EXPERIMENTAL
42, 363-373
PARASITOLOGY
Trichinella
spiralis: WILLIAM
(1977)
Taxes of First-Stage
Migratory
Larvae
L. HUGHES AND JOHN P. HARLEY
Department of Biological Sciences, Eastern Kentucky University, Richmond, Kentucky 40475, U.S.A. (Accepted
for publication
28 February
1977)
HUGHES, W. L., AND HARLEY, J, P. 1977. Trichinella spiTa&: Taxes of first-stage migratory larvae. Expe&zentaZ Parasitology 42, 363-373. Male albino mice were infected orally with 450 2 10 excysted Trichinellu s&&s larvae. Between Days 6 and 11 after infection, adults were collected from the intestine and incubated in &TO. The females shed first-stage migratory larvae which were collected and placed in a migratory chamber. The larvae showed a definite ability to respond to a microenvironmental stimulus. The taxis was positive (p < 0.025) to a 120-mV stimulus simulating the skeletal muscle action potential and to a 120-mV polar stimulus (p < 0.005). There was a negative taxis (p < 0.005) to a 95-mV stimulus simulating the resting skeletal muscle potential and to a KC1 gradient. A significant (p < 0.05) distribution ‘occurred to a lactic acid gradient, and there were no taxes ( p > 0.05) to either glycogen or phosphocreatine. INDEX DESCRIPTORS: Trichinella spiralis; Nematode; First-stage larva; Mouse; Larval taxes; Migratorv chamber: mV = millivolt stimulus; Polar stimulus; KC1 gradient; Lactic acid; Glycogen; Phosphocreatine. v
~
IXTRODUCTION
Recently, Farris and Harley (1977) stated that Trichinella spiralis first-stage migratory larvae do “not utilize the myoneural junction as a ‘homing signal’ . . . prior to fiber invasion.” The words “homing signal” imply a taxis. From an experimental standpoint, it is of interest to know if T. spiralis first-stage migratory larvae (Harley 1972) are capable of responding to a microenvironmental stimulus as presented for other parasites. For example, Ronald (1960, 1962, 1963) demonstrated positive taxes in Terranova decipiens larvae. Croll (1975) has published an excellent review of the responses of several nematodes on agar plates. Vogel reported that Capillaria hepatica larvae were influenced by an organotropism in their migration to the liver. Bonner and Etges (1967) reported a sexual attraction
Copyright All rights
0 1977 by Academic of reproduction in any
Press, form
between the male and female adults of T. spiralis based on an unidentified chemical. But, to date, no definitive reports have been published in the literature specifically relating to the taxes of the first-stage migratory larvae of T. spiralis. Due to the major theories of T. spiralis specificity assuming a chemotaxis for skeletal muscle and assuming taxes of the firststage larvae, this study was undertaken to determine: (a) the ability of the first-stage migratory larvae to respond to microenvironmental stimuli; and (b) to advance what is known about the selectivity of the first-stage migratory larvae for skeletal muscle. MATERIALSAND
METHODS
The Parasite The strain of the nematode Trichinella spiralis used in this investigation has been
Inc.
reserved.
ism
0014-4894
364
HUGHES
AND HART.,EY
maintained in laboratory rats and mice for the past 40 years at the Department of Parasitology and Laboratory Practice, University of North Carolina at Chapel Hill. Seven infected male mice were given to Eastern Kentucky University for use in this investigation. This T. spiralis strain has been subsequently maintained by passage in HA/ICR (Sprague-Dawley) male albino mice. Maintenance of Experimental, and Stock Mice
Control,
Sprague-Dawley male HA/ICR white mice were used throughout this investigation. All mice were maintained in groups of six in clean wire cages without bedding. The animal room was kept at a constant temperature of 23 + 4 C, and a 14-hr light and IO-hr dark diurnal cycle was utilized. Purina Laboratory Chow and fresh water were provided ad libitum. All mice were acclimated to animal room conditions for at least 1 week prior to infection. Larsh (1963) has shown that a week’s acclimation was sufficient for mice prior to infection with T. spiralis. Infection
of Mice
All larvae used for infections were obtained from stock mice that had been infected for at least 60 days. Recovery of these larvae from mice skeletal muscle was by standard techniques. All mice were 34 * 3 days of age when infected and weighed 20-22 g. All mice were inoculated by gavage with 450 f 10 excysted muscle larvae.
RQCOVQTY of the First-Stage Migratory
Larvae
The first-stage migratory larvae (hereafter simply referred to as larvae) used in this investigation were recovered by using a modification of the Dennis, Despommier,
and Davis (1970) technique. Food was removed from the infected mice 24 hr prior to their being killed. Harley and Gallicchio (1971) have shown that larvae were first recoverable at 4 days PI. There was then a gradual increase in mean numbers recoverable, reaching a peak on the ninth day PI and then declining through the 15th day PI, Thus, Days 6-11 PI were chosen for killing the infected mice used in this investigation. The infected mice were killed with chloroform fumes in a killing jar. The entire small intestine of each animal was removed, slit longitudinally, and cut into 2-cm sections. Each section was placed in a modified Baermann apparatus containing 0.85% saline solution at 37 C. Adult worms were collected over a 2-hr period. The adult worms were washed three times with O.SS% saline at 37 C. Worms thus treated appeared free of debris and were viable. One hundred adult female T. spiralis were then transferred to ,a sterile 250-ml Erlenmeyer flask containing 15 ml of the following culture medium: Medium 199 with sodium bicarbonate (Microbiological Associates, Baltimore, Maryland) (70% v/v) ; dialyzed calf serum ( Microbiological Associates, Baltimore, Maryland) (29% v/v); and VCN Inhibitor (BBL, Cockeysville, Maryland) (l%, v/v). The adult worms were incubated for 5 hr in this medium. Temperature, being a critical factor ( Despommier, personal communication), was maintained at 37 * 0.1 C. During this incubation period larvae were shed. The medium containing adults and larvae was then passed through an S-in, lOOmesh brass sieve into a sterile 50-ml glass beaker. The brass sieve effectively eliminated the adults from the medium. A 0.5ml aliquot of medium was removed using a l-ml tuberculin syringe. The suspension was spread on a large glass slide, and using 100~ magnification, larvae viability was determined. In all cases 98% of the larvae were viable prior to experimentation.
Trichirda The Migratory
spiralis:
365
LARVAL TAXES
Chamber
An experimental migratory chamber (Fig, 1) designed after a similar chamber developed by Bonner and Etges (1967) was constructed for the in vitro study of taxes responses of the larvae. Preparation of the Migratory
23.5
Chamber
A permeable barrier, Whatman No. 1 filter paper, was cut and placed over the hole in the diffusion barrier. Glass slides were inserted in the grooves adjacent to the 0 migratory zone of the migratory chamber. Fifteen milliliters of medium containing viable larvae were transferred from the culture flask to the 0 migratory zone. Since the total number of larvae introduced into the 0 zone varied with the different experiments, all results from the individual zones were expressed as a mean percentage of the total number of larvae in all zones (see Statistical Treatment). The remaining migratory zones and the chemical holding chamber were filled with the culture medium. The migratory chamber was then placed in an incubation oven (Imperial II Incubator, Chicago Surgical and Electrical Co., Chicago, Illinois) set at 37 * 0.1 C and allowed to equilibrate to oven conditions. The glass slides inserted in the grooves adjacent to the 0 migratory zone were removed, and migration of larvae was allowed for 2 hr. In all cases migration occurred in the incubation oven with temperature maintained at 37 I+ 0.1 C. This eliminated light and temperature as variables. Likewise, care was taken to insure that the zones were level with the horizontal as indicated by a bubble level. Chemotmis In the chemotaxis investigation, a series of chemicals associated with the energy source for muscular contraction were used. These chemicals (from Sigma Chemical
FIG. 1. Migratory holding slide.
