Nippostrongylus brasiliensis and Haemonchus contortus: Function of the excretory ampulla of the third-stage larva

EXPERIMENTAL

PARASITOLOGY

52,

191-198(1981)

Nippostrongylus brasiliensis and Haemonchus contortus: Function of the Excretory Ampulla of the Third-Stage Larva H. J. ATKINSON Depurtment

of Pure

und Applied

Zoology,

University

of Leeds,

Leeds

LS2 9JT, England,

U.K.

AND

C. 0. E. ONWULIRI Depurtment

of Zoology,

University

of Nigeriu,

(Accepted for publication

Nsukku,

Nigeria

12 August 1980)

ATKINSON, H. J., AND ONWULIRI, C. 0. E. 1981. Nippostrongylus brusiliensis and Huemonchus contortus: Function of the excretory ampulla of the third-stage larva. Experimental Purusitology 52, 00-00. An improved technique for measuring the water content of

nematodes is described using an electronic interferometer. Changes in phase of a laser beam passing through a known pathlength of the nematode have been used to measure the refractive index and hence the water content and relative volume of the animal. Third-stage larvae of Nippostrongylus brusiliensis and Huemonchus contortus which possess an excretory ampulla, differed from second-stage larvae lacking this ampulla in requiring a greater fall in the osmolarity of artificial tap water before there was a significant increase in their water content. Increases in the pulsation frequency of the ampulla also occurred in less hypotonic solutions than those required to increase the water content of the third-stage larvae. The ampulla pulsation frequency of third-stage larvae of N. brusiliensis increased after locomotor activity in hypotonic tap water and locomotory wave frequency of third-stage larvae of N. brusiliensis was independent of the extent of hypotonicity for a range of solutions that reduced wave propogation by its second-stage larva. The results suggest that the ampulla is an adaptation to hypotonic conditions favouring a volume homeostasis that is required for optimal locomotor activity of the third-stage infective larvae of these nematodes. INDEX DESCRIPTORS: Nippostrongylus brusiliensis; Huemonchus contortus; nematodes, parasitic; Excretory ampulla; Osmoregulation; Hypotonicity; Body water content; Microinterferometry; Locomotion; Physiology. INTRODUCTION

Weinstein (1952) cited reports of a contractile ampulla in the so-called excretory system of certain nematodes and gave a detailed description of this structure for Nippostrongylus brasifiensis (muris). He showed in the infective larvae of N. brasiliensis and Ancylostoma caninum that there is an inverse relationship between the osmolarity of hypotonic conditions and the pulsation of the ampulla. Results for Ancylostoma tubaeforme were similar but in addition the pulsation of the ampulla was

shown to be dependent on temperature and oxygen (Croll et al. 1972). The volume of water expelled by the ampulla of N. brasiliensis has been calculated from estimates of both its volume and its pulsation frequency (Weinstein 1952; Wright and Newall 1976) but the influence that this has in reducing increases in tissue hydration or body volume is not known. Many nematodes show increases in length under hypotonic conditions (Stephenson 1942; Lee 1960; Anya 1966; Croll and Viglierchio 1969; Wright and Newall 1976) but changes in volume are not readily estimated from 191 0014-4894/81/050191-08$02.00/O Copyright All rights

@ 1981 by Academic Press, Inc. of reproduction in any form reserved.

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measurements of body dimensions (Wright and Newall 1976). The water content of nematodes can be measured by interferometry (Ellenby 1968a, b) and this technique has been applied to larvae of H. contortus during dessication (Ellenby 1968~) and to Nematodirus battus during the hatching process (Perry 1977). This work describes an improved interferometric technique for measuring the water content of nematodes that has enabled changes in hydration of larval N. brasiliensis and larval H. contortus under hypotonic conditions to be measured. This approach, and some additional experiments, have enabled the function of the excretory ampulla of the third-stage larvae of N. brasiliensis and H. contortus to be examined more closely than in previous studies.

