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Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus
‘C 1982
0020-7519/82/060503-05$03.CW0 Pergamon Press Ltd. Society for Parasrrology
Auslralian
CHANGES IN OPTICAL PATH DIFFERENCE IN THE OESOPHAGEAL REGION AND THE EXCRETORY CELLS DURING EXSHEATHMENT IN HAEMONCHUS CONTORTUS and R. I. SOMMERVILLE
K. G. DAVEY* Department
of Zoology,
University
of Adelaide,
Adelaide,
S.A.,
Australia
(Received 22 March 1982) Abs1rac1-DAvEY K. G. and SOMMERVILLE R. 1. 1982. Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Huernonchus contortus. Inrernntional Journalfor Parasitology 12: 503-507. Changes in the optical path difference (opd) between various parts of the worm and the medium in which the worms were immersed were determined by quantitative interference microscopy. The opd of the oesophagus and the excretory cells both increased upon stimulation of the worms with CO, at 38.5”C, suggesting a decrease in volume of those structures. The oesophagus decreased markedly in length and slightly in diameter, yielding a decrease in volume of approximately 15 to 17 pi. Desheathing the worms with NaOCl produced changes in the oesophagus but not the excretory cells. This confirms previous findings that exsheathment involves at least two parallel processes, both of which are initiated by CO, and only one of which is stimulated by exposure to NaOCl. INDEX
KEY WORDS:
Nematode
development;
water content;
*Present
address: Downsview,
Department Ontario,
of Biology, M3J 1P3 Canada.
microscopy.
MATERIALS
INTRODUCTION
EARLIER PAPER (Davey & Rogers, 1982) demonstrated that Huemonchus lost water and volume during exsheathment. These losses were not exclusively associated with the sheath and occurred in two phases, both of which were set in train by exposure to CO, at 38”C, but only one of which was induced by desheathing in NaOCl. The present paper seeks to identify the sources of these losses by employing quantitative interference microscopy to measure changes in the optical path difference of various regions of the worms. The optical path difference (opd) of the light passing through an object compared to the medium in which it is immersed can be measured in the quantitative interference microscope. For biological materials, opd is proportional to the concentration of materials in the object (Davies, 1958). It therefore follows that changes in the opd will reflect changes in concentration and if changes in concentration are not the result of synthesis of additional material, then changes in opd will be inversely proportional to changes in volume. These considerations have !ed to the use of the interference microscope in determining changes in volumes of follicle cells in insects (AbuHakima and Davey, 1977). Interference microscopy has also been used to determine water content in nematodes (Ellenby, 1968 a,b). AN
University,
interference
AND METHODS
The worms were maintained and treated with CO, for exsheathing or with NaOCl for desheathing as described previously (Davey & Rogers, 1982). The opd was measured using the Jamin-Lebedeff system as manufactured by Carl Zeiss (Carl Zeiss Canada Ltd., Toronto). In this instrument, measuring and reference beams are polarized and the measurement of the opd emerges in terms of degrees of rotation of a polarising analyser. This reading, R, is directly proportional to the opd, such that opd = R x &, where L represents the wavelength of the light used. The system was illuminated by a tungsten light source through an interference filter at 546 nm. The data presented in this paper are the readings of R in degrees and have not been converted to other units. It is important to emphasise that the data deal with changes in the opd, rather than with absolute values. The JaminLebedeff system permits an arbitrary setting for the 0 position and one complete revolution (360”) of the analyser is equivalent to an opd of 1 1. The opd between the worm and the aqueous medium is certainly greater than 1 A,due to the thickness of the worm. Comparison of the interference colours generated by the worm with a Michel-Levy colour chart (Carl Zeiss, Toronto, Canada) suggested that at least a second order and possibly a third order spectrum was produced, indicating a total opd of at least 2 I; a similar total opd is suggested by the interference microscopy observations of Ellenby (1968b). For the purposes to which the measurements are put in this paper, the absolute values of opd are of no consequence, since changes of less than 1 A are involved. Where values of opd are quoted, it should be understood that the 0 value has been arbitrarily set. While the changes that are observed are too rapid to involve synthesis and must therefore involve changes in fluid