chamber;
CM
chamber. CHC, DB, diffusion barrier;
t
chemical GS, glass
Co., St. Louis, Missouri) were glycogen, lactic acid, and phosphocreatine. In each case a 1% (v/v) solution was prepared using the specific chemical and the Dennis, Despommier, and Davis (1970) medium. Each chemical-medium solution was prepared and allowed to equilibrate in the incubation oven for at least 1 hr. After this period the medium in the chemical holding chamber was replaced with the 1% chemical-medium solution. Preliminary experiments indicated that soluble chemicals placed in the chemical holding chamber would immediately begin to diffuse across the permeable barrier, thus forming a congradient in the migratory centration chamber, Galvan80taxis In the first part of the galvanotaxis investigation a 0.9% KC1 medium solution was prepared and allowed to equilibrate in the incubation oven for 1 hr. The medium in the chemical holding chamber was then replaced with the 0.9% KC1 medium. Again the glass slides adjacent to the 0 migratory zone were removed, and migration of the larvae was allowed for 2 hr. In the second part of the galvanotaxis investigation, an attempt was made to reproduce the membrane potential of skeletal muscle, using the permeable barrier to represent the membrane. Two electrical leads were attached to the positive and negative terminals of an electrical stimu-
366
HUGHES
TABLE
AND
I
Migration of Trichinella spiralis Larvae under Normal Conditions Zone
Percentage migrated Run
+2 +1 0 -1 -2
5.74 15.93 59.99 13.89 4.43
1
of larvae per zone
Run 24.02 23.24 40.42 5.95 6.36
2
Run3 14.90 19.87 27.82 24.89 12.52
Mt!an percentage of migration 14.89 19.68 42.74 14.91 7.77
Standard deviation
zt9.1400 13.6587 f16.2104 zt9.5111 14.2253
lator (SO5 Stimulator, Grass Medical Instruments, Quincy, Massachusetts). Simulating the diaphragm (Guyton 1976), duration was set at 10 msec, frequency at 0.2 pulses/set, and delay at 0.2 msec. During the 95mV resting potential investigation, the positive lead from the electrical stimulator was placed in the +2 migratory zone at the face of the permeable barrier. The negative lead was secured in the chemical holding chamber on the opposite side of the permeable barrier. The 95mV electrical potential was then calibrated with a potentiometric strip chart recorder (Model SRLG, E. H. Sargent and Co., Chicago, Illinois). In the 120-mV action potential investigation, both electrical leads were again placed on opposite sides of the permeable barrier but the positive and negative poles were reversed. The strip chart recorder was again used to calibrate the 120-mV potential. In the positive-negative polar investigation, the positive lead was placed in the -2 migratory zone, and the negative lead was placed in the +2 migratory zone. The electrical stimulus remained at 120 mV. In all three cases the glass slides adjacent to the 0 migratory zone were removed, and larval migration was allowed for 2 hr.
HARLEY
were inserted in the grooves between migratoly zones, This assured a cessation of larval migration and prevented any medium leakage between adjacent zones. All the medium in each migratory zone was removed using a lo-ml syringe, measured, and recorded. This medium was discharged into a 50-ml glass beaker with a magnetic stirring bar and agitated on a magnetic stirring device for uniform suspension of any larvae present. A l-ml aliquot of the suspension was removed from the beaker using a l-ml tuberculin syringe. This aliquot was spread on a large (2 x 2-in.) glass slide. Using a hand tally counter, all migratory larvae were counted under 100~ magnification with a compound research microscope,
ZONE,+2 Collecting and Counting the First-Stage Migratory Larvae
In all taxes investigations, after the 2-hr migration period, the four glass slides
-+I
0
-1
-2
FIG. 2. Graphical histogram of the mean percentage of migration of Trickella spiralis under normal conditions. Straight lines above and below._ the indicate standard deviation. _.--- mean -.
Trichinella
spiralis:
LARVAL
367
TAXES
and the number was recorded. Four more l-ml aliquots were counted and recorded in the same manner. Statistical Treatment From the five counts a mean number of T. SpiraUs larvae was determined for each migratory zone. The total number of larvae in each zone was calculated. Then the percentage of larvae per zone compared to the total number in all zones was determined. In all cases a minimum of two investigations per taxis response was performed. From these the mean percentage of migration and the standard deviation for each migratory zone were calculated. Chi-square distribution ( Sokal and Rohlf 1969) was used to determine any statisti-
I
ZONE ,+2
+1
0
-1
-2
FIG. 4. Graphical histogram ‘of the mean percentage of migration of Trichinellu spidis in a 1% phosphocreatine gradient. Straight lines above and below the mean indicate standard deviation. df = 4;x==
cal significance of observed migration of the larvae. A probability of 0.05 or less was considered significant. All data were processed on a Honeywell 2050 computer for statistical comparison of experimental and control data.
t
5
8.328; p > 0.05.
RESULTS z
Distribution of First-Stage Migratory Larvae under Normal Conditions
L
ZONE --++2
$1
0
-1
-2
FIG. 3. Graphical histogram of the mean centage of migration of Trichinellu spiralis 1% glycogen gradient. Straight lines above below the mean indicate standard deviation. 4; x2 = 2.007; p > 0.05.
perin a and df =
In order to determine the distribution of the first-stage Trichinella spiralis larvae under normal conditions, three experimental runs were made without a chemical gradient or electrical stimulus. The distribution of larvae per migratory zone expressed as a percentage of the total number
HUGHES
AND HARLEY
ergy source for muscular contraction with the distribution under normal conditions. Data on the mean percentage of migration per zone for all experimental runs in a 1% glycogen gradient are shown in Fig. 3. These results were not significant (p > 0.05), indicating no taxis of the larvae to glycogen. Data on larval distribution in a 1% phosphocreatine gradient are shown in Fig. 4. These results were not significant (p > 0.05), indicating no taxis of the larvae to phosphocreatine. The mean percentage of migration per zone of the larvae in a 1% lactic acid gradient was statistically significant (p < 0.05). However, Fig. 5 showed approximately equal numbers of larvae migrating
ZONE -
’
FIG. 5. Graphical histogram of the mean percentage of migration of Trichinellu spidis in a 1% lactic acid gradient. Straight lines above and below the mean indicate standard deviation. df = 4; x2 = 10.235; p < 0.05.
of larvae in the chamber for each of the three experimental runs is shown in Table I. The mean percentage of migration for each zone is shown in Table I and a histogram of the mean percentage of migration per zone is shown in Fig. 2. Response of the First-Stage Migratory Larvae to Chemicak Associated with the Energy Source for Muscular Contraction One parameter used in this investigation of the taxis response of the larvae was the comparison of the distribution (or mean percentage of migration of larvae) in chemical gradients associated with the en-
ZONE--,+2
+I
0
-2
FIG. 6. Graphical histogram of the mean percentage of migration of Trichinella spiralis in a 0.9% KC1 gradient. Straight lines above and below the mean indicate standard deviation. & = 4;
x2 = 46.880; p < 0.005.
