohydrate may be absorbed by the parasites (Mettrick 1973). Given that 94% of an oral glucose load normally appears in the mammalian portal vein as glucose (Kuyumjian and Kalant 1986), it is not surprising that in some studies parasitzied rats lose weight (Mettrick 1972), although weight loss is atypical (see Insler and Roberts 1976). H. diminuta is also capable of assimilating nutrients from the circulatory system of the rat (Chandler et al. 1950; Kilejian et al. 1968; Platzer and Roberts 1969; Castro et al. 1976; Cornford 1977), but the tape-

Glucose uptake in the rat tapeworm Hyis via a stereospecific, active transporter, which up-regulates in response to substrate depletion (Phifer 1960b; Pappas 1975). This tapeworm undergoes a diurnal migration within the rat small intestine, which has been correlated with the feeding regime of the rat host (Hopkins 1969; Read and Kilejian 1969). An experimental liquid meal will perturb this migratory rhythm, causing the parasites to migrate proximally in the luminal small intestine (Mettrick and Podesta 1974). Orally administered histamine induces distal migration of these parasites, while oral glucose maximally induces proximal migration (Mettrick and Podesta 1982). Intestinal tapeworms are highly sensitive to dietary carbohydrate deficiencies (Read menolepis diminuta

25 0014-4894190$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

26

EAIN M.CORNFORD

worms do not survive for long in parenterally fed hosts (Castro ef al. 1974, 1976; Cornford 1977). Studies of monosaccharide uptake in the rodent have demonstrated a proximal-to-distal gradient in glucose transport along the small intestine (Fisher and Parsons 1950; Baker et al. 1961; Diamond and Karasov 1984), which is modified in tapeworm parasitized rats (Podesta and Mettrick 1977). Although the effects of substrate concentration, pH, and sodium on intestinal vis-a-vis parasite glucose uptake have been studied in detail (Podesta and Mettrick 1974), it is not known whether or not regional surface responses of the parasite to the proximal-to-distal intestinal glucose gradient occur. The rostral-to-caudal orientation of tapeworms typically opposes intestinal flow and peristalsis (regardless of proximal-distal position along the small intestine). Thus the present study was initiated to determine if effects of the intestinal glucose gradient in vivo (Karasov et al. 1983) were apparent in H. diminuta. MATERIALS

AND METHODS

Rearing and infection of intermediate insect hosts and collection of cysticercoids are described elsewhere (MacInnis and Voge 1970). Male Wistar rats (270-330 g, from Hilltop Laboratories, Chatsworth, CA) were lightly anesthetized with halothane and fed exactly 5 (or 10) cysticercoids (suspended in OS-l.0 ml of saline). The cysticercoids had been freshly collected from macerated flour beetles. Rats were fed water and Purina lab chow (No. 5001) ad lib throughout the entire study and were maintained in an AAALAC accredited animal facility at 70-74°F. There were equal light-dark cycles, changing each day at 0700 and 1900 hr, and the animals had a minimum of 12 complete air changes per hour. The collection of tapeworms from infected hosts was as described by MacInnis and Voge (1970). except that RPM1 1640 was used in place of normal saline and the rats were not fasted overnight prior to experimental studies. In vivo studies. Rats were anesthetized 14days after exposure to cysticercoids. The femoral vein was exposed and injected with 0.2 ml of isotopic solution. Each injection syringe had been prewarmed (38°F) and contained approximately 2.5 pCi of 3-0-[‘4C]methylglucose and 10 pCi of [‘HIwater. The rats were placed on a warmed platform to maintain body temperature