York
503
K. G.
504
DAVEY
I.J.P. VOL. 12. 1982
and R. I. SOMMERVILLE
volume, it is important to realise that changes in thickness of the specimen will alter the opd. The diameter of the worm does indeed change by about 15% during exsheathment (Davey & Rogers, 1982), but it should be noted that a decrease in thickness of the specimen will tend to decrease the opd. Since increases in the opd are in fact observed, assuming that the thickness remains constant tends to strengthen the validity of the observations (AbuHakima & Davey, 1977). Where values of “P” are quoted, these have been determined by simple “t” tests. Preliminary observations demonstrated that changes in opd which could be correlated with the exsheathing process were observed in only two areas: the oesophagus and the excretory system. No other systematic changes in the opd occurred in other tissues. In particular, the opd of the intestinal or central region of the worm did not alter during
exsheathment. Observations on the oesophageal region were carried out under a 10 x objective, for a total magnification of 125 x. To observe the excretory cells, it was necessary to use a 40 x or 100 x (oil immersion) objective, for a total magnification of 500 x or 1250 x respectively. RESULTS
Changes in opd in the oesophageal region The “oesophageal region” of the worm is here defined as that region of the worm from the anterior end to the base of the oesophagus. In general changes in opd were uniform throughout this region when viewed through the 10 x objective. When larvae were suspended in 0.05 M sodium borate in 0.9% sodium chloride and exposed to CO,at 38.5”C, theopd of the oesophageal region increased (Fig. 1). This change was clearly associated with exsheathment and showed its sharpest increase at a time when many worms were exsheathing. Moreover, treated worms could be divided into three populations on the basis of the condition of their sheaths. Those worms which, as a result of exposure to CO, at 38*5”C, exsheathed, exhibited an
opd of 126 ? 6.5 (SEM; n = 18). Those treated worms which failed to ecdyse and over which the sheath remained tightly adherent, particularly at the anterior end, exhibited an opd of 66k3.0 (n = 29), a figure which is not markedly different from untreated larvae (57k2.8). Some worms failed to ecdyse, but exhibited a loosening of the cuticle, particularly at the anterior end. These exhibited an opd of 103 * 3.6 (n = 30), a figure which is considerably greater than that for untreated worms. When worms were desheathed by exposure to NaOCl, there was an increase in opd. Thus, for the batch of worms used in this experiment, the opd of the oesophageal region of untreated worms was 49 -t4.4 (n = 15). For 15 worms desheathed by exposure to 0.1% NaOCl for 10 min, the opd was 104 ? 4.0. Some worms treated with NaOCl failed to lose their sheaths. Of these, some had “loose” cuticles as described above; the opd of 15 such worms was 67 f 5.7, a relatively small but significant increase (0.05 > P > 0.01). Others had cuticles which had not become loose; the opd for 15 such worms was 55 f 3.5”. This increase is not significant (P > 0.05). Does the opd of worms desheathed by exposure to NaOCl further increase when the worms are subjected to an exsheathing stimulus of CO, at 38.5”C? The opd of 15 untreated worms was 47 ? 3.3. For 15 worms from the same batch desheathed by exposure to 0.1% NaOCl for 10 min, the opd for the oesophageal region rose to 93 * 5.1. For 15 desheathed worms which had been treated with CO, at 38.5”C for 10 min and held at 38.5”C for 2 h, the opd was 97 + 3.7, a value which does not differ significantly from desheathed, unstimulated worms (P> 0.1). For 15 worms exsheathed by CO, without prior exposure to NaOCl, the opd of the
140
I20 i
0
t 20
40
60 Tlmr
80
I
120
00
of exposure
to
CO,
140
160
180
2cQ
(mid
FIG. 1. The time-course of increase in opd of the oesophageal region of Haemonchus contortus exposed to CO, at 38.5”C. Each point is the mean of determinations of 15 worms and the vertical lines indicate the S.E. of the means.
I.J.P. VOL.12. 1982
Optical path difference and exsheathment
oesophageal region was 108 + 4.9, a level which is not significantly different from that of worms desheathed by NaOCl (P> 0.05).