Trich,inella
spiralis:
369
LARVAL TAXES
to the negative and positive sides of the chamber. The distribution appeared to be more or less equal among all five migratory zones. Thus, the distribution itself indicated neither a positive nor negative taxis to lactic acid. Response of the First-Stage Migratory Larvae to Electrical Stimuli Associated with Membrane Potentials and to a KC1 Gradient The data from this investigation indicated that all migrations associated with electrical stimuli and the distribution in a 0.9% KC1 gradient were statistically significant (p < 0.05). Giese (1973) points out that if a membrane is equally perme-
ZONE -
t’r
i-1
0
-1
-2
FIG. 8. Graphical histogram of the mean percentage of migration of Trichindla spiralis to a 120-mV stimulus. Straight lines above and below the mean indicate standard deviation. df = 4; xa = 11.694; p < 0.025.
FIG. 7. Graphical histogram of the mean percentage of migration of Trichinella spiralis to a 95-mV stimulus. Straight lines above and below the mean indicate standard deviation. df = 4; x2 = 24.026; p < 0.005.
able to two ions, any potential developed would be transitory. The mobility of the ions determines how long that transitory potential will last. Potassium ions and chloride ions have almost equal mobilities. Therefore, any potential developed in this experiment with the KC1 medium solution would be transitory and slight. However, Fig. 6 showed a highly significant p < 0.005) distribution of the mean percentage of migration per zone in a 0.9% KC1 gradient. Figure 6 indicated that this distribution was weighted toward the negative zones of the migratory chamber. The histogram also showed a sharp reduction in the percentage of migration to the $2 migratory zone when compared to the normal distribution. Thus, the data indicated
HUGHES
AND HARLEY
The larval distribution (Fig. 8) in a 120mV stimulus was also significant (p < 0.025). The histogram showed a shift in the mean percentage of migration toward the positive zones of the migratory chamber. This indicated a taxis of larvae to a 120-mV electrical stimulus simulating the action potential of skeletal muscle, and in this case the taxis was positive. Distribution of larvae in a positivenegative polar stimulus (Fig. 9) was also highly significant (p < 0.005). The histogram showed a shift in the migration toward the 120-mV negative lead. This indicated a positive larval taxis to a I2O-mV stimulus and negative pole, DISCUSSION
ZONE-t2
+I
0
-1
-2
FIG. 9. Graphical histogram of the mean percentage of migration of Trichinella spiralis to a positive-negative polar stimulus. Straight lines above and below the mean indicate standard deviation. df = 4; x2 = 19.812; p < 0.005.
a highly significant (p < 0.005) taxis to potassium ions, chloride ions, or both. Since both ions will diffuse with equal mobility across the permeable barrier, it was impossible to determine if the T. spiralis larval taxis was to potassium ions, chloride ions, or both ions. It was obvious, nevertheless, that the taxis was negative. The data (Fig. 7) for larval distribution to a 95mV stimulus was also highly significant (p < O.OOS), and again the histogram was weighted toward the negative zones of the migratory chamber. There was a decrease in the mean percentage of migration of larvae to the +2 and +l zones. Thus, the data supported a negative taxis to a 95mV stimulus simulating the resting membrane potential of skeletal muscle.
In effect, the data indicated that the firststage Trichindla spiralis migratory larvae had taxes. Askanazy (per Gould 1970) first assumed that the larvae were attracted to skeletal muscle by a chemotaxis. Vogel (1930) suggested that the larvae were influenced by a special organotropism in their migration to skeletal muscle. Since then, three major hypotheses have emerged explaining the specificity of the larvae for skeletal muscle. Flury and Groll (1913) proposed that the larvae invade glycogenrich muscles. Lewis (1928) believed that the larvae invade only glycogen-poor muscles. Staubli (1950a, b) and Ogielski (1949) proposed that those muscles with the richest blood supply and most active are favored by the larvae. Bristol, Harley, and Gallicchio (1972) supported the blood supply theory and refuted both Lewis (1928) and Flury and Groll (1913) by reporting no difference in the number of larvae in glycogen-rich or glycogen-poor diaphragms. Current data on glycogen thus supports Bristol, Harley, and Gallicchio (1972), for Fig. 3 showed no significant (p > 0.05) taxis of the larvae in response to a glycogen gradient. Our current data can neither conclusively support nor refute the rich blood
Trichindla
spirah:
supply theory of Staubli (1905a, b) and Ogielski ( 1949). Because specificity caused by a rich blood supply in the active muscles would be merely a mechanical invasion, there would be no taxis responsible for that specificity. The data in this investigation did indicate that the larvae had taxes, and on this basis, this investigation supports a chemotaxis theory of specificity for skeletal muscle. There is an apparently striking difference between the biochemical metabolism of resting and active muscles. Bell, Davidson, and Emslie-Smith (1972) reported that muscle carbohydrate or glycogen is the major source of fuel during muscular activity, while in the resting state, mammalian skeletal muscle uses fatty acids and acetoacetate as sources of fuel. Oser (1965) reported that muscles in intense activity will synthesize high energy phosphate bonds as much as possible by glycolysis and will deplete any high energy phosphate bonds stored in phosphocreatine. Bell, Davidson, and Emslie-Smith (1972) observed that phosphocreatine cannot be used as a direct source of energy but serves as an emergency mechanism to regenerate ATP in active muscles. Considering the above facts, data on larval distribution in a 1% phosphocreatine gradient (Fig. 4) were not significant (p > 0.05), indicating no taxis of the larvae to phosphocreatine, an energy mechanism for muscular contraction. Lewis (1928) observed that lactic acid, specific in muscles, may be that causative factor for T. s-piralis specificity. Bell, Davidson, and Emslie-Smith (1972) observed that pyruvic acid is the normal end product of muscle glycolysis, but lactic acid may form and accumulate in the muscles as the result of repeated contractions. Oser (1965) reported that lactic acid levels are low in resting muscle but increase in activity in anaerobiosis. Figure 5 showed a significant (p < 0.05) distribution of the larvae in a 1% lactic acid gradient. It was
LARVAL
TAXES
371
impossible to determine, however, from Fig. 5 whether this taxis was positive or negative. The data from Fig. 5 cannot conclusively support or refute lactic acid as the causative factor in larval specificity. The 0.9% KC1 gradient represented the diffusion of potassium and chloride ions across the cell membrane after the action potential begins in skeletal muscle. Figure 6 indicated a highly significant (p < 0.005) negative taxis. It was impossible to determine conclusively whether the negative taxis of the T. spiralis larvae was to the potassium ions, chloride ions, or both. But, based on the negative taxis in the 95mV stimulus, it can be assumed that the larval negative taxis was to the potassium ions with their concurrent positive charge. Many investigators (Osterhout 1936a, b; Bennett, Ware, Dunn, and McIntyre 1953; White, Handler, and Smith 1973; Giese 1973; Guyton 1976) have reported that the average resting membrane potential of skeletal muscle is 95 mV, and the outside of the cell membrane is positive relative to the negative inside in a resting state. The polarity of the cell membrane reverses during the action potential (Hodgkin and Huxley 1939, 1945; Curtis and Cole 1942; Graham and Gerard 1946; Nastuk and Hodgkin 1950; Davson 1959; Giese 1973) and the average value for the action potential ranges from 100 to 120 mV (Kuffler 1942; Katz 1948; Giese 1973). Hodgkin (1951) observed that the action potential is considerably larger than the resting potential. Both Giese (1973) and Guyton (1976) reported that the outward diffusion of potassium ions during any action potential repolarizes the cell membrane. The data in Figs. 8 and 9 showed that the larvae had a positive taxis to a 120-mV stimulus simulating the action potential. The data in Figs. 6 and 7 showed that the larvae had a negative taxis to a positive charge and to a 95mV stimuhrs simulating the resting membrane potential. Considering the positive taxis of the T. spiralis lar-
372
HUGHES
PIND
vae to a 12O-mV stimulus, there is the possibility that the predilection of the larvae for skeletal muscle m’ay, be due to either the considerably higher charge of the action potential or the reversed potential of the cell membrane during the action potential (or both). If it is assumed that there are more action potentials per minute, and thus more negativity on the outside of the cell membrane per minute in more active muscles when compared to less active muscles, there is the possibility that the larval predilection for the more active muscles can be explained by this increased negativity. Also, the negative taxis of the larvae to a 95mV stimulus simulating the resting membrane potential, where the outside of the membrane is positive, can possible explain the decreased infection in less active muscles. These muscles would have more positivity on the outside of the cell membrane per minute. It is well know that larvae ‘only invade smooth and cardiac muscles in aberrant cases. Bell, Davidson, and Em&e-Smith (1972) reported that the resting and action potentials of both smooth and cardiac muscles are considerably less than in skeletal muscle. Thus, this could possibly explain the nonspecificity of the larvae for both smooth and cardiac muscle. These possibilities are not conclusively supported by data from this investigation nor are they entirely refuted. In essence they are possibilities that do exist, based on the taxes of the Trichindla spiralis firststage larvae shown in this investigation. ACKNOWLEDGMENT Supported in part by a faculty No. 03-07 from Eastern Kentucky
research Grant University.
