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Partial-pupae of the greater wax moth, Galleria mellonella (Linnaeus)
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Partial-Pupae Galleria
7, 489-492
(1965)
of
the
mellonella RONALD
Division
of Biological
Greater
Sciences,
L.
University
Accepted
April
Wax
(Linnaeus)
Moth, ’
TAYLOR of California,
if-vine,
California
28, 1965
Under laboratory culture, individuals of the greater wax moth, Galleria mellonella (Linnaeus), are occasionally observed which only partially complete the larval-pupal transformation and then die. In form they appear as larvae, however, a large portion of the body is hard and dark and thereby appears pupal. Histological sections clearly reveal such individuals to be in a late pharate stage. The last larval cuticle is equally digested over both “larval” and “pupal” areas and appears fully prepared for ecdysis. The new (pupal) cuticle in the hard and dark areas appears normal. The main abnormality is in the light-colored areas where the new cuticle appears to have been partially laid down and then its development arrested. The induction mechanism of G. mellonella partial-pupae is unknown, though it appears to be an injury-induced phenomenon.
1956)) formaldehyde-cetyl pyridinium chloride (Williams and Jackson, 1956), and In laboratory cultures of the greater wax Zenker’s fluid. All specimenswere dehydrated moth, Galleria mellonella (Linnaeus) , indivia the butanol series (Lees, 1950), embedded viduals are occasionally observed which only in tissue-mat (m.p. 61’C) containing SC/( partially complete the larval-pupal transforbayberry wax and 5% beeswax, and cut at mation; they die before the ecdysis to the 8 microns. Cross sections were made of some pupal instar. In external morphology, the individuals whereas others were cut in the animals appear larval in shape, however, a frontal and sagittal planes. large portion of the body is hard (tanned) and Somepartial-pupae were stained with alcian dark (melanized) thereby appearing pupal. blue following the method of Baldwin and For convenience, we refer to these individuals Salthouse (1959) except for using alcian blue which appear part-larval and part-pupal as at a pH of 2.7 and omitting metanil yellow. ‘Lpartial-pupae.” Other individuals were stained with aldehyde The histological picture of the integument in fuchsin according to Halmi’s modification the “larval” and “pupal” regions, as well as (1952), following the precautions listed by other incidental observations relating to this Elftman (1959), but omitting the useof hemastrange occurrence, are describedin this paper. toxylin. These two staining procedures have MATERIALS AND METHODS been found quite valuable in studies on the Seven partial-pupae were fixed alive in integument (Taylor and Richards, 1963; formaldehyde-saline (Williams and Jackson, 1965). INTRODUCTION
RESULTS 1 This investigation was supported by Public Health Service Research Grant No. 5358 (to E. A. Steinhaus) from National Institute of Allergy and Infectious Diseases.
There is a considerableamount of variation among partial-pupae, that is, variation as to which regions appear larval and which appear 489
490
TAYLOR
pupal. The head, prothorax, mesothorax, dorsum of the metathorax, and last few abdominal segments are nearly always “pupal.” The ventral surface of the metathorax and first three abdominal segments are always “larval” in coloration and hardness. The variation occurs, therefore, in the dorsum of the first three abdominal segments and in the entire fourth through sixth abdominal segments. These latter areas are sometimes “larval” and sometimes “pupal” or any degree in between. The region of demarcation between the two regions is occasionally sharp, but generally diffused. It is usually very sharp where the “larval” areas of the metathorax separate from the “pupal” areas. With fine forceps, the last larval cuticle can be dissected off the entire partial-pupa leaving an organism basically pupal in external morphology, the main morphological abnormality being that the wing cases and the antenna1 and proboscis sheaths have not expanded. The observation noted earlier that the ventral surface of the metathorax and first three abdominal segments in partialpupae are always “larval” in coloration is probably explained by the fact that these are regions which, in a normal pupa, are covered by the wing pads and which do not darken. One is still left to explain, however, the variation in coloration that occurs in the dorsum of the first three abdominal segments and in the entire fourth through sixth abdominal segments. After the last larval cuticle is removed, the cuticle in the nondarkened (“larval”) areas is white, delicate, and soft. There appears to be a direct relationship between hardening and darkening of the cuticle-the darker the cuticle, the harder it is. The latter is determined crudely by probing the cuticle with dissecting needles. Histological sections clearly reveal the partial-pupae to be in a late pharate stage, that is, two distinct cuticles are present (Fig. 1).