The Pulsation Frequency of Third-Stage Larvae

of the Ampulla of Both Species

Experiments used artificial tap water made up in double-distilled water as described by Greenaway (1974). Analytical grade reagents were used and the artificial tap water contained 0.35 mA4 NaCl, 0.044 mM KCl, 1.0 mM Ca(HCO& and 0.4 ti Mg(HCO,),. This solution had an osmotic pressure of about 5 mOsm and throughout this work it has been termed 100% artificial tap water (100% ATW). Other solutions were made up by diluting this primary standard in double-distilled water. All solutions were equilibrated with air to ensure that each reached pH 7.5 After at least 12 hr in 100% ATW, about 50 third-stage larvae of N. brasiliensis were transferred by fine handling needles to 10% ATW on a dry microscope slide that had MATERIALS AND METHODS been previously rinsed in ethanol and then Animals in distilled water. Each slide had a paint Larvae of Nippostrongylus brasiliensis ring to which a coverslip could be sealed were cultured in faeces taken daily from with a quick-drying paint so that a cavity rats (Wistar) 5- 12 days after their infection was formed enclosing the nematodes withwith 2000 third-stage larvae of this out compression. The pulsation rate of the nematode. The faeces were mixed with an ampulla of 20 individuals chosen at random equal volume of charcoal and cultured in was counted for 1 min at 25 C under a glass crystallizing dishes at 27 C. Second- phase-contrast microscope (Zeiss photostage larvae were recovered from this mixmicroscope II) at a magnification of 400X. ture 2 days later using a Baermann ap- The rate of pulsation of the ampullae were paratus (Anonymous 1977) and exsheathed measured at intervals of time from 5 min to third-stage larvae were collected from the 8 hr after sealing the coverslip to the slide. walls of the dishes after a further 2 days. In a second experiment, third-stage larThe larvae were stored in 100% artificial tap vae of N. brasiliensis were transferred from water for at least 12 hr at 20 C before allo100 to 20% ATW and sealed into a cavity cation to a particular experiment; surplus slide as before. After 30 min the pulsation animals were discarded after 7 days. Lar- rate of the ampullae of 10 individuals was vae of Haemonchus contortus were cul- measured just after they had been observed tured in the faeces of a lamb which had to complete a short period of activity and of been infected with 30,000 infective larvae of 11 larvae which showed prolonged activity. this species alone and reared without acciThe pulsation frequency of the ampullae dental infection with other nematodes. The of 20 third-stage larvae of both H. contortus faeces were cultured in glass jars (Kilner) at and N. brasiliensis was measured 30 min 27 C and second- and ensheathed thirdafter their transfer from 100% ATW to one stage larvae were collected as before after 3 of a series of dilutions from 90 to 10% and 6 days, respectively. These animals ATW. Similar experiments were also carwere stored as described for N. brasiliensis. ried out in which larvae were transferred to

Nippostrongylus

AND

Haemonchus:

double-distilled water, but the precise osmolarity of this solution was probably influenced to some extent by the leaching of salts from the glass slide and coverslip during the experiment. The Frequency

of Locomotory under Conditions

Waves of

N. brasiliensis Hypotonic

Groups of second- and third-stage larvae were transferred from 100% ATW to solutions ranging from 20 to 100% ATW in plastic (polystyrene) dishes for 30 min at room temperature. They were warmed to about 25 and 35 C for second- and thirdstage larvae, respectively, and viewed under a stereobinocular microscope at constant, transmitted illumination. The frequency of sinusoidal waves was estimated for 20 individuals of each stage in all solutions over a 2-min period after 30 min in the test solution. H. contortus was not used on this and some other occasions because a limited number of individuals of this species were available. Measurement of the Water the Nematodes

Content

of

EXCRETORY

AMPULLA

193

axis of the nematode was estimated using a vernier eyepiece (Olympus) previously calibrated with a stage micrometer (Zeiss). The retardation of the laser beam could also be recorded by connecting a potentiometric recorder (Servoscribe 1s) in parallel to the phase meter. An example of the phase differences obtained on the recorder as the laser beam traversed the diameter of a third-stage larva of N. brasiliensis is given in Fig. 1. Any initial value between the phase of the beam which is to pass through the nematode and a reference signal can be obtained and in Fig. 1 this was 0.5 wave. This remains constant after the start (s) of the scan until the beam begins to traverse the diameter of the nematode (d). The phase difference then approaches 1.0 wave but the instrument cannot detect values greater than this and therefore the reading appears to fall signalling the start of a second wave of retardation. The value then continues to rise but less rapidly as the rate of change of the optical pathlength decreases towards the centre of the nematode. There is sometimes a small fall in the reading when much of the pathlength is within the pseudocoelomic fluid which probably has a lower solid content than other tissues. The maximum reading is obtained at the axis of the nematode in the pharynx (Fig. 1, p). The changes in reading