REFERENCES BELL, G. H., DAVIDSON, J. N., AND EMSLIE-SMITH, D. 1972. “Textbook of Physiology and Biochemistry,” pp. 825-859. Williams and Wilkins, Baltimore, Maryland.
HARLEY
BENNETT, A. L., WARE, F., JR., DUNN, A. L., AND MCINTYRE, A. R. 1953. The normal membrane resting potential of mammalian skeletal muscle measured in vivo. Journal of Cellular and Comparative Physiology 42, 343-357. BONNER, T. P., AND ETGES, F. J. 1967. Chemically mediated sexual attraction in Trichinella spiralis. Experimental Parasitology 21, 53-60. BRISTOL, J., HARLEY, J. P., AND GALLICCHI.O, V. 1972. Infectivity of Trichinella spiralis (Nematoda) muscle larvae in normal vs. alloxantreated rats. Experientia 28, 102. CROLL, N. A. 1975: Behavioural analysis of nematode movement. Advances in Parasitology 13, 71-122. CURTIS, H. J., AND COLE, K. S. 1942. Membrane resting and action potentials from the squid giant axon. Journal of Cellular and Comparative Physiology 19, 135-144. DAVSON, H. 1959. “Testbook of General Physiol,> ogy. Brown, Boston, Massachusetts. DENNIS, D. T., DESPOMMIER, D. D., AND DAVIS, N. 1970. Infectivity of the newborn larva of Trichinella spiralis in the rat. journal of Parasitology 56, 974-977. FARRIS, K. N., AND HARLEY, J. P. 1977. Trichinella spiralis: Alteration of gastrocnemius muscle kinetics in the mouse. Experimental Parasitology 41,11-70. FLURY, F., AND GROLL, H. 1913. Stoffwechseluntersuchungen an trichinosen Tieren. Archiw fur Experimentelle Pathologie und Pharmakologie 73,214-232. GIESE, A. C. 1973. “Cell Physiology.” Saunders, Philadelphia, Pennsylvania. GOULD, S. E. 1970. “Trichinosis in Man and Animals.” Thomas, Springfield, Illinois. GRAHAM, J., AND GERARD, R. W. 1946. Membrane potentials and excitation of impaled single muscle fibers. Journal of Cellular and Comparative Physiology 28, 99-117. GUYTON, A. 1976. “Textbook of Medical Physiol,, ogy. Saunders, Philadelphia, Pennsylvania. HARLEY, J. P. 1972. Clarification of Trichinella spiralis (Nematode) life cycle terminology. Acta Parasitologica Polo&a 3’8, 463467. HARLEY, J. P., AND GALLICCHIO, V. 1971. Trichinellu spiralis: Migration of larvae in the rat. Experimental Parasitology 30, 11-21. HODGKIN, A. L. 1951. The ionic basis for electrical activity in nerve and muscle. Biological Review 26, 339. HODGKIN, A. L., AND HUXLEY, A. F. 1939. Action potentials recorded from inside a nerve fiber. Nature (London) 144,710-711. HODGKIN, A. L., AND HUXLEY, A. F. 1945. Resting and action potentials in single nerve fibers. Journal of Physiology 104, 176-195.
Trichimlla KATZ, B. 1948. The electrical
spiralis:
properties of the muscle fiber membrane. Proceedings of the Royal Society of Biology 135, 506-534. KUFFLER, S. W. 1942. Electric potential changes at an isolated nerve-muscle junction. Journal of Neurophysiology 5, 18-26. LARSH, J. E. 1963. Experimental trichinosis. Advances in Parasitology 1, 213-286. LEWIS, J. H. 1928. Influence of glycogen on the infection of muscle with Trichinae. Transactions of the Chicago Pathological Society 13, 12-15. NASTUK, W. L., AND HODGKIN, A. L. 1950. The electrical activity of single muscle fibers. Journal of Cellular and Comparative Physiology 35, 39-73. OGIELSKI, L. 1949. Reaction of the vascular vessels against an invasion of the larvae of Trichinella spiralis. Zoologica Poloniae 5, 3542, OSER, B. L. 1965. “Hawk’s Physiological ChemisMcGraw-Hill, New York, try,” PP. 213-232. New York. OSTERHOUT,W. J. V. 1936a. The absorption of electrolytes in large plant cells. Botanical Revieu> 2, 283. OSTERHOUT,W. J. V. 1936b. Electrical phenomena in large plant cells. Physiological Reviews 16, 216. RONALD, K. 1960. The effects of physical stimuli
LARVAL
TAXES
373
on the larval stage of Terranova decipiens. I. Temperature. Canadian Journal of Zoology 38, 623-642. RONALD, K. 1962. The effects of physical stimuli on the larval stages of Terranooa decipiens. II. Relative humidity, pressure, and gases. Canadian Jotma of Zoology 40, 1223-1227. RONALD, K. 1963. The effects of physical stimuli on the larval stage of Terranova decipiens. III. Electromagnetic spectrum and galvanotaxis. Canadian Journal of Zoology 41, 197-217. SOKAL, R. R., AND ROHLF, F. J. 1969. “Biometry.” Freeman, San Francisco, California. STAUBLI, C. 1905a. Klinische unde experimentelle Untersuchungen uber Trichinosis. Verhandlungen des Kongresses fur Innere Medizin 22, 353362. STAUBLI, C. 1905b. Klinishe und experimentelle Untersuchungen uber Trichinose und uber die Eosinophilie im allgemeinen. V&how, Archiv fur Pathologische Anatomie und Phgsiologie und fur Rlinische Medizin 85, 286-341. VOGEL, H. 1930. Ueber die Organotropie von Hepaticola hepatica. Zeithschrifi fur Parasitenkunde 2, 502-505. WHITE, A., HANDLER, P., AND SMITH, E. L. 1973. “Principles of Biochemistry.” hlcGraw-Hill, New York, New York.
