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Cuticular abnormality of an axenically cultured strain of the nematode Neoaplectana glaseri
JOURXAL
OF JSVERT2~%3RbTE
wticular
PeATHOLOGY
19,
-Abnormality the
405-408
of an Axenically
Nematode J.
GEORGE The
(1972)
JACKSON
Rockefeller Received
Cultured
Neoaplectana AND University, Januaq
!~IARIA Xew
Strai.n
glaseri A. York
RUDZiNSKA 10021
10, 1972
Cuticular bubbling, a growth abnormality, was noticed in one of several lines of the nematode ~Veoaplectana glaseri after 23 years of axenic cultivation apart from t,he source host, Popilliajaponica grubs. The abnormality occurs in all developmental stages, is particularly apparent in old or undernourished cultures, and does not significantly interfere with the reproduction potential or infectivity of well nourished cultures. Bubbling has not been cured by temperature shock or antibiotics, nor bee]: successfully transferred to unaffect~ed culture lines. Electron microscopy shows that the bubble envelope consists of one or more cuticles which, in different bubbles, contain tissue components in various stages of degeneration. This suggests that bubbles arise from an excessive elaboration of cuticle leading to an outpocketing and a pinching off of parts of the nematode’s body wall and the subsequent decomposition of e:!trapped tissues.
in the 23rd year of growth apart from the source host. Cuticular bubbles are visible with the light microscope and the appearance of normally smooth and “bubbly” nematodes in contrasted in Figs. 1 and 2,
;l*eoa;nlectana glaseri, was the first paranematode to be cultivated completely, z.e., through all stages of t,he life cycle, for continuous generations, in the absence of other living species (Glaser, 1931, 1940). Several successive generations can even be grown in a chemically defined medium (Jackson, 1962, 1969). Originally found in dead and dying grubs of the so-called Japanese beetle, Popillia japonica, the nematode is infectious as well for other insect pests of plant life (Glaser and Fox, 1930; Stoll, 1953; Jackson and Moore, 1969; Turco et al., 1970). This relative ease of culturing N. glaseri and the pat’hogenic potential have led to a worldwide dissemination of cultures for use in physiological, immunological, and insect control st’udies. Only one abnormality, a transient “gigant#ism”, has been recorded previously by McCoy et al. (1938) prior to the establishment of present culture strains in 1944. The abnorma,lity, which we call “cuticular bubbling”, was noticed in several cult’ures lat’e shit
I\$ATERIALS A3’eoaplectana
0
1972 by Academic
Press, Inc.
glaseri
METHODS has
been
cultured
axenically since 1944 on enriched agar slants with sterile slices of rabbit, kidney, Three to 5 weeks after inoculation of a culture, new third-stage larvae are harvest’ed from thin films of moist’ure on i-he upper slant and inside walls of the containing glass tube. These are routinely stored under ea. 2 cm of refrigerated water for several weeks or months and then used to start new cultures. This culture system was devised by Glaser (1940) and has been described in derail wit#h slight modifications by Stoii (1939) and Jackson (1969). The latt,er aut’hnrs also describe liquid culture media, which are more convenient in testing for the effects oi antibiot,ics and nutrient,s. Sterility test,ing was done routinely in semisolid thioglycolate medium, in 50 % w/v beef heart infusion 405
Copyright
ASD
406
JACKSON
AND
broth with 1% Pfanstiehl peptone, on dextrose agar slants, and on serum agar plates. For electron microscopy, the nematodes were fixed for 1 hr or more at 4°C in 3 % glutaraldehyde buffered with 0.1 M cacodylate to pH 7.3. Some nematodes were processed whole and others were cut in half. Both the intact and cut worms were washed for 1 hr in several changes of 0.1 M cacodylate buffer t)hat contained 4% sucrose. Postfixation in t’he buffer was with 2% OsO+ The worms were subsequently dehydrated in an ascending series of et’hyl alcohol concentrations (70%, 90%, 100%) and in propyline oxide, before being treated with mixtures of Epon and propyline oxide (1: 1, 1: 2). At, room temperature they were placed in fresh Epon daily for several days and embedded at 56°C in Epon within Beam capsules or vinyl cups, the cups already having a polymerized (at 56°C) layer of Epon at the bottom. From such preparations, blocks were cut with a diamond knife at the desired orientation for cross or longitudinal sections. The sections were double stained with many1 acetate and lead citrate before micrographs were taken with a RCA-EMU3F electron microscope. The structural details of normal Neoaplectana glaseri third-stage cuticle have been described previously (Jackson and Bradbury, 1970). RESULTS