Changes in the anatomy of the oesoDhagus The lumen of the oesophagus of an unstimulated worm follows an approximately straight course (Fig. 2). On the other hand, in worms exsheathed by CO* or desheathed by NaOCl, the lumen develops a threedimensional kink (Fig. 3). This implies a decrease in length of the oesophagus. Using procedures outlined in an earlier paper (Davey & Rogers, 1982), the length of the oesophagus was determined for normal larvae, desheathed larvae and exsheathed larvae. For 5 unstimulated larvae the length was 161 ? 5.0 pm. For 4 larvae desheathed by NaOCl, the length had decreased to 142 +- 2.0 and for 4 larvae exsheathed by COz, the figure was 140 ‘_ 3.7.
Changes in the volume of the oesophagus For purposes of calculating the volume, the oesophagus can be treated as a cylinder. The diameter of the oesophagus of worms subjected to from various treatments determined was photographs as previously described (Davey & Rogers, 1982) and found to be 17 ? 0.4 m for 5 untreated worms, 13 * 0.8 pm for 5 NaOCl-treated worms and 14 + O-7 pm for 4 worms exsheathed by CO,. Using the lengths as determined above, the volumes of the oesophagus therefore became 37 pl for unstimulated worms, 19 pl for worms desheathed by NaOCl and 22 pl for worms exsheathed by CO,. Thus, the changes in linear dimensions suggest that 15-18 pl of volume was lost from the oesophagus as result of exsheathment with CO, or desheathing with NaOCl.
Changes in opd of the excretory cells The
excretory cells can be identified in living under the 100x objective (Fig. 4). There was a marked increase in opd in the excretory cells soon after stimulation with CO, at 38.5”C (Fig. 5). Because the excretory cells are more difficult to visualise than the oesophagus, it was not possible to accumulate data earlier than 15 min after exposure to COz. Nevertheless, it is clear that the increase in opd of the excretory cells follows a time-course very much like that in the oesophagus and that the change is complete at about the time that exsheathment is achieved in most worms. The mean opd for the excretory cells before stimulation was 55 k2.2 (n = 34), while the mean opd of exsheathed worms which had been stimulated at least 60 min previously was 132 rt 1.6 (n = 38). Worms which remained ensheathed after 60 min, regardless of whether they possessed “tight” or “loose” sheaths failed to exhibit the same marked increase in opd. For 13 such worms, the opd was 53 * 6.2. Worms exposed to CO* at room temperature yielded an opd of 49 24.3 (n = 16), and those exposed to 38.5”C without CO2 had an opd of 57 ? 3.6 (n = 13).
Haemonchus
505
Worms desheathed by exposure to NaOCl failed to exhibit the increase in opd which is characteristic of worms activated by CO1. Thus 15 worms desheathed by NaOCl exhibited a mean opd for the excretory cells of 51 f 4.9. For 14 worms from the same batch which were desheathed by NaOCl, activated by CO, and incubated at 38.5”C for 60 min, the opd was 133 ? 2.7. DISCUSSION
The data presented here are consistent with the conclusions reached in an earlier paper (Davey & Rogers, 1982). It is clear that stimulation with CO2 at 38.5”C leads to increases in the opd in the oesophageal region and in the excretory cells and that these increases have a time course which is consistent with their possible involvement in the exsheathment process. Moreover, desheathing by treatment with NaOCl produces the same changes in the oesophageal region, but does not affect the excretory cell. Thus, an independent experimental approach confirms that there are two parallel processes set in train by activation with CO, at 38.5”C and that only one of these processes is mimicked by exposure to NaOCl. The changes in opd and conformation of the oesophagus suggest strongly that the loss of fluid from the larva as a result of NaOCl treatment, described in the earlier paper (Davey & Rogers, 1982), emanates from the oesophagus. This is also suggested by the fact that the calculated reduction in the volume of the oesophagus, which is in the range of 15-18 pl, coincides closely with the estimated reduction in fluid volume of the larva of 17-20 pl calculated in the earlier paper (Davey & Rogers, 1982). The possible involvement of the oesophagus in the exsheathment process in Haemonchus gives further weight to the suggestion that the pharyngeal glands might secrete components of a moulting fluid in Caenorhabditis (Singh & Sulston, 1978). The changes in the excretory cells are not surprising in view of the involvement of that structure in the ecdysial process in Phocanema (Davey & Kan, 1968). While it is true that the earliest events in the excretory cell of Phocanema involve an increase in water content (Davey, 1979), there must be an eventual reduction in volume associated with the secretion of material from the gland. The fact that the excretory cell constitutes only a small part of the light path through the whole nematode renders precise quantitation of the change in volume of the cells difficult. About all that can be said is that their opd increases. Since changes in the surrounding tissues of the worm have never been observed, the changes observed in the opd of the excretory cells are due to changes in the cells. The excretory system of Haemonchus has yet to be described in detail, but examination of unpublished electron micrographs provided by Prof. W. P. Rogers has revealed that the dimensions of the excretory cells are simply too small
I.J.P. VOL. 12. 1982
K. G. DAVEY and R. 1. S~M~ERVILLE
FIG. 2. Photograph
of the oesophageal
region of an untreated larva. oesophagus is relatively straight.