42, 363-373
PARASITOLOGY
Trichinella
spiralis: WILLIAM
(1977)
Taxes of First-Stage
Migratory
Larvae
L. HUGHES AND JOHN P. HARLEY
Department of Biological Sciences, Eastern Kentucky University, Richmond, Kentucky 40475, U.S.A. (Accepted
for publication
28 February
1977)
HUGHES, W. L., AND HARLEY, J, P. 1977. Trichinella spiTa&: Taxes of first-stage migratory larvae. Expe&zentaZ Parasitology 42, 363-373. Male albino mice were infected orally with 450 2 10 excysted Trichinellu s&&s larvae. Between Days 6 and 11 after infection, adults were collected from the intestine and incubated in &TO. The females shed first-stage migratory larvae which were collected and placed in a migratory chamber. The larvae showed a definite ability to respond to a microenvironmental stimulus. The taxis was positive (p < 0.025) to a 120-mV stimulus simulating the skeletal muscle action potential and to a 120-mV polar stimulus (p < 0.005). There was a negative taxis (p < 0.005) to a 95-mV stimulus simulating the resting skeletal muscle potential and to a KC1 gradient. A significant (p < 0.05) distribution ‘occurred to a lactic acid gradient, and there were no taxes ( p > 0.05) to either glycogen or phosphocreatine. INDEX DESCRIPTORS: Trichinella spiralis; Nematode; First-stage larva; Mouse; Larval taxes; Migratorv chamber: mV = millivolt stimulus; Polar stimulus; KC1 gradient; Lactic acid; Glycogen; Phosphocreatine. v
~
IXTRODUCTION
Recently, Farris and Harley (1977) stated that Trichinella spiralis first-stage migratory larvae do “not utilize the myoneural junction as a ‘homing signal’ . . . prior to fiber invasion.” The words “homing signal” imply a taxis. From an experimental standpoint, it is of interest to know if T. spiralis first-stage migratory larvae (Harley 1972) are capable of responding to a microenvironmental stimulus as presented for other parasites. For example, Ronald (1960, 1962, 1963) demonstrated positive taxes in Terranova decipiens larvae. Croll (1975) has published an excellent review of the responses of several nematodes on agar plates. Vogel reported that Capillaria hepatica larvae were influenced by an organotropism in their migration to the liver. Bonner and Etges (1967) reported a sexual attraction
Copyright All rights
0 1977 by Academic of reproduction in any
Press, form
between the male and female adults of T. spiralis based on an unidentified chemical. But, to date, no definitive reports have been published in the literature specifically relating to the taxes of the first-stage migratory larvae of T. spiralis. Due to the major theories of T. spiralis specificity assuming a chemotaxis for skeletal muscle and assuming taxes of the firststage larvae, this study was undertaken to determine: (a) the ability of the first-stage migratory larvae to respond to microenvironmental stimuli; and (b) to advance what is known about the selectivity of the first-stage migratory larvae for skeletal muscle. MATERIALSAND
METHODS
The Parasite The strain of the nematode Trichinella spiralis used in this investigation has been
Inc.
reserved.
ism
0014-4894
364
HUGHES
AND HART.,EY
maintained in laboratory rats and mice for the past 40 years at the Department of Parasitology and Laboratory Practice, University of North Carolina at Chapel Hill. Seven infected male mice were given to Eastern Kentucky University for use in this investigation. This T. spiralis strain has been subsequently maintained by passage in HA/ICR (Sprague-Dawley) male albino mice. Maintenance of Experimental, and Stock Mice
Control,
Sprague-Dawley male HA/ICR white mice were used throughout this investigation. All mice were maintained in groups of six in clean wire cages without bedding. The animal room was kept at a constant temperature of 23 + 4 C, and a 14-hr light and IO-hr dark diurnal cycle was utilized. Purina Laboratory Chow and fresh water were provided ad libitum. All mice were acclimated to animal room conditions for at least 1 week prior to infection. Larsh (1963) has shown that a week’s acclimation was sufficient for mice prior to infection with T. spiralis. Infection
of Mice
All larvae used for infections were obtained from stock mice that had been infected for at least 60 days. Recovery of these larvae from mice skeletal muscle was by standard techniques. All mice were 34 * 3 days of age when infected and weighed 20-22 g. All mice were inoculated by gavage with 450 f 10 excysted muscle larvae.
RQCOVQTY of the First-Stage Migratory
Larvae
The first-stage migratory larvae (hereafter simply referred to as larvae) used in this investigation were recovered by using a modification of the Dennis, Despommier,
and Davis (1970) technique. Food was removed from the infected mice 24 hr prior to their being killed. Harley and Gallicchio (1971) have shown that larvae were first recoverable at 4 days PI. There was then a gradual increase in mean numbers recoverable, reaching a peak on the ninth day PI and then declining through the 15th day PI, Thus, Days 6-11 PI were chosen for killing the infected mice used in this investigation. The infected mice were killed with chloroform fumes in a killing jar. The entire small intestine of each animal was removed, slit longitudinally, and cut into 2-cm sections. Each section was placed in a modified Baermann apparatus containing 0.85% saline solution at 37 C. Adult worms were collected over a 2-hr period. The adult worms were washed three times with O.SS% saline at 37 C. Worms thus treated appeared free of debris and were viable. One hundred adult female T. spiralis were then transferred to ,a sterile 250-ml Erlenmeyer flask containing 15 ml of the following culture medium: Medium 199 with sodium bicarbonate (Microbiological Associates, Baltimore, Maryland) (70% v/v) ; dialyzed calf serum ( Microbiological Associates, Baltimore, Maryland) (29% v/v); and VCN Inhibitor (BBL, Cockeysville, Maryland) (l%, v/v). The adult worms were incubated for 5 hr in this medium. Temperature, being a critical factor ( Despommier, personal communication), was maintained at 37 * 0.1 C. During this incubation period larvae were shed. The medium containing adults and larvae was then passed through an S-in, lOOmesh brass sieve into a sterile 50-ml glass beaker. The brass sieve effectively eliminated the adults from the medium. A 0.5ml aliquot of medium was removed using a l-ml tuberculin syringe. The suspension was spread on a large glass slide, and using 100~ magnification, larvae viability was determined. In all cases 98% of the larvae were viable prior to experimentation.
Trichirda The Migratory
spiralis:
365
LARVAL TAXES
Chamber
An experimental migratory chamber (Fig, 1) designed after a similar chamber developed by Bonner and Etges (1967) was constructed for the in vitro study of taxes responses of the larvae. Preparation of the Migratory
23.5
Chamber
A permeable barrier, Whatman No. 1 filter paper, was cut and placed over the hole in the diffusion barrier. Glass slides were inserted in the grooves adjacent to the 0 migratory zone of the migratory chamber. Fifteen milliliters of medium containing viable larvae were transferred from the culture flask to the 0 migratory zone. Since the total number of larvae introduced into the 0 zone varied with the different experiments, all results from the individual zones were expressed as a mean percentage of the total number of larvae in all zones (see Statistical Treatment). The remaining migratory zones and the chemical holding chamber were filled with the culture medium. The migratory chamber was then placed in an incubation oven (Imperial II Incubator, Chicago Surgical and Electrical Co., Chicago, Illinois) set at 37 * 0.1 C and allowed to equilibrate to oven conditions. The glass slides inserted in the grooves adjacent to the 0 migratory zone were removed, and migration of larvae was allowed for 2 hr. In all cases migration occurred in the incubation oven with temperature maintained at 37 I+ 0.1 C. This eliminated light and temperature as variables. Likewise, care was taken to insure that the zones were level with the horizontal as indicated by a bubble level. Chemotmis In the chemotaxis investigation, a series of chemicals associated with the energy source for muscular contraction were used. These chemicals (from Sigma Chemical
FIG. 1. Migratory holding slide.