Cultures with abnormal worms were traced to a common source culture made earlier in the year, but it is likely that cuticular bubbling arose previously in this line and was not detected. Other than cuticular bubbling, nothing unusual was noticed with light microscopy. Tests for contaminants were negative. The reproductive and infective potentials of the affected line were normal. All cultures derived from the common source contained some nematodes with cuticular bubbles. The precentage of af-
RUDZINSKA
fected individuals was ~12% in well nourished, act,ively reproducing cultures. As many as 43 % of nematodes showed the abnormality after the third week, when such cultures have passed their reproductive peak. A high incidence was also seen earlier in cultures raised in nonoptimal media. The low incidence of bubbling during growth and reproduction phases in adequate media may explain the otherwise apparent normality of affected cultures. However, even in a thriving culture, if the individual nematode that is affected by severe bubbling is a larval stage, it may be prevented from successful molting. With light microscopy it is also possible to observe the effect of pressure on the bubbles and the whole organism. When a suspension of affected nematodes is placed between a glass slide and coverslip and pressure is applied gradually, the bubbles burst to the outside but the nematode remains intact and active until additional pressure is exerted. This implies that there is cuticle or a strong musculature beneath the bubbles, otherwise the nematodes’ turgor would cause internal organs to protrude. This finding, general curiosity about the fine structure of abnormal cuticle, and the possiblity that an infeet,ious agent not detected by sterility tests might be located, led to electron microscopy. Abnormal divisions and fusions of cuticle make it appear that bubbles, like the main body, are entirely surrounded by cuticle (Fig. 3). With electron microscopy no connection has, so far, been found between the int’erior of bubbles and the soft tissues of the main body. That such connect’ions may have existed is suggested by bubble contents, for while the material inside some bubbles is disorganized beyond identification (Fig. 4), the contents of other bubbles look like degenerate tissue. Muscle cells can on occasion be recognized, as can osmiophilic droplets. The number of droplets tends to be greater in bubbles than in the main body.
FIG. 1. Light micrograph of midportion of adult female Xeoaplectana glaseri from culture line not affected by cuticular bubbling. Cuticular surface is smooth except for vulva (long arrow). X108. FIG. 2. Comparable light micrograph of midportion of adult female N. glaseri. Cuticdar bubbkx (short arrows) on the dorsal surface; ventral surface is normally smoot,h except for vulva (long arrow) X180. FIG. 3. Electron micrograph of partial section through a third-stage i\;. glaseri affected by cnt’icular bubbling. Bubble (B) and main body (M) are surrounded and separated by a branched sheath of secondstage cuticle (s). Inside this sheath, both main body and bubble are surrounded by third-stage cuticle (61 and c2). Granular material (g) between the sheath and cuticles is more abundant than in unaffected nematodes. Bubble and main body contain muscle cells (m), and osmiophilic droplets (0). There are also many vacuoles (v) in the bubble, and at the center a large homogeneous (h) area of ~~niform density. x4,440. FIG. 4. Electron micrograph of partial section through a third-st,age AT. glaseri affected by cnticidar bubbling. This bubble (B) contains no recognizable tissue components. X3040. FIG. 5. Higher magnification of vacuoles shown in Fig. 3. X17,200. 407
408
JACKSON
AND
Bubbles also contain numerous vacuoles filled with membranous material (Figs. 3, 5). The vacuoles are probably autophagic, and they resembie sequestered cell organelles in advanced stages of degradation.