Individuals were transferred from 100% ATW to a test solution into a cavity slide and measurement was made 30 min later using a microinterferometer (Vickers M86). The optical system of this equipment is unsuitable for measuring phase retardations greater than 1.0 wave (Goldstein 1977) and so its operation was modified for this work. The interference optics were adjusted and the microinterferometer was focussed on the anterior parts of pharyngeal region of the nematode using a water immersion objective of 75 and 10x ocular lenses. The manual controls of the scanning unit were FIG. 1. A potentiometric pen recording of the outadjusted in the Y axis so that the laser light source beam of 632.5 nm passed through a put of a microinterferometer. This shows changes in, the phase of a laser beam as it traverses the cross cross section of the animal. The retardation section of the pharyngeal region of the third-stage of the beam at the axis of the nematode was larva of Nippostongylus brasiliensis. Start of the scan obtained from observing the phase meter of (s); finish of the scan (f); diameter of the nematode (d); the instrument. The pathlength through the diameter of the pharynx (p).

194

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AND

are repeated as the beam passes out of the animal and the scan is completed (f). The complete scan takes about 6 set and precalibration allows the diameter (d) of the nematode to be determined directly from the recording. The water content of the nematode can be calculated using a specific refractive increment for living cells (Barer and Joseph 1954; Davies et al. 1954) in a manner similar to that described by Ellenby (1968a, b): RI = 0.6325P,,,ld,

water content (%) = 100 - (R,/0.0018), where RI is refractive index difference between the nematode and the medium (water); 0.6325 pm is the wavelength of the laser beam; P,,, is the maximum retardation of the laser beam; d (pm) is the diameter of the nematode; and 0.0018 is the specific refractive increment for living cells. For the example of a third-stage larva of N. brasifiensis in 10% ATW Fig. 1, P,,, = 1.125 waves, d = 18.0 Fm, and the water content is 78.0%. The water content of 10 individuals was measured, as described, for both second- and third-stage larvae of N. brasiliensis and H. contortus using a range of solutions from 0 to 100% ATW. RESULTS

Amp&a Pulsation Frequency of the Third-Stage Larvae of Nippostrongylus brasiliensis and Time under Hypotonic Conditions

The infrequent mean pulsation rate of the ampulla of the third-stage larva of N. brasiliensis in 100% ATW of 0.05 k 0.015 Hz (mean ? SE are given throughout the results) increased significantly to 1.59 & 0.062 and 1.77 -C 0.073 Hz after transfer to 10% ATW for 10 and 30 min, respectively (P < 0.05; sequential Q test, Snedecor and Cochran (1967)). The values gradually fell after 30 min (Fig. 2) until after 6 and 8 hr the pulsation rates were no longer greater than before transfer of the larvae to hypotonic conditions (P > 0.05). The results show that

ONWULIRI

?-

‘.i! 0

FIG. 2. Changes third-stage larva of time after transfer small sealed volume Each mean is given

4

8

Time(h)

in the ampulla Nippostrongylus

from an artificial of a 10% dilution with its standard

pulsation

of the with tap water to a of this solution. error.

hrusiliensis

changes in the pulsation of the ampulla are rapid after transfer to 10% ATW. The change is greatest after 30 min under hypotonic conditions and this has been used as a standard interval for subsequent experiments. Mechanical disturbance was not responsible for a prolonged increase in ampulla activity because this effect did not occur for larvae transferred to 100% ATW for 30 min. Ampulla Pulsation Frequency and Activity of the Third-Stuge Larvae of

N. brasiliensis The inactive larvae in 20% ATW had a mean ampulla pulsation frequency of 0.15 +- 0.04 Hz but active individuals under the same conditions showed a much higher mean rate of pulsation of 0.699 ? 0.15 Hz (t test; P < 0.01). This suggests that the pumping of the ampulla under hypotonic conditions is influenced by the activity of the nematode and so individuals showing prolonged inactivity were avoided in subsequent experiments. Amp&la Pulsation Frequency of Third-Stage Larvae of Both Species under Hypotonic Conditions

The pulsation rate of the ampulla of the third-stage larvae of N. brasiliensis in-

Nippostrongylus

AND Haemonchus: EXCRETORY

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19.5

creased as the osmolarity of the bathing medium was decreased (Fig. 3) and the pulsation rate in 55% ATW and more hypotonic conditions was significantly greater than in 100% ATW (P < 0.05; sequential Q test). Similar results were also obtained for third-stage larvae of H. contortus (Fig. 4) and in this case 50% ATW was the least hypotonic condition under which the mean rate of pulsation (1.29 k 0.11 Hz) was significantly greater than that in 100% ATW (0.35 + 0.38 Hz; P < 0.05). Frequency of Locomotory Waves of Second- and Third-Stage Larvae of N. brasiliensis under Hypotonic Conditions