chamber;
CM
chamber. CHC, DB, diffusion barrier;
t
chemical GS, glass
Co., St. Louis, Missouri) were glycogen, lactic acid, and phosphocreatine. In each case a 1% (v/v) solution was prepared using the specific chemical and the Dennis, Despommier, and Davis (1970) medium. Each chemical-medium solution was prepared and allowed to equilibrate in the incubation oven for at least 1 hr. After this period the medium in the chemical holding chamber was replaced with the 1% chemical-medium solution. Preliminary experiments indicated that soluble chemicals placed in the chemical holding chamber would immediately begin to diffuse across the permeable barrier, thus forming a congradient in the migratory centration chamber, Galvan80taxis In the first part of the galvanotaxis investigation a 0.9% KC1 medium solution was prepared and allowed to equilibrate in the incubation oven for 1 hr. The medium in the chemical holding chamber was then replaced with the 0.9% KC1 medium. Again the glass slides adjacent to the 0 migratory zone were removed, and migration of the larvae was allowed for 2 hr. In the second part of the galvanotaxis investigation, an attempt was made to reproduce the membrane potential of skeletal muscle, using the permeable barrier to represent the membrane. Two electrical leads were attached to the positive and negative terminals of an electrical stimu-
366
HUGHES
TABLE
AND
I
Migration of Trichinella spiralis Larvae under Normal Conditions Zone
Percentage migrated Run
+2 +1 0 -1 -2
5.74 15.93 59.99 13.89 4.43
1
of larvae per zone
Run 24.02 23.24 40.42 5.95 6.36
2
Run3 14.90 19.87 27.82 24.89 12.52
Mt!an percentage of migration 14.89 19.68 42.74 14.91 7.77
Standard deviation
zt9.1400 13.6587 f16.2104 zt9.5111 14.2253
lator (SO5 Stimulator, Grass Medical Instruments, Quincy, Massachusetts). Simulating the diaphragm (Guyton 1976), duration was set at 10 msec, frequency at 0.2 pulses/set, and delay at 0.2 msec. During the 95mV resting potential investigation, the positive lead from the electrical stimulator was placed in the +2 migratory zone at the face of the permeable barrier. The negative lead was secured in the chemical holding chamber on the opposite side of the permeable barrier. The 95mV electrical potential was then calibrated with a potentiometric strip chart recorder (Model SRLG, E. H. Sargent and Co., Chicago, Illinois). In the 120-mV action potential investigation, both electrical leads were again placed on opposite sides of the permeable barrier but the positive and negative poles were reversed. The strip chart recorder was again used to calibrate the 120-mV potential. In the positive-negative polar investigation, the positive lead was placed in the -2 migratory zone, and the negative lead was placed in the +2 migratory zone. The electrical stimulus remained at 120 mV. In all three cases the glass slides adjacent to the 0 migratory zone were removed, and larval migration was allowed for 2 hr.
HARLEY
were inserted in the grooves between migratoly zones, This assured a cessation of larval migration and prevented any medium leakage between adjacent zones. All the medium in each migratory zone was removed using a lo-ml syringe, measured, and recorded. This medium was discharged into a 50-ml glass beaker with a magnetic stirring bar and agitated on a magnetic stirring device for uniform suspension of any larvae present. A l-ml aliquot of the suspension was removed from the beaker using a l-ml tuberculin syringe. This aliquot was spread on a large (2 x 2-in.) glass slide. Using a hand tally counter, all migratory larvae were counted under 100~ magnification with a compound research microscope,
ZONE,+2 Collecting and Counting the First-Stage Migratory Larvae
In all taxes investigations, after the 2-hr migration period, the four glass slides
-+I
0
-1
-2
FIG. 2. Graphical histogram of the mean percentage of migration of Trickella spiralis under normal conditions. Straight lines above and below._ the indicate standard deviation. _.--- mean -.
Trichinella
spiralis:
LARVAL
367
TAXES
and the number was recorded. Four more l-ml aliquots were counted and recorded in the same manner. Statistical Treatment From the five counts a mean number of T. SpiraUs larvae was determined for each migratory zone. The total number of larvae in each zone was calculated. Then the percentage of larvae per zone compared to the total number in all zones was determined. In all cases a minimum of two investigations per taxis response was performed. From these the mean percentage of migration and the standard deviation for each migratory zone were calculated. Chi-square distribution ( Sokal and Rohlf 1969) was used to determine any statisti-
I
ZONE ,+2
+1
0
-1
-2
FIG. 4. Graphical histogram ‘of the mean percentage of migration of Trichinellu spidis in a 1% phosphocreatine gradient. Straight lines above and below the mean indicate standard deviation. df = 4;x==
cal significance of observed migration of the larvae. A probability of 0.05 or less was considered significant. All data were processed on a Honeywell 2050 computer for statistical comparison of experimental and control data.
t
5
8.328; p > 0.05.
RESULTS z
Distribution of First-Stage Migratory Larvae under Normal Conditions
L
ZONE --++2
$1
0
-1
-2
FIG. 3. Graphical histogram of the mean centage of migration of Trichinellu spiralis 1% glycogen gradient. Straight lines above below the mean indicate standard deviation. 4; x2 = 2.007; p > 0.05.
perin a and df =
In order to determine the distribution of the first-stage Trichinella spiralis larvae under normal conditions, three experimental runs were made without a chemical gradient or electrical stimulus. The distribution of larvae per migratory zone expressed as a percentage of the total number
HUGHES
AND HARLEY
ergy source for muscular contraction with the distribution under normal conditions. Data on the mean percentage of migration per zone for all experimental runs in a 1% glycogen gradient are shown in Fig. 3. These results were not significant (p > 0.05), indicating no taxis of the larvae to glycogen. Data on larval distribution in a 1% phosphocreatine gradient are shown in Fig. 4. These results were not significant (p > 0.05), indicating no taxis of the larvae to phosphocreatine. The mean percentage of migration per zone of the larvae in a 1% lactic acid gradient was statistically significant (p < 0.05). However, Fig. 5 showed approximately equal numbers of larvae migrating
ZONE -
’
FIG. 5. Graphical histogram of the mean percentage of migration of Trichinellu spidis in a 1% lactic acid gradient. Straight lines above and below the mean indicate standard deviation. df = 4; x2 = 10.235; p < 0.05.
of larvae in the chamber for each of the three experimental runs is shown in Table I. The mean percentage of migration for each zone is shown in Table I and a histogram of the mean percentage of migration per zone is shown in Fig. 2. Response of the First-Stage Migratory Larvae to Chemicak Associated with the Energy Source for Muscular Contraction One parameter used in this investigation of the taxis response of the larvae was the comparison of the distribution (or mean percentage of migration of larvae) in chemical gradients associated with the en-
ZONE--,+2
+I
0
-2
FIG. 6. Graphical histogram of the mean percentage of migration of Trichinella spiralis in a 0.9% KC1 gradient. Straight lines above and below the mean indicate standard deviation. & = 4;
x2 = 46.880; p < 0.005.