Bubbling of N. glaseri differs morphologically from changes caused in this nematode’s cuticle by prolonged contact with rat peritoneal exudate (Jackson and Bradbury, 1970). It also differs from bacterial lesions reported for Ascaris cuticle (Weinberg and Keilin, 1912; Manter, 1928; St,ewart and Godwin, 1963; McKinnon and Lubinsky, 1966; Anderson et al., 1971). As far as we know, no abnormal ‘(growths” of nematodes have been described previously, which is not surprising in view of nematodes’ constant somatic cell number. That the cuticular bubbles of N. glaseri contain tissue components in various stages of degeneration suggests that initially the bubbles are not walled off from the main body but become so. Thus, bubble formation may involve an excessive elaborat’ion of cuticle leading to a bulging-out and then a pinching-off of part of the body wall, with a subsequent degeneration of cells. An alternative hypothesis, that cellular elements laid down in cuticle during its formation are later activated to grow, seems unlikely considering the poor nutrient transport in cuticle and lack of space in the already formed and hardened secretion. ACKNOWLEDGMENTS This work, done in the department William Trager, with the technical Erminio Gubert, was supported U.S. Public Health Service AI-04842, AI-09522, and AI-08989. Despommier participated in the electron microscopy.
of Professor assistance of in part by the through grants Dr. Dickson 1). initial stages of
REFERENCES ANDERSON, F. G.
W. K.., MADDEN, P. A., 1971. Histopathologic
AND and
TROMBA, bacterio-
RUDZINSKA logic examination of cuticular lesions of Ascaris swum. J. Parasitol., 57,1010-1014. GLASER, R. W. 1931. The cultivation of a nematode parasite of an insect. Science, 73,614-615. GLASER, R. W. 1940. The bacteria-free culture of a nematode parasitic in the Japanese beetle. Proc. Sot. Exp. Biol. Med., 43, 512-514. GLOSSER, R. W., AND Fox, H. 1930. A nematode parasite of the Japanese beetle (Popillia japonica Newm.). Science, 71, 16-17. JUXSON, G. J. 1962. The parasitic nematode, Neoaplectana glaseri, in axenic culture. II. Initial results with defined media. Exp. Parasitol., 12, 25-32. JACKSON, G. J. 1969. Nutritional control of nematode development. Advan. Exp. Med. Biol., 3, 333341. JACICSON, G. J., AND BRADBURY,~. C. 1970. Cutitular fine structure and molting of Neoaplectana glaseri (Nematoda), after prolonged contact with rat peritoneal exudate. J. Parasitol., 56, 108115. JXI~SON; G. J., AND MOORE, G. E. 1969. Infectivity of nematodes, Neoaplectana species, for the larvae of the weevil Hylobius pales, after rearing in species isolation. J. Invertebr. Pathol., 14, 194-198. MCCOY, E. E., GIRTH, H. B., AND GLASER, R. W. 1938. Notes on a giant form of the nematode Neoaplectana glaseri. J. Parasitol., 24,471-472. MCKINNON, G. A., AND LUBINSKY, G. A. 1966. The occurrence of dermomyositis of ascarids in Canada. Can. J. Zool., 44, 1090-1091. MANTER, II. W. 1928. A disease of Ascaris lumbricoides. J. Parasitol., 16, 101. STEWART, J. B., .~ND GODWIN, H. J. 1963. Cutitular lesions of Ascaris suum caused by Pseudomonas sp. J. Parasitol., 49, 231-234. STOLL, N. R. 1953. Continued infectivity for Japanese beetle grubs of Neoaplectana glaseri (Nematoda) after seven years axenic cultures. In “Thapar Commemoration Volume” (J. Dayal and K. S. Singh, eds.) pp. 259-268. Univ. of Lucknow, India. STOLL, N. R. 1959. Conditions favoring the axenic culture of Neoaplectana glaseri, a nematode parasite of certain insect grubs. Annals N. Y. Acad. Sci. 77 (Art. 2), 126-136. TURCO, C. P., HOPKINS, S. H., AND TKAMES, W. H., JR. 1970. Susceptibility of five insect pests to Neoaplectana glaseri Steiner, 1929. J. Parasitol., 56, 277-280. WEINBERG, M., AND KEILIN, C. R. 1912. Une maladie de l’ilscaris megalocephala. C. R. Sot. Biol., 73, 260-262.