Note that the lumen,
1, of the
FIG 3. As in Fig. 2, but from an animal stimulated by CO2 at 38.5”C. Note the kink (arrow) in the lumen, 1 of the oesophagus. Because the kink is three-dimensional, some of it lies out of the plane of focus of the rest of the lumen. The course followed by the lumen is indicated by the solid black line above the figure. FIG. 4. Photograph
to show the excretory
cells (arrow) as seen in the interference excretory duct (d).
microscope.
Note
I.J.P.
VOL.
Optical path difference and exsh~thment
12. 1982
0
507
I
2x2
40
Bo
60
Time of cxposun
120
100 to
140
160
180
200
COafminl
FIG. 5. The time-course of increase in opd of the excretory cells of Haemonrhus confortus exposed to CO, at 389C. Each point is the mean of determinations on 15 worms and the vertical lines indicate the S.E. of the means.
to account for the additional reduction in fluid volume of 15 pl experienced by a larva activated by CO2 as compared to one desheathed by NaOCI. While precise measurements were not made, each excretory cell is no more than 2 m in diameter, suggesting that each cell would have to be approximately 2.5 mm long in order to accommodate 15 pl. Thus, while the excretory cells are potentially implicated in exsheathment in Huemonchus, they are not in themselves the source of the remaining water loss. Either there is another, as yet unidentified, source contributing to fluid loss, or the fluid involved passes through the excretory system. The absence of detailed information on the structure of the excretory cells and their relationship to the excretory duct in Haemonchus renders difficult the interpretation of the data presented here.
Acknowledgements-These experiments were carried out while KGD, on study leave from York University, was the recipient of a Distinguished Visiting Scholarship from the Universiry of Adelaide. The research was supported by the Australian Research Grants Committee and the Natural Sciences and Engineering Research Council of Canada.
REFERENCES ABU-HAKIMAR, & DAVEYK. G. 1977. The action of juvenile hormone on the follicle cells of ~~o~~~~~ prolixta: the importance of volume changes. Journal of Experimental Bioiogy 69: 33-44. DAVEYK. 0. 1979. Molting in a parasitic nematode, Phocanema decipiens: the role of water uptake. International Journal for Parasitology 9: 121-125. DAVEYK. G. & KAN S. P. 1968. Molting in a parasitic nematode, Phocanema decipiens IV. Ecdysis and its control. Canadian Journal of Zoology 46: 893-898. DAVEYK. G. & ROGERSW. P. 1982. Changes in water content and volume accompanying exsheathment of Haemonchus contortus. International Journal for Parasitology 12: 93-96. DAVIESH. G. 1958. The determination of mass and concentration by microscope interferometry. In Generai Cytochemical Merhods (Edited by DANIELLIJ. F.) Academic Press, New York. ELLENBYC. 1968a. Determination of the water content of nematode worms by interference microscopy. Experientia 24: 84-85. ELLENBYC. 196813.Desiccation survival of the infective larva of Haemonchus contortus. Journal of Experimen&at Biology 49: 469-475. SINCH R. N. d SULSTONJ. E. 1978. Some observations on moulting in ~aenorha~dit~ etegans. Nematoiogica 24: 63-71.