Trich,inella
spiralis:
369
LARVAL TAXES
to the negative and positive sides of the chamber. The distribution appeared to be more or less equal among all five migratory zones. Thus, the distribution itself indicated neither a positive nor negative taxis to lactic acid. Response of the First-Stage Migratory Larvae to Electrical Stimuli Associated with Membrane Potentials and to a KC1 Gradient The data from this investigation indicated that all migrations associated with electrical stimuli and the distribution in a 0.9% KC1 gradient were statistically significant (p < 0.05). Giese (1973) points out that if a membrane is equally perme-
ZONE -
t’r
i-1
0
-1
-2
FIG. 8. Graphical histogram of the mean percentage of migration of Trichindla spiralis to a 120-mV stimulus. Straight lines above and below the mean indicate standard deviation. df = 4; xa = 11.694; p < 0.025.
FIG. 7. Graphical histogram of the mean percentage of migration of Trichinella spiralis to a 95-mV stimulus. Straight lines above and below the mean indicate standard deviation. df = 4; x2 = 24.026; p < 0.005.
able to two ions, any potential developed would be transitory. The mobility of the ions determines how long that transitory potential will last. Potassium ions and chloride ions have almost equal mobilities. Therefore, any potential developed in this experiment with the KC1 medium solution would be transitory and slight. However, Fig. 6 showed a highly significant p < 0.005) distribution of the mean percentage of migration per zone in a 0.9% KC1 gradient. Figure 6 indicated that this distribution was weighted toward the negative zones of the migratory chamber. The histogram also showed a sharp reduction in the percentage of migration to the $2 migratory zone when compared to the normal distribution. Thus, the data indicated
HUGHES
AND HARLEY
The larval distribution (Fig. 8) in a 120mV stimulus was also significant (p < 0.025). The histogram showed a shift in the mean percentage of migration toward the positive zones of the migratory chamber. This indicated a taxis of larvae to a 120-mV electrical stimulus simulating the action potential of skeletal muscle, and in this case the taxis was positive. Distribution of larvae in a positivenegative polar stimulus (Fig. 9) was also highly significant (p < 0.005). The histogram showed a shift in the migration toward the 120-mV negative lead. This indicated a positive larval taxis to a I2O-mV stimulus and negative pole, DISCUSSION
ZONE-t2
+I
0
-1
-2
FIG. 9. Graphical histogram of the mean percentage of migration of Trichinella spiralis to a positive-negative polar stimulus. Straight lines above and below the mean indicate standard deviation. df = 4; x2 = 19.812; p < 0.005.
a highly significant (p < 0.005) taxis to potassium ions, chloride ions, or both. Since both ions will diffuse with equal mobility across the permeable barrier, it was impossible to determine if the T. spiralis larval taxis was to potassium ions, chloride ions, or both ions. It was obvious, nevertheless, that the taxis was negative. The data (Fig. 7) for larval distribution to a 95mV stimulus was also highly significant (p < O.OOS), and again the histogram was weighted toward the negative zones of the migratory chamber. There was a decrease in the mean percentage of migration of larvae to the +2 and +l zones. Thus, the data supported a negative taxis to a 95mV stimulus simulating the resting membrane potential of skeletal muscle.
In effect, the data indicated that the firststage Trichindla spiralis migratory larvae had taxes. Askanazy (per Gould 1970) first assumed that the larvae were attracted to skeletal muscle by a chemotaxis. Vogel (1930) suggested that the larvae were influenced by a special organotropism in their migration to skeletal muscle. Since then, three major hypotheses have emerged explaining the specificity of the larvae for skeletal muscle. Flury and Groll (1913) proposed that the larvae invade glycogenrich muscles. Lewis (1928) believed that the larvae invade only glycogen-poor muscles. Staubli (1950a, b) and Ogielski (1949) proposed that those muscles with the richest blood supply and most active are favored by the larvae. Bristol, Harley, and Gallicchio (1972) supported the blood supply theory and refuted both Lewis (1928) and Flury and Groll (1913) by reporting no difference in the number of larvae in glycogen-rich or glycogen-poor diaphragms. Current data on glycogen thus supports Bristol, Harley, and Gallicchio (1972), for Fig. 3 showed no significant (p > 0.05) taxis of the larvae in response to a glycogen gradient. Our current data can neither conclusively support nor refute the rich blood
Trichindla
spirah:
supply theory of Staubli (1905a, b) and Ogielski ( 1949). Because specificity caused by a rich blood supply in the active muscles would be merely a mechanical invasion, there would be no taxis responsible for that specificity. The data in this investigation did indicate that the larvae had taxes, and on this basis, this investigation supports a chemotaxis theory of specificity for skeletal muscle. There is an apparently striking difference between the biochemical metabolism of resting and active muscles. Bell, Davidson, and Emslie-Smith (1972) reported that muscle carbohydrate or glycogen is the major source of fuel during muscular activity, while in the resting state, mammalian skeletal muscle uses fatty acids and acetoacetate as sources of fuel. Oser (1965) reported that muscles in intense activity will synthesize high energy phosphate bonds as much as possible by glycolysis and will deplete any high energy phosphate bonds stored in phosphocreatine. Bell, Davidson, and Emslie-Smith (1972) observed that phosphocreatine cannot be used as a direct source of energy but serves as an emergency mechanism to regenerate ATP in active muscles. Considering the above facts, data on larval distribution in a 1% phosphocreatine gradient (Fig. 4) were not significant (p > 0.05), indicating no taxis of the larvae to phosphocreatine, an energy mechanism for muscular contraction. Lewis (1928) observed that lactic acid, specific in muscles, may be that causative factor for T. s-piralis specificity. Bell, Davidson, and Emslie-Smith (1972) observed that pyruvic acid is the normal end product of muscle glycolysis, but lactic acid may form and accumulate in the muscles as the result of repeated contractions. Oser (1965) reported that lactic acid levels are low in resting muscle but increase in activity in anaerobiosis. Figure 5 showed a significant (p < 0.05) distribution of the larvae in a 1% lactic acid gradient. It was
LARVAL
TAXES
371
impossible to determine, however, from Fig. 5 whether this taxis was positive or negative. The data from Fig. 5 cannot conclusively support or refute lactic acid as the causative factor in larval specificity. The 0.9% KC1 gradient represented the diffusion of potassium and chloride ions across the cell membrane after the action potential begins in skeletal muscle. Figure 6 indicated a highly significant (p < 0.005) negative taxis. It was impossible to determine conclusively whether the negative taxis of the T. spiralis larvae was to the potassium ions, chloride ions, or both. But, based on the negative taxis in the 95mV stimulus, it can be assumed that the larval negative taxis was to the potassium ions with their concurrent positive charge. Many investigators (Osterhout 1936a, b; Bennett, Ware, Dunn, and McIntyre 1953; White, Handler, and Smith 1973; Giese 1973; Guyton 1976) have reported that the average resting membrane potential of skeletal muscle is 95 mV, and the outside of the cell membrane is positive relative to the negative inside in a resting state. The polarity of the cell membrane reverses during the action potential (Hodgkin and Huxley 1939, 1945; Curtis and Cole 1942; Graham and Gerard 1946; Nastuk and Hodgkin 1950; Davson 1959; Giese 1973) and the average value for the action potential ranges from 100 to 120 mV (Kuffler 1942; Katz 1948; Giese 1973). Hodgkin (1951) observed that the action potential is considerably larger than the resting potential. Both Giese (1973) and Guyton (1976) reported that the outward diffusion of potassium ions during any action potential repolarizes the cell membrane. The data in Figs. 8 and 9 showed that the larvae had a positive taxis to a 120-mV stimulus simulating the action potential. The data in Figs. 6 and 7 showed that the larvae had a negative taxis to a positive charge and to a 95mV stimuhrs simulating the resting membrane potential. Considering the positive taxis of the T. spiralis lar-
372
HUGHES
PIND
vae to a 12O-mV stimulus, there is the possibility that the predilection of the larvae for skeletal muscle m’ay, be due to either the considerably higher charge of the action potential or the reversed potential of the cell membrane during the action potential (or both). If it is assumed that there are more action potentials per minute, and thus more negativity on the outside of the cell membrane per minute in more active muscles when compared to less active muscles, there is the possibility that the larval predilection for the more active muscles can be explained by this increased negativity. Also, the negative taxis of the larvae to a 95mV stimulus simulating the resting membrane potential, where the outside of the membrane is positive, can possible explain the decreased infection in less active muscles. These muscles would have more positivity on the outside of the cell membrane per minute. It is well know that larvae ‘only invade smooth and cardiac muscles in aberrant cases. Bell, Davidson, and Em&e-Smith (1972) reported that the resting and action potentials of both smooth and cardiac muscles are considerably less than in skeletal muscle. Thus, this could possibly explain the nonspecificity of the larvae for both smooth and cardiac muscle. These possibilities are not conclusively supported by data from this investigation nor are they entirely refuted. In essence they are possibilities that do exist, based on the taxes of the Trichindla spiralis firststage larvae shown in this investigation. ACKNOWLEDGMENT Supported in part by a faculty No. 03-07 from Eastern Kentucky
research Grant University.