and the isotope was recirculated to reach equilibrium. Twenty-five microcuries of [“3mIn], chelated to EDTA (see below), was injected 40 min later; this isotope binds to circulating transfenin and distributes to the intravascular space, much like radioiodinated serum albumin. At 45 min after the first injection, 2.0 ml of blood was withdrawn and the rat was killed. Representative tissues were dissected, weighed, and processed for scintillation counting, along with triplicate samples of weighed plasma. Tissue:plasma ratios of the nonmetabolized 3-O-[‘4C]methylglucose were determined as described previously (Comford et al. 1979). Three aliquots of the blood plasma were also saved for plasma glucose determinations. Radioisotopes. Isotopes were purchased from New England Nuclear Corporation (Boston, MA). Specific activities (in mCi/mmol) were: 3-O-[methyl-‘YZ] methylglucose 55 and [3H]water 1 mCi/g; the “‘“In generator was purchased from NEN Radiopharmaceuticals (North Billerica, MA). To each 1.0 ml of indium eluted, 10 pl of sterile disodium edetate solution (150 mg/ml; Endrate, Abbott Laboratories, North Chicago, IL) was added. The pH of the solution was adjusted to about 7.0 by the dropwise addition of sterile 0.1 N NaOH (about 40 pl), and the pH was fixed at 7.55 by adding 100 mM Hepes (N-2-hydroxyethyl piperazine N-2-sulfonic acid; obtained from Calbiochem, LaJolla, CA) to give a final buffer concentration of 10 mM. In vitro studies. The distribution volume of 3-O-[‘4C]methylglucose, at equilibrium, was determined to ascertain free glucose concentrations within the worms. The culture medium employed in these studies was RPM1 1640, buffered with 10 mM Sorenson’s phosphate buffer, 2.0 mM glucose plus a trace concentration (~7 pm) of radiolabeled 3-O-methyl glucose. Because of their potential effects on glucose utilization, bicarbonate buffers (and an enriched CO, atmosphere) were avoided. Tapeworms were incubated in individual petri dishes at 38°C. At 15,30, and 40 min after starting the incubation, 50% (volume) of the medium (2.0-2.5 ml) was withdrawn and replaced with a fresh aliquot. to maintain the substrate and isotopic concentrations at the original levels. [(“3mIn), chelated to EDTA, was added to the medium 2-3 min before the end of the incubation period to estimate the fraction of extrategumental isotope in order to make appropriate corrections. No significant quantities of the [‘lsm In] chelate were retained in any of the four tapeworm quartiles examined.] At the end of the incubation period (45 mitt), worms were removed from the medium and briefly rinsed in silicone oil to remove any surface-adherent isotopes. The tapeworm was cut into four quartiles of equal length (approx 10 cm), the first quartile being the scolex-neck, with immature proglottids, the fourth quartile the most mature (i.e., gravid) proglottids, and each piece of tissue weighed. Samples

TAPEWORM

GLUCOSE

CONTENT

were digested in an organic base and prepared for liquid scintillation counting. This technique assumes that 3-O-methyl glucose is not metabolized by helminths in the time required to achieve equilibrium. In other studies (Comford and Fitzpatrick 1985)it was demonstrated that this hexose could be reextracted from schistosome tissue, separated in column chromatography with the neutral eluate, and it cochromatographed with the original sample as a single peak on TLC silica gel plates. Two other assumptions are made. The transport of glucose and methylglucose is assumed to be symmetrical, or any asymmetries are proportionally the same for the two hexoses. Second, it is assumed that these two hexoses behave similarly at internal barriers. Thus if the glucose transporter on the tapeworm internal membranes is different from the tegumenta! sodium-dependent active transporter, the assumptions made with respect to the behavior at the tegumenta! transporter must also be made for such internal glucose transporters. The S-set tissue uptake indices of a tracer (0.010 mM) concentration of 3-O-methyl glucose and 3O-methylglucose + 5 mM unlabeled glucose were measured in triplicate in IO-day H. diminuta as described by Comford et al. (1982). These studies demonstrated that 5 rnM glucose caused an 83% reduction in 30methylglucose uptake. This observation confirms that these two hexoses share the same tegumenta! glucose transporter (see Pappas 1975) and contradicts the reports of Starling (1975) and Rosen and Uglem (1988). Glucose utilization rates. Hymenolepids were perfused from rat hosts 20 days after feeding cysticercoids. RPM1 1640was prepared as above with varying concentrations of glucose (2.5. and IO mM) and maintained at 37-38°C throughout the experiment. Each tapeworm was placed in a single dish of the appropriate glucose concentration to equilibrate for about IO min. The experiment began when the tapeworm was cut into four equal quartiles, each quartile removed to another (separate) petri dish, and each sectioned quartile incubated in exactly 5.00 ml of medium. A time zero aliquot of the medium was taken from each dish for glucose analysis. At 30 and 60 min. aliquots were taken from every dish for glucose analyses, and the worm sections were weighed after 60 min. Glucose content was also measured in each “equilibration” dish immediately after removal of the worm sections and compared 1 hr later. No significant ( second > third > fourth quartiles) in [‘4C]methyl glucose distribution (Fig. 1). (Note that although tissue: plasma ratios are reported for tapeworm regions, gut luminal glucose concentrations were not determined, even though plasma-borne [‘4C]methylglucose presumably moved by way of the gut lumen to the tapeworm.)