0020-7519/82/060503-05$03.CW0 Pergamon Press Ltd. Society for Parasrrology
Auslralian
CHANGES IN OPTICAL PATH DIFFERENCE IN THE OESOPHAGEAL REGION AND THE EXCRETORY CELLS DURING EXSHEATHMENT IN HAEMONCHUS CONTORTUS and R. I. SOMMERVILLE
K. G. DAVEY* Department
of Zoology,
University
of Adelaide,
Adelaide,
S.A.,
Australia
(Received 22 March 1982) Abs1rac1-DAvEY K. G. and SOMMERVILLE R. 1. 1982. Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Huernonchus contortus. Inrernntional Journalfor Parasitology 12: 503-507. Changes in the optical path difference (opd) between various parts of the worm and the medium in which the worms were immersed were determined by quantitative interference microscopy. The opd of the oesophagus and the excretory cells both increased upon stimulation of the worms with CO, at 38.5”C, suggesting a decrease in volume of those structures. The oesophagus decreased markedly in length and slightly in diameter, yielding a decrease in volume of approximately 15 to 17 pi. Desheathing the worms with NaOCl produced changes in the oesophagus but not the excretory cells. This confirms previous findings that exsheathment involves at least two parallel processes, both of which are initiated by CO, and only one of which is stimulated by exposure to NaOCl. INDEX
KEY WORDS:
Nematode
development;
water content;
*Present
address: Downsview,
Department Ontario,
of Biology, M3J 1P3 Canada.
microscopy.
MATERIALS
INTRODUCTION
EARLIER PAPER (Davey & Rogers, 1982) demonstrated that Huemonchus lost water and volume during exsheathment. These losses were not exclusively associated with the sheath and occurred in two phases, both of which were set in train by exposure to CO, at 38”C, but only one of which was induced by desheathing in NaOCl. The present paper seeks to identify the sources of these losses by employing quantitative interference microscopy to measure changes in the optical path difference of various regions of the worms. The optical path difference (opd) of the light passing through an object compared to the medium in which it is immersed can be measured in the quantitative interference microscope. For biological materials, opd is proportional to the concentration of materials in the object (Davies, 1958). It therefore follows that changes in the opd will reflect changes in concentration and if changes in concentration are not the result of synthesis of additional material, then changes in opd will be inversely proportional to changes in volume. These considerations have !ed to the use of the interference microscope in determining changes in volumes of follicle cells in insects (AbuHakima and Davey, 1977). Interference microscopy has also been used to determine water content in nematodes (Ellenby, 1968 a,b). AN
University,
interference
AND METHODS
The worms were maintained and treated with CO, for exsheathing or with NaOCl for desheathing as described previously (Davey & Rogers, 1982). The opd was measured using the Jamin-Lebedeff system as manufactured by Carl Zeiss (Carl Zeiss Canada Ltd., Toronto). In this instrument, measuring and reference beams are polarized and the measurement of the opd emerges in terms of degrees of rotation of a polarising analyser. This reading, R, is directly proportional to the opd, such that opd = R x &, where L represents the wavelength of the light used. The system was illuminated by a tungsten light source through an interference filter at 546 nm. The data presented in this paper are the readings of R in degrees and have not been converted to other units. It is important to emphasise that the data deal with changes in the opd, rather than with absolute values. The JaminLebedeff system permits an arbitrary setting for the 0 position and one complete revolution (360”) of the analyser is equivalent to an opd of 1 1. The opd between the worm and the aqueous medium is certainly greater than 1 A,due to the thickness of the worm. Comparison of the interference colours generated by the worm with a Michel-Levy colour chart (Carl Zeiss, Toronto, Canada) suggested that at least a second order and possibly a third order spectrum was produced, indicating a total opd of at least 2 I; a similar total opd is suggested by the interference microscopy observations of Ellenby (1968b). For the purposes to which the measurements are put in this paper, the absolute values of opd are of no consequence, since changes of less than 1 A are involved. Where values of opd are quoted, it should be understood that the 0 value has been arbitrarily set. While the changes that are observed are too rapid to involve synthesis and must therefore involve changes in fluid