Third-stage larvae were more active than the second-stage larvae in all of solutions which were tested. The relationship for both stages between the logarithm of wave frequency (I’) and the logarithm of osmolarity (X) of the solution was fitted using regression analysis (Snedecor and Cochran 1967) and the curves fitted from this trans-

FIG. 3. Changes in the relative body volume for second-stage larvae of Nippostrongylus brusiliensis 30 min after transfer from artificial tap water (100% ATW) to percentage dilutions of this solution. Similar results are given for the third-stage larvae of this nematode together with changes in the pulsation frequency of its ampulla under these conditions. Mean and SE for the ampulla pulsation frequency of the third-stage larvae (0). Relative volume of secondstage larvae (B) and third-stage larvae (0) have been calculated from changes in water content (see text). The smallest significant increase in volume or ampulla pulsation rate from that in 100% ATW (a).

FIG. 4. Changes in relative body volume for second-stage larvae of Huemonchus c~ntortus 30 min after transfer from artificial tap water to percentage dilutions of this solution. Similar results are given for the third-stage larvae of this nematode and also changes in the pulsation frequency of its ampulla under these conditions. See legend to Fig. 3 for further details.

formation have been plotted in Fig. 5. The curve for second-stage larvae showed a significant change with osmolarity (P < 0.05; t test with a null hypothesis for the slope) but the same approach showed that the locomotion of the third-stage larvae was independent of the dilution of the tap water. Covariance analysis of the two regression lines confirmed this difference in the response of the two stages of N. brasiliensis to hypotonic conditions. (P < 0.05).

FIG. 5. Changes in frequency of locomotory waves of second- (m) and third-stage (0) larvae of Nipposrrongylus brasiliensis after transfer from artificial tap water to percentage dilutions of this solution. Each mean is given with its SE and the curves have been fitted to the data by regression analysis after logarithmic transformation of both axes.

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ATKINSON

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Chnnges in Body Volume under Hypotonic Conditions The mean percentage water content of secondand third-stage larvae of N. brasiliensis increased progressively with increasing hypotonicity of the tap water, with a total change from 77.0 ? 0.47 and 73.4 ? 0.33 in 100% ATW to 82.51 + 0.80 and 81.7 ? 0.56 in distilled water for second-stage and the third-stage larvae, respectively. However, the change for the third stage in the less hypotonic solutions was less than for the second stage, with a significant change occurring first in 50% for the second-stage and 20% for the thirdstage larvae (P < 0.05; sequential Q test). Assuming no change in body solids during the experiments, the change in ratio of solids:water on a weight-to-weight basis can be used to estimate changes in volume once allowance has been made for the specific volume of 0.75 for protoplasmic solids (Barer and Joseph 1954; Hale 1958). Estimates of changes in volume on this basis are given for N. brasiliensis in Fig. 3. These results show that the change in volume was less for third-stage than for second-stage larvae except under the extremely hypotonic conditions. The results for H. contortus were similar to those for N. brasifiensis. The mean water content of the third-stage larvae increased from 71.81 + 0.34 in 100% ATW to 75.89 t 0.51 in distilled water and the corresponding values for the second-stage larvae were 74.08 + 0.31 and 76.75 ? 0.50, respectively. Except under extremely hypotonic conditions, the water content of third-stage larvae was less affected by hypotonic conditions than that of the second-stage larvae, with a significant increase in water content first occurring at 30 and 60% ATW, respectively (P < 0.05; sequential Q test). The results are plotted as changes in body volume in Fig. 4 and they are similar to those for N. brasiliensis except that the changes in volume are greater for the latter species.

ONWULIRI

DISCUSSION The estimated water content of thirdstage larvae in 100% ATW was comparable to values for nematodes obtained previously using an interferometer and a fringe field eyepiece (Ellenby 1968a, b) including value in tap water of about 75% for the infective stage of Haemonchus contortus (Ellenby 1968~) and 74.5% for Nematodirus battus (Perry 1977). The results suggest that the new technique is at least as accurate as the earlier approach and it has the advantage of being more rapid because it does not require subsequent processing of photographs. The pulsation frequency of the ampulla of the third-stage larva of Nippostrongylus brusiliensis increased soon after transfer of the animals to hypotonic conditions (Fig. 2). The subsequent reduction of pulsation rate with time could be related to osmoregulation but comparison with previous results for Ancylostoma tubaeforme obtained by Croll et uf. (1972) suggests that it may have resulted from lack of oxygen after sealing the nematodes into a cavity slide. Croll et al. (1972) also concluded that mobility was an essential part of the mechanism of water expulsion in the hypotonic osmoregulation of A. tubaeforme. The results for N. brusiliensis suggest a relationship between activity and the pulsation of the ampulla but it seems more likely that the expulsion of water by the ampulla facilitates locomotion rather than vice versa. Changes in volume were less for third-stage larvae of both species than for their second stage except under extreme conditions where further reductions in osmolarity were not matched adequately by additional increases in the pulsation frequency of the ampulla. It seems that under these conditions, the ampulla was unable to cope fully with the influx of water and consequently hydration increased. Assuming body length and radius increase in proportion with body volume, it can be shown by a standard for-