28

EAIN

RENAL

M.

CORNFORD

CORTEX

-I

LIVER

I

I

DUODENUM

I

ILEUM TAPEWORM ABDOMINAL SKELETAL

I

4

.:.:.:.‘.‘.‘.

I

FAT MUSCLE

m

.4

.2

TISSUE-PLASMA 3-O-METHYL

H. DIMINUTA

.6 RATIO

OF

.a

, 1

14-C

GLUCOSE

FIG. I. Tissue:plasma distribution of 14C 45 min after intravenous administration of 3-O[‘4C]methylglucose. Data are from four rats (mean body weight 285 f 17 g, and mean plasma glucose level 8 + 1 mM at autopsy), each of which was infected with five cysticercoids 14 days prior to analysis. (Tapeworms were dissected free of the gut and rinsed in ice-cold silicone oil to effect removal of surface-borne radioactivity.) The data demonstrate that systemically administered hexose can equilibrate across mucosal barriers and into the tapeworm during the 45min experimental period. Note also that the relative mean fraction of glucose in the first quartile of the tapeworm is intermediate between the duodenum and ileum, with the second, third, and fourth quartiles demonstrating progressively less free hexose. Although regional differences are not statistically significant (except in contrasting first and fourth quartiles, where P < 0.05), a gradient in glucose content across the quartiles is suggested by the correlation coeffkient (R = 0.72; P < 0.05) determined from linear regression analysis.

Because isotope distribution was measured at equilibrium, it is not possible that the observed effect was attributable to the relatively greater surface area and reduced cross-sectional volume in the anterior regions of the tapeworm. Furthermore, analyses of the concomitant distribution of tritiated water indeed established that equilibrium conditions were attained. Tissue: plasma ratios measured in the first to fourth quartiles were 0.80 + 0.04, 0.82 ? 0.06, 0.81 + 0.04, and 0.79 + 0.05, consistent with previous reports indicating that in intact worms, water content represents 77-79% of the total mass (Lee and Ip 1986). The increased t4C uptake in the scolexneck region was a function of increased glucose distribution and the concomitant absence of increased water influx. That is, tritiated water distribution was equal in all quartiles examined, while glucose distribu-

tion appeared to correlate with the intestinal glucose gradient. Glucose utilization rates, determined from the loss of substrate from the medium over a 60-min incubation period, are presented in Table I. These data confirm that in addition to increased glucose content in the anterior regions, there are parallel gradients in glucose utilization rate. The first quartile consumes greater quantities than the second, and this gradient continues (second > third, third > fourth) caudally. Furthermore, the glucose utilization rate in vitro is a function of substrate concentration; the greatest utilization rates were observed in the presence of 10 mM glucose and reduced rates with 5 and 2 mM glucose, respectively (Table I). Analysis of variance indicates a significant (F = 10.9, P < 0.05) effect of concentration across all quartiles examined. [The substrate concentrations