York
503
K. G.
504
DAVEY
I.J.P. VOL. 12. 1982
and R. I. SOMMERVILLE
volume, it is important to realise that changes in thickness of the specimen will alter the opd. The diameter of the worm does indeed change by about 15% during exsheathment (Davey & Rogers, 1982), but it should be noted that a decrease in thickness of the specimen will tend to decrease the opd. Since increases in the opd are in fact observed, assuming that the thickness remains constant tends to strengthen the validity of the observations (AbuHakima & Davey, 1977). Where values of “P” are quoted, these have been determined by simple “t” tests. Preliminary observations demonstrated that changes in opd which could be correlated with the exsheathing process were observed in only two areas: the oesophagus and the excretory system. No other systematic changes in the opd occurred in other tissues. In particular, the opd of the intestinal or central region of the worm did not alter during
exsheathment. Observations on the oesophageal region were carried out under a 10 x objective, for a total magnification of 125 x. To observe the excretory cells, it was necessary to use a 40 x or 100 x (oil immersion) objective, for a total magnification of 500 x or 1250 x respectively. RESULTS
Changes in opd in the oesophageal region The “oesophageal region” of the worm is here defined as that region of the worm from the anterior end to the base of the oesophagus. In general changes in opd were uniform throughout this region when viewed through the 10 x objective. When larvae were suspended in 0.05 M sodium borate in 0.9% sodium chloride and exposed to CO,at 38.5”C, theopd of the oesophageal region increased (Fig. 1). This change was clearly associated with exsheathment and showed its sharpest increase at a time when many worms were exsheathing. Moreover, treated worms could be divided into three populations on the basis of the condition of their sheaths. Those worms which, as a result of exposure to CO, at 38*5”C, exsheathed, exhibited an
opd of 126 ? 6.5 (SEM; n = 18). Those treated worms which failed to ecdyse and over which the sheath remained tightly adherent, particularly at the anterior end, exhibited an opd of 66k3.0 (n = 29), a figure which is not markedly different from untreated larvae (57k2.8). Some worms failed to ecdyse, but exhibited a loosening of the cuticle, particularly at the anterior end. These exhibited an opd of 103 * 3.6 (n = 30), a figure which is considerably greater than that for untreated worms. When worms were desheathed by exposure to NaOCl, there was an increase in opd. Thus, for the batch of worms used in this experiment, the opd of the oesophageal region of untreated worms was 49 -t4.4 (n = 15). For 15 worms desheathed by exposure to 0.1% NaOCl for 10 min, the opd was 104 ? 4.0. Some worms treated with NaOCl failed to lose their sheaths. Of these, some had “loose” cuticles as described above; the opd of 15 such worms was 67 f 5.7, a relatively small but significant increase (0.05 > P > 0.01). Others had cuticles which had not become loose; the opd for 15 such worms was 55 f 3.5”. This increase is not significant (P > 0.05). Does the opd of worms desheathed by exposure to NaOCl further increase when the worms are subjected to an exsheathing stimulus of CO, at 38.5”C? The opd of 15 untreated worms was 47 ? 3.3. For 15 worms from the same batch desheathed by exposure to 0.1% NaOCl for 10 min, the opd for the oesophageal region rose to 93 * 5.1. For 15 desheathed worms which had been treated with CO, at 38.5”C for 10 min and held at 38.5”C for 2 h, the opd was 97 + 3.7, a value which does not differ significantly from desheathed, unstimulated worms (P> 0.1). For 15 worms exsheathed by CO, without prior exposure to NaOCl, the opd of the
140
I20 i
0
t 20
40
60 Tlmr
80
I
120
00
of exposure
to
CO,
140
160
180
2cQ
(mid
FIG. 1. The time-course of increase in opd of the oesophageal region of Haemonchus contortus exposed to CO, at 38.5”C. Each point is the mean of determinations of 15 worms and the vertical lines indicate the S.E. of the means.
I.J.P. VOL.12. 1982
Optical path difference and exsheathment
oesophageal region was 108 + 4.9, a level which is not significantly different from that of worms desheathed by NaOCl (P> 0.05).