OF JSVERT2~%3RbTE
wticular
PeATHOLOGY
19,
-Abnormality the
405-408
of an Axenically
Nematode J.
GEORGE The
(1972)
JACKSON
Rockefeller Received
Cultured
Neoaplectana AND University, Januaq
!~IARIA Xew
Strai.n
glaseri A. York
RUDZiNSKA 10021
10, 1972
Cuticular bubbling, a growth abnormality, was noticed in one of several lines of the nematode ~Veoaplectana glaseri after 23 years of axenic cultivation apart from t,he source host, Popilliajaponica grubs. The abnormality occurs in all developmental stages, is particularly apparent in old or undernourished cultures, and does not significantly interfere with the reproduction potential or infectivity of well nourished cultures. Bubbling has not been cured by temperature shock or antibiotics, nor bee]: successfully transferred to unaffect~ed culture lines. Electron microscopy shows that the bubble envelope consists of one or more cuticles which, in different bubbles, contain tissue components in various stages of degeneration. This suggests that bubbles arise from an excessive elaboration of cuticle leading to an outpocketing and a pinching off of parts of the nematode’s body wall and the subsequent decomposition of e:!trapped tissues.
in the 23rd year of growth apart from the source host. Cuticular bubbles are visible with the light microscope and the appearance of normally smooth and “bubbly” nematodes in contrasted in Figs. 1 and 2,
;l*eoa;nlectana glaseri, was the first paranematode to be cultivated completely, z.e., through all stages of t,he life cycle, for continuous generations, in the absence of other living species (Glaser, 1931, 1940). Several successive generations can even be grown in a chemically defined medium (Jackson, 1962, 1969). Originally found in dead and dying grubs of the so-called Japanese beetle, Popillia japonica, the nematode is infectious as well for other insect pests of plant life (Glaser and Fox, 1930; Stoll, 1953; Jackson and Moore, 1969; Turco et al., 1970). This relative ease of culturing N. glaseri and the pat’hogenic potential have led to a worldwide dissemination of cultures for use in physiological, immunological, and insect control st’udies. Only one abnormality, a transient “gigant#ism”, has been recorded previously by McCoy et al. (1938) prior to the establishment of present culture strains in 1944. The abnorma,lity, which we call “cuticular bubbling”, was noticed in several cult’ures lat’e shit
I\$ATERIALS A3’eoaplectana
0
1972 by Academic
Press, Inc.
glaseri
METHODS has
been
cultured
axenically since 1944 on enriched agar slants with sterile slices of rabbit, kidney, Three to 5 weeks after inoculation of a culture, new third-stage larvae are harvest’ed from thin films of moist’ure on i-he upper slant and inside walls of the containing glass tube. These are routinely stored under ea. 2 cm of refrigerated water for several weeks or months and then used to start new cultures. This culture system was devised by Glaser (1940) and has been described in derail wit#h slight modifications by Stoii (1939) and Jackson (1969). The latt,er aut’hnrs also describe liquid culture media, which are more convenient in testing for the effects oi antibiot,ics and nutrient,s. Sterility test,ing was done routinely in semisolid thioglycolate medium, in 50 % w/v beef heart infusion 405
Copyright
ASD
406
JACKSON
AND
broth with 1% Pfanstiehl peptone, on dextrose agar slants, and on serum agar plates. For electron microscopy, the nematodes were fixed for 1 hr or more at 4°C in 3 % glutaraldehyde buffered with 0.1 M cacodylate to pH 7.3. Some nematodes were processed whole and others were cut in half. Both the intact and cut worms were washed for 1 hr in several changes of 0.1 M cacodylate buffer t)hat contained 4% sucrose. Postfixation in t’he buffer was with 2% OsO+ The worms were subsequently dehydrated in an ascending series of et’hyl alcohol concentrations (70%, 90%, 100%) and in propyline oxide, before being treated with mixtures of Epon and propyline oxide (1: 1, 1: 2). At, room temperature they were placed in fresh Epon daily for several days and embedded at 56°C in Epon within Beam capsules or vinyl cups, the cups already having a polymerized (at 56°C) layer of Epon at the bottom. From such preparations, blocks were cut with a diamond knife at the desired orientation for cross or longitudinal sections. The sections were double stained with many1 acetate and lead citrate before micrographs were taken with a RCA-EMU3F electron microscope. The structural details of normal Neoaplectana glaseri third-stage cuticle have been described previously (Jackson and Bradbury, 1970). RESULTS