The larval procuticle is almost completely digested over the entire organism-the unshed cuticle consisting of epicuticle and some exocuticle; it appears fully prepared for ecdysis. A distinct ecdysial membrane adheres to the inner surface of the old cuticle (Fig. 2). (A more detailed account of the ecdysial membrane and subcuticle of G. mellonella is presented in Taylor and Richards, 1965). In general, the last larval cuticle in a normal pharate pupa appears identical to that described above in partial-pupae. From these observations one can conclude that whatever it is that causes partial-pupa formation, it has little effect, if any, upon the last larval cuticle. The new cuticle in the nondarkened regions is a fraction of the thickness of the cuticle in the darkened regions (Fig. 1). Its thinness and lack of melanization as compared to the hard and dark regions is what gives it a “larval” appearance. That it is basically of a pupal nature, however, is deduced from the fact that new prolegs or other structures diagnostic of a larval stage are not present. The epidermis under the nondarkened cuticle is always thinner than the epidermis beneath the melanized cuticle (Fig. 1). An unusual feature is that the old cuticle (with its ecdysial membrane) is generally in juxtaposition with the new cuticle in the nondarkened regions. The two cuticles are clearly separated, however, by the alcian blue- and aldehyde fuchsin-positive ecdysial membrane. The normal situation occurs in the darkened regions, that is, the old cuticle is separated from the new cuticle by a distinct exuvial space filled with molting fluid. DISCUSSION
The general picture is as follows: The physiological processes for the separation and digestion of the old cuticle function normally. Synthesis of pupal cuticle begins at some stage during the digestion process, but for some reason it is arrested or otherwise ceases or proceeds too slowly in those regions
FE. I. Cross section of Gallevia ?nellonella partial-pupa showing (1) the differences in thickness of the pupal cuticle and epidermis between the “larval” and “pupal” areas, (2) the constant thickness of the last larval cuticle in both regions, and (3) the adherence of the last larval cuticle to the pupal cuticle in the “larval” area. 375 X. The symbols are UC, last larval cuticle; pc, pupal cuticle; em, ecdysial membrane; epid, epidermis; es, exuvial space with molting fluid. FIG.
2.
Came
as Fig.
1 only
at greater
magnification
in order 491
to show
the ecdysial
membrane,
1,joo
x,
492
TAYLOR
destined to becomethe light, larval-appearing areas. Ecdysis is prevented, and death ensues. (In a few casesthe old cuticle had actually split along the ecdysial line and moved one to two body segmentsposteriorly before death.) Although ecdysis is prevented, the normally developed pupal cuticle darkens and hardens. The coloration provided by the last larval cuticle plus the normal coloration of the new pupal cuticle accounts for the darker color of the partial-pupae in the darkened regions as compared to a normal pupa. This is shownby the fact that partial-pupae from which the larval cuticle has been manually stripped are of normal pupal color. Richards (1958) reported a similar pathological condition in a single specimen of another pyralid moth, Anagasta Kiihniella (Zeller) . Although the external gross appearance he reported was apparently similar to that in G. mellonella partial-pupae, the histological picture is quite different. Richards reports that in A. kiihniella the pathology residesin the old cuticle-the procuticle being normally digested in certain regions and totally undigested in other areas. As noted above in G. mellonella, the old cuticle is perfectly normal whereas it is the new cuticle which developsabnormally. The induction of this pathological state is unknown. Its low but constant occurrence in cultured animals at first suggesteda possible genetic basis, but then it was discovered that cautery and handling (Barbara Thorsen, personal communication) and injections of hemolymph (Suresh Shrivastava, personal communication) cause high percentages of partial-pupa formation. Such observations indicate an injury-induced phenomenon. rnder the culture conditions employed, injury to a small percentage of the animals is of common occurrence. If we had clearer concepts of the nature of the pattern formation, especially of the control
of the development of sclerites sharply delimited from membrane (e.g., intersegmental membrane), it might be possible to speak in terms of upsetting the mechanismof pattern control. One can imagine partial-pupae as resulting from deformation of the pattern mechanism. But when the pattern-forming mechanism is still a mystery speculations along such lines are hardly useful. Clearly, more work remains to be done in order to elucidate the induction mechanism of G. melt’onella partial-pupae. ACKNOWLEDGMEKTS
To ProfessorA. Glenn Richards,Departmentof Entomology,Fisheries,and Wildlife, University of Minnesota,I expressmy appreciationfor pointing out the problemand for his carefulreadingof the manuscript. REFERENCES
BALDWIN, W. F., AND SALTHOUSE, T. N. 1959. Derma1 glandsand mucinin the molting cycle of Rhodnius prolizus Stol. 1. Insect Physiol., 3, 345348. ELFTMAN, H. 1959. Aldehyde-fuchsin for pituitary cytochemistry.J. Histochem. Cytochem., ‘7, 9% 100. HALMI, N. S. 1952. Differentiationof two types
of basophils in the adenohypophysis of the rat andthe mouse.Stain Technol., 27, 61-64. LEES, B. 1950. “The Microtomist’sVade-Mecum” (J. B. Gatenby and H. W. Beams,eds.),11th ed., 753 pp. Blakiston, Philadelphia,Pennsylvania. RICHARDS, A. G. 1958. The ecdysialmembrane of the moth, Ephestia kiihniella Z. Z. Natwforsch., 13b, 811-812. TAYLOR, R. L., AND RICHARDS, A. G. 1963. The subimaginal cuticle of the mayfly Callibaetis sp. (Ephemeroptera). Ann. Entonzol. Sot. Am., 56, 418-426. TAYLOR, R. L., AND RICHARDS, A. G. 1965. Integumentary changes during moulting of arthropods with special reference to the subcuticle and ecdysial membrane. J. Morphol., 116,1-22. WII.LIAMS, G., AND JACKSON, D. S. 1956. Two organic fixatives for acid mucopolysaccharides. Stain. Technol., 31, 189-191.