REFERENCES BELL, G. H., DAVIDSON, J. N., AND EMSLIE-SMITH, D. 1972. “Textbook of Physiology and Biochemistry,” pp. 825-859. Williams and Wilkins, Baltimore, Maryland.
HARLEY
BENNETT, A. L., WARE, F., JR., DUNN, A. L., AND MCINTYRE, A. R. 1953. The normal membrane resting potential of mammalian skeletal muscle measured in vivo. Journal of Cellular and Comparative Physiology 42, 343-357. BONNER, T. P., AND ETGES, F. J. 1967. Chemically mediated sexual attraction in Trichinella spiralis. Experimental Parasitology 21, 53-60. BRISTOL, J., HARLEY, J. P., AND GALLICCHI.O, V. 1972. Infectivity of Trichinella spiralis (Nematoda) muscle larvae in normal vs. alloxantreated rats. Experientia 28, 102. CROLL, N. A. 1975: Behavioural analysis of nematode movement. Advances in Parasitology 13, 71-122. CURTIS, H. J., AND COLE, K. S. 1942. Membrane resting and action potentials from the squid giant axon. Journal of Cellular and Comparative Physiology 19, 135-144. DAVSON, H. 1959. “Testbook of General Physiol,> ogy. Brown, Boston, Massachusetts. DENNIS, D. T., DESPOMMIER, D. D., AND DAVIS, N. 1970. Infectivity of the newborn larva of Trichinella spiralis in the rat. journal of Parasitology 56, 974-977. FARRIS, K. N., AND HARLEY, J. P. 1977. Trichinella spiralis: Alteration of gastrocnemius muscle kinetics in the mouse. Experimental Parasitology 41,11-70. FLURY, F., AND GROLL, H. 1913. Stoffwechseluntersuchungen an trichinosen Tieren. Archiw fur Experimentelle Pathologie und Pharmakologie 73,214-232. GIESE, A. C. 1973. “Cell Physiology.” Saunders, Philadelphia, Pennsylvania. GOULD, S. E. 1970. “Trichinosis in Man and Animals.” Thomas, Springfield, Illinois. GRAHAM, J., AND GERARD, R. W. 1946. Membrane potentials and excitation of impaled single muscle fibers. Journal of Cellular and Comparative Physiology 28, 99-117. GUYTON, A. 1976. “Textbook of Medical Physiol,, ogy. Saunders, Philadelphia, Pennsylvania. HARLEY, J. P. 1972. Clarification of Trichinella spiralis (Nematode) life cycle terminology. Acta Parasitologica Polo&a 3’8, 463467. HARLEY, J. P., AND GALLICCHIO, V. 1971. Trichinellu spiralis: Migration of larvae in the rat. Experimental Parasitology 30, 11-21. HODGKIN, A. L. 1951. The ionic basis for electrical activity in nerve and muscle. Biological Review 26, 339. HODGKIN, A. L., AND HUXLEY, A. F. 1939. Action potentials recorded from inside a nerve fiber. Nature (London) 144,710-711. HODGKIN, A. L., AND HUXLEY, A. F. 1945. Resting and action potentials in single nerve fibers. Journal of Physiology 104, 176-195.
Trichimlla KATZ, B. 1948. The electrical
spiralis:
properties of the muscle fiber membrane. Proceedings of the Royal Society of Biology 135, 506-534. KUFFLER, S. W. 1942. Electric potential changes at an isolated nerve-muscle junction. Journal of Neurophysiology 5, 18-26. LARSH, J. E. 1963. Experimental trichinosis. Advances in Parasitology 1, 213-286. LEWIS, J. H. 1928. Influence of glycogen on the infection of muscle with Trichinae. Transactions of the Chicago Pathological Society 13, 12-15. NASTUK, W. L., AND HODGKIN, A. L. 1950. The electrical activity of single muscle fibers. Journal of Cellular and Comparative Physiology 35, 39-73. OGIELSKI, L. 1949. Reaction of the vascular vessels against an invasion of the larvae of Trichinella spiralis. Zoologica Poloniae 5, 3542, OSER, B. L. 1965. “Hawk’s Physiological ChemisMcGraw-Hill, New York, try,” PP. 213-232. New York. OSTERHOUT,W. J. V. 1936a. The absorption of electrolytes in large plant cells. Botanical Revieu> 2, 283. OSTERHOUT,W. J. V. 1936b. Electrical phenomena in large plant cells. Physiological Reviews 16, 216. RONALD, K. 1960. The effects of physical stimuli
LARVAL
TAXES
373
on the larval stage of Terranova decipiens. I. Temperature. Canadian Journal of Zoology 38, 623-642. RONALD, K. 1962. The effects of physical stimuli on the larval stages of Terranooa decipiens. II. Relative humidity, pressure, and gases. Canadian Jotma of Zoology 40, 1223-1227. RONALD, K. 1963. The effects of physical stimuli on the larval stage of Terranova decipiens. III. Electromagnetic spectrum and galvanotaxis. Canadian Journal of Zoology 41, 197-217. SOKAL, R. R., AND ROHLF, F. J. 1969. “Biometry.” Freeman, San Francisco, California. STAUBLI, C. 1905a. Klinische unde experimentelle Untersuchungen uber Trichinosis. Verhandlungen des Kongresses fur Innere Medizin 22, 353362. STAUBLI, C. 1905b. Klinishe und experimentelle Untersuchungen uber Trichinose und uber die Eosinophilie im allgemeinen. V&how, Archiv fur Pathologische Anatomie und Phgsiologie und fur Rlinische Medizin 85, 286-341. VOGEL, H. 1930. Ueber die Organotropie von Hepaticola hepatica. Zeithschrifi fur Parasitenkunde 2, 502-505. WHITE, A., HANDLER, P., AND SMITH, E. L. 1973. “Principles of Biochemistry.” hlcGraw-Hill, New York, New York.