Cultures with abnormal worms were traced to a common source culture made earlier in the year, but it is likely that cuticular bubbling arose previously in this line and was not detected. Other than cuticular bubbling, nothing unusual was noticed with light microscopy. Tests for contaminants were negative. The reproductive and infective potentials of the affected line were normal. All cultures derived from the common source contained some nematodes with cuticular bubbles. The precentage of af-
RUDZINSKA
fected individuals was ~12% in well nourished, act,ively reproducing cultures. As many as 43 % of nematodes showed the abnormality after the third week, when such cultures have passed their reproductive peak. A high incidence was also seen earlier in cultures raised in nonoptimal media. The low incidence of bubbling during growth and reproduction phases in adequate media may explain the otherwise apparent normality of affected cultures. However, even in a thriving culture, if the individual nematode that is affected by severe bubbling is a larval stage, it may be prevented from successful molting. With light microscopy it is also possible to observe the effect of pressure on the bubbles and the whole organism. When a suspension of affected nematodes is placed between a glass slide and coverslip and pressure is applied gradually, the bubbles burst to the outside but the nematode remains intact and active until additional pressure is exerted. This implies that there is cuticle or a strong musculature beneath the bubbles, otherwise the nematodes’ turgor would cause internal organs to protrude. This finding, general curiosity about the fine structure of abnormal cuticle, and the possiblity that an infeet,ious agent not detected by sterility tests might be located, led to electron microscopy. Abnormal divisions and fusions of cuticle make it appear that bubbles, like the main body, are entirely surrounded by cuticle (Fig. 3). With electron microscopy no connection has, so far, been found between the int’erior of bubbles and the soft tissues of the main body. That such connect’ions may have existed is suggested by bubble contents, for while the material inside some bubbles is disorganized beyond identification (Fig. 4), the contents of other bubbles look like degenerate tissue. Muscle cells can on occasion be recognized, as can osmiophilic droplets. The number of droplets tends to be greater in bubbles than in the main body.
FIG. 1. Light micrograph of midportion of adult female Xeoaplectana glaseri from culture line not affected by cuticular bubbling. Cuticular surface is smooth except for vulva (long arrow). X108. FIG. 2. Comparable light micrograph of midportion of adult female N. glaseri. Cuticdar bubbkx (short arrows) on the dorsal surface; ventral surface is normally smoot,h except for vulva (long arrow) X180. FIG. 3. Electron micrograph of partial section through a third-stage i\;. glaseri affected by cnt’icular bubbling. Bubble (B) and main body (M) are surrounded and separated by a branched sheath of secondstage cuticle (s). Inside this sheath, both main body and bubble are surrounded by third-stage cuticle (61 and c2). Granular material (g) between the sheath and cuticles is more abundant than in unaffected nematodes. Bubble and main body contain muscle cells (m), and osmiophilic droplets (0). There are also many vacuoles (v) in the bubble, and at the center a large homogeneous (h) area of ~~niform density. x4,440. FIG. 4. Electron micrograph of partial section through a third-st,age AT. glaseri affected by cnticidar bubbling. This bubble (B) contains no recognizable tissue components. X3040. FIG. 5. Higher magnification of vacuoles shown in Fig. 3. X17,200. 407
408
JACKSON
AND
Bubbles also contain numerous vacuoles filled with membranous material (Figs. 3, 5). The vacuoles are probably autophagic, and they resembie sequestered cell organelles in advanced stages of degradation.