TAPEWORM

GLUCOSE

CONTENT

AND

29

UTILIZATION

TABLE I Effects of Regional Quartile and Substrate Levels on Glucose Utilization Rates in 21-Day H. diminuta Glucose utilized nmoles . min- ’ mg- ’ Substrate concentration (mM) Quartile

2.0

First Second Third Fourth

2.574 + 0.262 2.013 + 0.724 1.981 2 0.810 1.716 rt_0.546"

5.0 3.450 2 3.229 + 2.601 + I.918 t

10.0

0.825 0.919 I.188 0.782"

7.646 2 5.711 k 3.248 + 4.023 2

2.426 0.921 1.397",b 2.233"

Now. N = 4-5 for each X * SD. ” P < 0.05, compared to first quartile. ’ P < 0.05, compared to the second quartile.

studied approximate luminal intestinal glucose levels; 12 mM in the anterior end after overnight feeding and 1.5 mM in the ileum (Starling 1975).] To determine if the gradient in free glucose observed in vivo (Fig. 1) persisted in vitro, a group of hymenolepids were perfused and incubated in RPM1 containing 2 mM glucose for 15 min, followed by a second 15min incubation with fresh media. They were removed to individual petri dishes containing fresh media and a tracer concentration of 3-O-[‘4C]methylglucose, with replenishment of the media as described in the methods. The free glucose level in vitro was estimated from the 15-min equilibrium distribution of [‘4C]methylglucose. (No correction for reductions in glucose concentration during the isotopic incubation was made since media were replenished as indicated above.) The glucose content of the first quartiles averaged 0.39 Fmol . gg’, the second quartiles 0.31, the third quartiles 0.28, and the fourth quartiles 0.25 pmol * gg ’ of glucose. As indicated in Fig. 2, in vitro-free glucose concentrations along the four quartiles are highly correlated (R = 0.98, P < 0.01) with utilization rates (determined for the same 2 mM substrate concentration in the respective quartiles). Collectively, these data indicate that in respect to glucose utilization, worm glucose levels are rate affecting. Linear regression analysis of the data in Fig. 2 indicates

the slope, k = 5.96 min-‘; i.e., glucose utilization exceeds free glucose content by a factor of almost six. Under these in vitro conditions, the half-life of a free glucose molecule in the tapeworm might be estimated from (T,,? = In 2/k; where k = the slope in min-‘). This exemplifies just how rapidly glucose is utilized by the H. diminuta; after translocation across the tegument, the half-life of a transported glucose molecule is about 0.12 min or < 10 sec. DISCUSSION

Phifer (1959) observed that glucose uptake was greatest in 12-day-old tapeworms, in his comparison of 8-, 12-, 16-, 20-, and 90-day-old H. diminuta. Other workers have also apparently made similar observations (see Pappas 1975). It was further suggested that glucose uptake may be greater in the anterior fourth of the rat tapeworm (Phifer 1960a). End products of metabolism were different in a comparative study of 6and ICday-old worms (Watts and Fairbairn 1974). Henderson (1977) noted that glucose uptake in this tapeworm was inversely proportional to age, but also observed equal uptake in comparing young and homologous anterior portions of older tapeworms. Higher oxygen uptake in the scolex, and a possible metabolic gradient, was suggested by Duwel and Kirsch (1971). Coles and Simpkin (1977) also suggested that the anteriormost 2 mm of this tapeworm had a