Changes in the anatomy of the oesoDhagus The lumen of the oesophagus of an unstimulated worm follows an approximately straight course (Fig. 2). On the other hand, in worms exsheathed by CO* or desheathed by NaOCl, the lumen develops a threedimensional kink (Fig. 3). This implies a decrease in length of the oesophagus. Using procedures outlined in an earlier paper (Davey & Rogers, 1982), the length of the oesophagus was determined for normal larvae, desheathed larvae and exsheathed larvae. For 5 unstimulated larvae the length was 161 ? 5.0 pm. For 4 larvae desheathed by NaOCl, the length had decreased to 142 +- 2.0 and for 4 larvae exsheathed by COz, the figure was 140 ‘_ 3.7.
Changes in the volume of the oesophagus For purposes of calculating the volume, the oesophagus can be treated as a cylinder. The diameter of the oesophagus of worms subjected to from various treatments determined was photographs as previously described (Davey & Rogers, 1982) and found to be 17 ? 0.4 m for 5 untreated worms, 13 * 0.8 pm for 5 NaOCl-treated worms and 14 + O-7 pm for 4 worms exsheathed by CO,. Using the lengths as determined above, the volumes of the oesophagus therefore became 37 pl for unstimulated worms, 19 pl for worms desheathed by NaOCl and 22 pl for worms exsheathed by CO,. Thus, the changes in linear dimensions suggest that 15-18 pl of volume was lost from the oesophagus as result of exsheathment with CO, or desheathing with NaOCl.
Changes in opd of the excretory cells The
excretory cells can be identified in living under the 100x objective (Fig. 4). There was a marked increase in opd in the excretory cells soon after stimulation with CO, at 38.5”C (Fig. 5). Because the excretory cells are more difficult to visualise than the oesophagus, it was not possible to accumulate data earlier than 15 min after exposure to COz. Nevertheless, it is clear that the increase in opd of the excretory cells follows a time-course very much like that in the oesophagus and that the change is complete at about the time that exsheathment is achieved in most worms. The mean opd for the excretory cells before stimulation was 55 k2.2 (n = 34), while the mean opd of exsheathed worms which had been stimulated at least 60 min previously was 132 rt 1.6 (n = 38). Worms which remained ensheathed after 60 min, regardless of whether they possessed “tight” or “loose” sheaths failed to exhibit the same marked increase in opd. For 13 such worms, the opd was 53 * 6.2. Worms exposed to CO* at room temperature yielded an opd of 49 24.3 (n = 16), and those exposed to 38.5”C without CO2 had an opd of 57 ? 3.6 (n = 13).
Haemonchus
505
Worms desheathed by exposure to NaOCl failed to exhibit the increase in opd which is characteristic of worms activated by CO1. Thus 15 worms desheathed by NaOCl exhibited a mean opd for the excretory cells of 51 f 4.9. For 14 worms from the same batch which were desheathed by NaOCl, activated by CO, and incubated at 38.5”C for 60 min, the opd was 133 ? 2.7. DISCUSSION
The data presented here are consistent with the conclusions reached in an earlier paper (Davey & Rogers, 1982). It is clear that stimulation with CO2 at 38.5”C leads to increases in the opd in the oesophageal region and in the excretory cells and that these increases have a time course which is consistent with their possible involvement in the exsheathment process. Moreover, desheathing by treatment with NaOCl produces the same changes in the oesophageal region, but does not affect the excretory cell. Thus, an independent experimental approach confirms that there are two parallel processes set in train by activation with CO, at 38.5”C and that only one of these processes is mimicked by exposure to NaOCl. The changes in opd and conformation of the oesophagus suggest strongly that the loss of fluid from the larva as a result of NaOCl treatment, described in the earlier paper (Davey & Rogers, 1982), emanates from the oesophagus. This is also suggested by the fact that the calculated reduction in the volume of the oesophagus, which is in the range of 15-18 pl, coincides closely with the estimated reduction in fluid volume of the larva of 17-20 pl calculated in the earlier paper (Davey & Rogers, 1982). The possible involvement of the oesophagus in the exsheathment process in Haemonchus gives further weight to the suggestion that the pharyngeal glands might secrete components of a moulting fluid in Caenorhabditis (Singh & Sulston, 1978). The changes in the excretory cells are not surprising in view of the involvement of that structure in the ecdysial process in Phocanema (Davey & Kan, 1968). While it is true that the earliest events in the excretory cell of Phocanema involve an increase in water content (Davey, 1979), there must be an eventual reduction in volume associated with the secretion of material from the gland. The fact that the excretory cell constitutes only a small part of the light path through the whole nematode renders precise quantitation of the change in volume of the cells difficult. About all that can be said is that their opd increases. Since changes in the surrounding tissues of the worm have never been observed, the changes observed in the opd of the excretory cells are due to changes in the cells. The excretory system of Haemonchus has yet to be described in detail, but examination of unpublished electron micrographs provided by Prof. W. P. Rogers has revealed that the dimensions of the excretory cells are simply too small
I.J.P. VOL. 12. 1982
K. G. DAVEY and R. 1. S~M~ERVILLE
FIG. 2. Photograph
of the oesophageal
region of an untreated larva. oesophagus is relatively straight.