Bubbling of N. glaseri differs morphologically from changes caused in this nematode’s cuticle by prolonged contact with rat peritoneal exudate (Jackson and Bradbury, 1970). It also differs from bacterial lesions reported for Ascaris cuticle (Weinberg and Keilin, 1912; Manter, 1928; St,ewart and Godwin, 1963; McKinnon and Lubinsky, 1966; Anderson et al., 1971). As far as we know, no abnormal ‘(growths” of nematodes have been described previously, which is not surprising in view of nematodes’ constant somatic cell number. That the cuticular bubbles of N. glaseri contain tissue components in various stages of degeneration suggests that initially the bubbles are not walled off from the main body but become so. Thus, bubble formation may involve an excessive elaborat’ion of cuticle leading to a bulging-out and then a pinching-off of part of the body wall, with a subsequent degeneration of cells. An alternative hypothesis, that cellular elements laid down in cuticle during its formation are later activated to grow, seems unlikely considering the poor nutrient transport in cuticle and lack of space in the already formed and hardened secretion. ACKNOWLEDGMENTS This work, done in the department William Trager, with the technical Erminio Gubert, was supported U.S. Public Health Service AI-04842, AI-09522, and AI-08989. Despommier participated in the electron microscopy.
of Professor assistance of in part by the through grants Dr. Dickson 1). initial stages of
REFERENCES ANDERSON, F. G.
W. K.., MADDEN, P. A., 1971. Histopathologic
AND and
TROMBA, bacterio-
RUDZINSKA logic examination of cuticular lesions of Ascaris swum. J. Parasitol., 57,1010-1014. GLASER, R. W. 1931. The cultivation of a nematode parasite of an insect. Science, 73,614-615. GLASER, R. W. 1940. The bacteria-free culture of a nematode parasitic in the Japanese beetle. Proc. Sot. Exp. Biol. Med., 43, 512-514. GLOSSER, R. W., AND Fox, H. 1930. A nematode parasite of the Japanese beetle (Popillia japonica Newm.). Science, 71, 16-17. JUXSON, G. J. 1962. The parasitic nematode, Neoaplectana glaseri, in axenic culture. II. Initial results with defined media. Exp. Parasitol., 12, 25-32. JACKSON, G. J. 1969. Nutritional control of nematode development. Advan. Exp. Med. Biol., 3, 333341. JACICSON, G. J., AND BRADBURY,~. C. 1970. Cutitular fine structure and molting of Neoaplectana glaseri (Nematoda), after prolonged contact with rat peritoneal exudate. J. Parasitol., 56, 108115. JXI~SON; G. J., AND MOORE, G. E. 1969. Infectivity of nematodes, Neoaplectana species, for the larvae of the weevil Hylobius pales, after rearing in species isolation. J. Invertebr. Pathol., 14, 194-198. MCCOY, E. E., GIRTH, H. B., AND GLASER, R. W. 1938. Notes on a giant form of the nematode Neoaplectana glaseri. J. Parasitol., 24,471-472. MCKINNON, G. A., AND LUBINSKY, G. A. 1966. The occurrence of dermomyositis of ascarids in Canada. Can. J. Zool., 44, 1090-1091. MANTER, II. W. 1928. A disease of Ascaris lumbricoides. J. Parasitol., 16, 101. STEWART, J. B., .~ND GODWIN, H. J. 1963. Cutitular lesions of Ascaris suum caused by Pseudomonas sp. J. Parasitol., 49, 231-234. STOLL, N. R. 1953. Continued infectivity for Japanese beetle grubs of Neoaplectana glaseri (Nematoda) after seven years axenic cultures. In “Thapar Commemoration Volume” (J. Dayal and K. S. Singh, eds.) pp. 259-268. Univ. of Lucknow, India. STOLL, N. R. 1959. Conditions favoring the axenic culture of Neoaplectana glaseri, a nematode parasite of certain insect grubs. Annals N. Y. Acad. Sci. 77 (Art. 2), 126-136. TURCO, C. P., HOPKINS, S. H., AND TKAMES, W. H., JR. 1970. Susceptibility of five insect pests to Neoaplectana glaseri Steiner, 1929. J. Parasitol., 56, 277-280. WEINBERG, M., AND KEILIN, C. R. 1912. Une maladie de l’ilscaris megalocephala. C. R. Sot. Biol., 73, 260-262.