OF INVERTEBRATE
PATHOLOGY
Partial-Pupae Galleria
7, 489-492
(1965)
of
the
mellonella RONALD
Division
of Biological
Greater
Sciences,
L.
University
Accepted
April
Wax
(Linnaeus)
Moth, ’
TAYLOR of California,
if-vine,
California
28, 1965
Under laboratory culture, individuals of the greater wax moth, Galleria mellonella (Linnaeus), are occasionally observed which only partially complete the larval-pupal transformation and then die. In form they appear as larvae, however, a large portion of the body is hard and dark and thereby appears pupal. Histological sections clearly reveal such individuals to be in a late pharate stage. The last larval cuticle is equally digested over both “larval” and “pupal” areas and appears fully prepared for ecdysis. The new (pupal) cuticle in the hard and dark areas appears normal. The main abnormality is in the light-colored areas where the new cuticle appears to have been partially laid down and then its development arrested. The induction mechanism of G. mellonella partial-pupae is unknown, though it appears to be an injury-induced phenomenon.
1956)) formaldehyde-cetyl pyridinium chloride (Williams and Jackson, 1956), and In laboratory cultures of the greater wax Zenker’s fluid. All specimenswere dehydrated moth, Galleria mellonella (Linnaeus) , indivia the butanol series (Lees, 1950), embedded viduals are occasionally observed which only in tissue-mat (m.p. 61’C) containing SC/( partially complete the larval-pupal transforbayberry wax and 5% beeswax, and cut at mation; they die before the ecdysis to the 8 microns. Cross sections were made of some pupal instar. In external morphology, the individuals whereas others were cut in the animals appear larval in shape, however, a frontal and sagittal planes. large portion of the body is hard (tanned) and Somepartial-pupae were stained with alcian dark (melanized) thereby appearing pupal. blue following the method of Baldwin and For convenience, we refer to these individuals Salthouse (1959) except for using alcian blue which appear part-larval and part-pupal as at a pH of 2.7 and omitting metanil yellow. ‘Lpartial-pupae.” Other individuals were stained with aldehyde The histological picture of the integument in fuchsin according to Halmi’s modification the “larval” and “pupal” regions, as well as (1952), following the precautions listed by other incidental observations relating to this Elftman (1959), but omitting the useof hemastrange occurrence, are describedin this paper. toxylin. These two staining procedures have MATERIALS AND METHODS been found quite valuable in studies on the Seven partial-pupae were fixed alive in integument (Taylor and Richards, 1963; formaldehyde-saline (Williams and Jackson, 1965). INTRODUCTION
RESULTS 1 This investigation was supported by Public Health Service Research Grant No. 5358 (to E. A. Steinhaus) from National Institute of Allergy and Infectious Diseases.
There is a considerableamount of variation among partial-pupae, that is, variation as to which regions appear larval and which appear 489
490
TAYLOR
pupal. The head, prothorax, mesothorax, dorsum of the metathorax, and last few abdominal segments are nearly always “pupal.” The ventral surface of the metathorax and first three abdominal segments are always “larval” in coloration and hardness. The variation occurs, therefore, in the dorsum of the first three abdominal segments and in the entire fourth through sixth abdominal segments. These latter areas are sometimes “larval” and sometimes “pupal” or any degree in between. The region of demarcation between the two regions is occasionally sharp, but generally diffused. It is usually very sharp where the “larval” areas of the metathorax separate from the “pupal” areas. With fine forceps, the last larval cuticle can be dissected off the entire partial-pupa leaving an organism basically pupal in external morphology, the main morphological abnormality being that the wing cases and the antenna1 and proboscis sheaths have not expanded. The observation noted earlier that the ventral surface of the metathorax and first three abdominal segments in partialpupae are always “larval” in coloration is probably explained by the fact that these are regions which, in a normal pupa, are covered by the wing pads and which do not darken. One is still left to explain, however, the variation in coloration that occurs in the dorsum of the first three abdominal segments and in the entire fourth through sixth abdominal segments. After the last larval cuticle is removed, the cuticle in the nondarkened (“larval”) areas is white, delicate, and soft. There appears to be a direct relationship between hardening and darkening of the cuticle-the darker the cuticle, the harder it is. The latter is determined crudely by probing the cuticle with dissecting needles. Histological sections clearly reveal the partial-pupae to be in a late pharate stage, that is, two distinct cuticles are present (Fig. 1).