30

EAIN M. CORNFORD

q

GLUCOSE UTILIZATION RATE hmol.min-‘. 1.0

mg-‘1

711

4th

0.1

0

0.2

0.3

0.4

0.5

0.6

FREE GLUCOSE CONCENTRATION (nmobmg-‘1 IN H. DIMINUTA

FIG. 2. An in vitro comparison of glucose utilization rates (shaded bars) and free glucose concentration (open bars) in the first, second, third, and fourth quartiles of the rat tapeworm, H. diminuta (20 days postinfection). Horizontal error bars = -t 1 SD. Internal glucose concentrations were determined (n = 8) in the respective quartiles from the distribution (tissue:medium) of 3-O- [“‘C]methylglucose, after equilibrating in culture medium containing 2 mM o-glucose. Surface-free hexose was minimized with a cold silicone oil rinse and estimated from the concomitant distribution of [“3mIn]EDTA. This polar chelate is excluded by intact membranes. Glucose utilization rates and glucose contents (independent variable) were subjected to linear regression analysis. Despite the uniform exogenous substrate, mean glucose utilization rates (from Table I) correlate highly (R = 0.98; P < 0.01) with mean glucose content of the respective quartiles, suggesting that utilization is linked to substrate concentrations.

higher rate of energy production than other parts of the tapeworm. Collectively, these studies predict greater glucose transporter activity (or density) in the anterior region of the tapeworm. Thus while these observations seem related to, and consistent with, the present work, the possibility of a gradient for glucose uptake along the long axis of H. diminuta was apparently not considered. Is the phenomenon described in this study intrinsic or an adaptation of the parasite to its host? Our data demonstrate that the glucose gradient in the tapeworm is not lost in the first few hours after removal to

culture medium. Persistance of the gradient in vitro does not necessarily suggest that the tapeworm glucose gradient is intrinsic. It should also be noted that the intestinal glucose gradient is lost in rodents maintained on a carbohydrate-free diet, but down-regulation (indicated by low V,,,,, and K, estimates) of the glucose transporter is not apparent for 3 days and requires almost 3 weeks for completion. Up-regulation, after returning animals to carbohydrate, is a much more rapid (24 hr) process (Diamond and Karasov 1984; Karasov et al. 1983). Thus the possible role of host induction of the parasite glucose gradient is unresolved.

TAPEWORM

GLUCOSE

CONTENT

Gradients of metabolites (Duwel and Kirsch 1971; Coles and Simpkin 1977) and nutrients have also been described in tapeworms. Cobalamin (vitamin B,,) is taken up by receptors distributed uniformly along the length of Spirometra mansonoides spargana (Friedman et al. 1983). However, the anteriormost portions of the tapeworm contain almost 50% of the total cobalamin, and it is believed that the cobalamin is transported (in a posterior-to-anterior direction) internally to the scolex for subsequent utilization during the rapid cellular proliferation associated with synthesis of strobila (Friedman et al. 1982). In H. diminuta, it has been suggested that the excretory-osmoregulatory ducts may function as a nutrient transport system (Webster 1972). The possibility of translocation by such a mechanism was not examined in the present study. However, the likelihood of increased glucose transporter activity in the tapeworm anterior seems a more attractive (albeit unproven) possibility, and supporting reports have been discussed above. Since worm fractions may have higher glucose utilization rates than intact helminths (Shapiro and Talalay 1982), the glucose utilization rates determined in the present report (Table I) seem consistent with previous studies. Read and Rothman (1957) reported 2.0 nmol * min- ’ * mg’, while Mettrick et a/. (1981) reported 1.2-l .5 nmol * min- ’ * mgg ’ were used by the rat tapeworm. In the latter study, a total of only 200 km01 of exogenous glucose were available for six 15day-old tapeworms, even though the initial substrate concentration was 10 mM, if the worms weighed 100 mg each, at the end of the 60-min incubation period probably less than 4 mM glucose remained. The rapid rate of glucose utilization seen here for the rat tapeworm is also comparable to that observed for schistosomes, rapidly growing tumor cells (see Shapiro and Talalay 1982; Cornford and Fitzpatrick 1987), and the cerebral cortex