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mula (Andrassy 195611967) that the vol- activity for rapid penetration of cat skin umes in 0% ATW correspond to an increase (Croll and Matthews 1972; Matthews 1975). in length of about 15 and 6% for third-stage It seems that the pulsating ampulla is an larvae of N. brasiliensis and H. contortus, homeostatic adaptation shared by third-s but by few respectively. Both of these estimates are stage larvae of trichostongyles other nematodes. The continual expulsion within the range of length changes recorded under similar conditions for animal para- of water shortly after exposure to hypositic nematodes (Lee 1960; Anya 1966). The tonic conditions lessens the volume change of the nematode sufficiently to leave its difference in volume changes for the thirdstage larvae may be correlated with the locomotion unimpaired by these condipresence of a sheath in H. contortus but not tions. Further work is necessary to study N. brasiliensis. The sheath may mechanithe mechanism of filling of the ampulla and cally oppose length changes or possibly the control of its pulsation. This may prove alter the permeability of the animal to to be similar to the contractile vacuole of water. protozoa such as Pelomyxa carolinensis in An increase in body volume would be which the vacuole apparently grows while a constant hypotonicity relaexpected to increase the length of most maintaining nematodes (Lee 1972; Lee and Atkinson tive to the cytoplasm (Riddick 1968). 1976) and this may stretch the longitudinal ACKNOWLEDGMENTS muscles sufficiently to impair their function We thank the British Council and the University of in locomotion. It is possible that nematodes Nigeria, Nsukka, for tinancing the visit of C.O.E.O. to may also partly oppose the influx of water Leeds, Professors D. L. Lee and A. 0. Anya for their by increasing their muscle tone and their support, and Dr. R. J. Thomas for providing H. conhydrostatic pressure. Second-stage larvae tortus. The techniques were developed for work of N. brasiliensis and H. contortus, and financed by the Agricultural Research Council many other nematodes, may respond in (AG241122). these ways to an initial exposure to hypoREFERENCES tonic conditions with a resultant effect ANDRASSY, I. 1956/1967. The determination of volume on wave propagation for locomotion. Norand weight of nematodes. In “English Translations mally this may be of little consequence to of Selected East European Papers in Nematology” the second stage of the two trichostrongyles (B. M. Zuckerman, M. W. Brzeski, and K. H. because as faecal dwellers they are unlikely Deubert, eds.). Univ. of Massachusetts Press, East Wareham. to experience a rapid fall in external osmotAnonymous. 1977. In “Manual of Veterinary ic pressure and they are also a feeding Parasitological Laboratory Techniques,” Ministry rather than dispersal stage of the life cycle. of Agriculture, Fisheries and Food Technical BulleHowever, the infective third-stage larvae of tin 18. H. M. Stationery Offtce, London. these two species, and related nematodes, ANYA, A. 0. 1966. Investigations on osmotic regulations in the parasitic nematode Aspiculuris fetrccpteru are migratory animals and may experience Shulz. Prrrositology 56, 583-588. a rapid fall in osmolarity on the soil surBARER, R., AND JOSEPH, A. 1954. Refractometry of face or on foliage during, or after, rainfall. living cells. Quurtrrly Journal of Microscopicul SciA loss of locomotor efficiency at these ence 95, 399-423. these times may lessen their opportunity for CROLL, N. A., AND VIGLIERCHIO, D. A. 1969. Osmoregulation and the uptake of ions in a marine dispersal. It may also extend the hazardous nematode. Proceedings of the Helminthologicnl Soperiod on the; surface of the host for skinciety of Washington 31, l-9. penetrating species such as N. brasifiensis CROLL, N. A., AND MATTHEWS, B. E. 1972. Activity and A. tubaeforme. The latter species is ageing and penetration of hookworm larvae. known to require a high level of locomotor Parasitology 66, 279-289.

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