Note that the lumen,
1, of the
FIG 3. As in Fig. 2, but from an animal stimulated by CO2 at 38.5”C. Note the kink (arrow) in the lumen, 1 of the oesophagus. Because the kink is three-dimensional, some of it lies out of the plane of focus of the rest of the lumen. The course followed by the lumen is indicated by the solid black line above the figure. FIG. 4. Photograph
to show the excretory
cells (arrow) as seen in the interference excretory duct (d).
microscope.
Note
I.J.P.
VOL.
Optical path difference and exsh~thment
12. 1982
0
507
I
2x2
40
Bo
60
Time of cxposun
120
100 to
140
160
180
200
COafminl
FIG. 5. The time-course of increase in opd of the excretory cells of Haemonrhus confortus exposed to CO, at 389C. Each point is the mean of determinations on 15 worms and the vertical lines indicate the S.E. of the means.
to account for the additional reduction in fluid volume of 15 pl experienced by a larva activated by CO2 as compared to one desheathed by NaOCI. While precise measurements were not made, each excretory cell is no more than 2 m in diameter, suggesting that each cell would have to be approximately 2.5 mm long in order to accommodate 15 pl. Thus, while the excretory cells are potentially implicated in exsheathment in Huemonchus, they are not in themselves the source of the remaining water loss. Either there is another, as yet unidentified, source contributing to fluid loss, or the fluid involved passes through the excretory system. The absence of detailed information on the structure of the excretory cells and their relationship to the excretory duct in Haemonchus renders difficult the interpretation of the data presented here.
Acknowledgements-These experiments were carried out while KGD, on study leave from York University, was the recipient of a Distinguished Visiting Scholarship from the Universiry of Adelaide. The research was supported by the Australian Research Grants Committee and the Natural Sciences and Engineering Research Council of Canada.
REFERENCES ABU-HAKIMAR, & DAVEYK. G. 1977. The action of juvenile hormone on the follicle cells of ~~o~~~~~ prolixta: the importance of volume changes. Journal of Experimental Bioiogy 69: 33-44. DAVEYK. 0. 1979. Molting in a parasitic nematode, Phocanema decipiens: the role of water uptake. International Journal for Parasitology 9: 121-125. DAVEYK. G. & KAN S. P. 1968. Molting in a parasitic nematode, Phocanema decipiens IV. Ecdysis and its control. Canadian Journal of Zoology 46: 893-898. DAVEYK. G. & ROGERSW. P. 1982. Changes in water content and volume accompanying exsheathment of Haemonchus contortus. International Journal for Parasitology 12: 93-96. DAVIESH. G. 1958. The determination of mass and concentration by microscope interferometry. In Generai Cytochemical Merhods (Edited by DANIELLIJ. F.) Academic Press, New York. ELLENBYC. 1968a. Determination of the water content of nematode worms by interference microscopy. Experientia 24: 84-85. ELLENBYC. 196813.Desiccation survival of the infective larva of Haemonchus contortus. Journal of Experimen&at Biology 49: 469-475. SINCH R. N. d SULSTONJ. E. 1978. Some observations on moulting in ~aenorha~dit~ etegans. Nematoiogica 24: 63-71.