AND

UTILIZATION

31

(Crane et al. 1980). Discrete regional differences in glucose utilization rates are readily demonstrable using the hexose analog 2deoxyglucose, which is (in most tissues) transported and phosphorylated, but not metabolized beyond 2-deoxyglucose 6phosphate. This technique utilizes the phosphorylation rate as an indicator of glucose utilization (Cornford and Fitzpatrick 1987). It is not applicable to H. diminuta, however, because the sodium-dependent active glucose transporter of the rat tapeworm does not transport 2-deoxy glucose (Phifer 1960b; Pappas 1975). Data in Fig. 2 indicate that the free glucose concentration within the tapeworm is a fraction (ranging from 0.1 to 0.2) of the external medium and displays an anterior to posterior gradient. In contrast, other workers suggest that the internal glucose concentration greatly exceeds that of the medium. Starling (1975) indicated that internal glucose concentrations were initially twofold higher than the (2 n&f) medium and rose by a factor of seven after a 60-min incubation in vitro. Methods were not given in this study. Pappas et al. (1974) reported that worms incubated in 5 mM medium initially had 4 mA4 glucose (in terms of worm water), but after 60 min the internal concentration had risen to 25 mM. These workers attribute their observations to the active glucose transporter and its accumulation of glucose against a concentration gradient. Pappas et al. (1974) used five worm aliquots of 200 mg wet weight; i.e., about 160 mg worm water. Fixation of the worms in 70% ethanol is not rapid, but relatively slow; it would require liberation of only 4 u.mol of glucose from glycogen in this time to produce the apparent 25 mM glucose level. The glucose assayed after overnight extraction in 70% ethanol was assumed to represent internal-free glucose-an assumption yet to be validated and not consistent with the data from the present study. In another study, Podesta er al. (1977) reported only a threefold increment in as-

EAIN M.CORNFORD

32

sayed glucose content. They homogenized the tapeworms in a deproteinizing solution. To test the possibility that glycogenolysis might be more promptly inhibited by this type of treatment, a group of tapeworms were incubated in RPM1 containing 7 mAI glucose. Worms rinsed in cold saline and extracted in 70% ethanol were assayed for glucose and contained an average of 10.7 ? 0.9 (n = 3) mM glucose (in terms of worm water). In contrast, worms treated with 0.9% perchloric acid and subjected to a similar extraction had only 5.9 + 1.1 (n = 3) mM glucose. Studies with 3-O-methylglucose would suggest an internal glucose concentration of about 1.5 mM for worms maintained in 7 m&I media, suggesting that perchloric acid inactivation is not as rapid as desired. This observation does indicate that glycogenolysis may be a rapid process, a finding which would be consistent with the short half-life (7 set) of free glucose in this worm. Since 3-0-methylglucose is not metabolized and distributes only to the free glucose pool, if infernalfree &cose levels are elevated, then proportionally more 30-methylglucose should also accumulate over time. Our results suggest that even though more glucose is taken up and utilized in the presence of increased substrate, the internal-free glucose concentration was not observed to exceed the exogenous substrate level. ACKNOWLEDGMENTS This work was supported by the Veterans Administration (Comprehensive Epilepsy Program, Neurology and Research Services, West Los Angeles V.A. Medical Center) and in part by NIH AI 15692.Thanks are due to Michael Fong, Dr. Xaioyan Zhou, George McGhee, and Lara Le for their assistance. REFERENCES BAKER, R. D., SEARLE, G. W., AND NUNN, A. S. 1961. Glucose and sorbose absorption at various levels of rat small intestine. American Journal of Physiology 200, 301-304. CASTRO,G. A., JOHNSON,L. R., COPELAND,E. M.,

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Biochemistry

and

Received 10April 1989;accepted with revision 25 July 1989

Glucose utilization rates are linked to the internal free glucose gradient in the rat tapeworm.

Hymenolepis diminuta is able to acquire plasma-borne glucose 3-O-[14C]methylglucose in vivo. Free glucose concentrations estimated for this helminth i...
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