The larval procuticle is almost completely digested over the entire organism-the unshed cuticle consisting of epicuticle and some exocuticle; it appears fully prepared for ecdysis. A distinct ecdysial membrane adheres to the inner surface of the old cuticle (Fig. 2). (A more detailed account of the ecdysial membrane and subcuticle of G. mellonella is presented in Taylor and Richards, 1965). In general, the last larval cuticle in a normal pharate pupa appears identical to that described above in partial-pupae. From these observations one can conclude that whatever it is that causes partial-pupa formation, it has little effect, if any, upon the last larval cuticle. The new cuticle in the nondarkened regions is a fraction of the thickness of the cuticle in the darkened regions (Fig. 1). Its thinness and lack of melanization as compared to the hard and dark regions is what gives it a “larval” appearance. That it is basically of a pupal nature, however, is deduced from the fact that new prolegs or other structures diagnostic of a larval stage are not present. The epidermis under the nondarkened cuticle is always thinner than the epidermis beneath the melanized cuticle (Fig. 1). An unusual feature is that the old cuticle (with its ecdysial membrane) is generally in juxtaposition with the new cuticle in the nondarkened regions. The two cuticles are clearly separated, however, by the alcian blue- and aldehyde fuchsin-positive ecdysial membrane. The normal situation occurs in the darkened regions, that is, the old cuticle is separated from the new cuticle by a distinct exuvial space filled with molting fluid. DISCUSSION
The general picture is as follows: The physiological processes for the separation and digestion of the old cuticle function normally. Synthesis of pupal cuticle begins at some stage during the digestion process, but for some reason it is arrested or otherwise ceases or proceeds too slowly in those regions
FE. I. Cross section of Gallevia ?nellonella partial-pupa showing (1) the differences in thickness of the pupal cuticle and epidermis between the “larval” and “pupal” areas, (2) the constant thickness of the last larval cuticle in both regions, and (3) the adherence of the last larval cuticle to the pupal cuticle in the “larval” area. 375 X. The symbols are UC, last larval cuticle; pc, pupal cuticle; em, ecdysial membrane; epid, epidermis; es, exuvial space with molting fluid. FIG.
2.
Came
as Fig.
1 only
at greater
magnification
in order 491
to show
the ecdysial
membrane,
1,joo
x,
492
TAYLOR
destined to becomethe light, larval-appearing areas. Ecdysis is prevented, and death ensues. (In a few casesthe old cuticle had actually split along the ecdysial line and moved one to two body segmentsposteriorly before death.) Although ecdysis is prevented, the normally developed pupal cuticle darkens and hardens. The coloration provided by the last larval cuticle plus the normal coloration of the new pupal cuticle accounts for the darker color of the partial-pupae in the darkened regions as compared to a normal pupa. This is shownby the fact that partial-pupae from which the larval cuticle has been manually stripped are of normal pupal color. Richards (1958) reported a similar pathological condition in a single specimen of another pyralid moth, Anagasta Kiihniella (Zeller) . Although the external gross appearance he reported was apparently similar to that in G. mellonella partial-pupae, the histological picture is quite different. Richards reports that in A. kiihniella the pathology residesin the old cuticle-the procuticle being normally digested in certain regions and totally undigested in other areas. As noted above in G. mellonella, the old cuticle is perfectly normal whereas it is the new cuticle which developsabnormally. The induction of this pathological state is unknown. Its low but constant occurrence in cultured animals at first suggesteda possible genetic basis, but then it was discovered that cautery and handling (Barbara Thorsen, personal communication) and injections of hemolymph (Suresh Shrivastava, personal communication) cause high percentages of partial-pupa formation. Such observations indicate an injury-induced phenomenon. rnder the culture conditions employed, injury to a small percentage of the animals is of common occurrence. If we had clearer concepts of the nature of the pattern formation, especially of the control
of the development of sclerites sharply delimited from membrane (e.g., intersegmental membrane), it might be possible to speak in terms of upsetting the mechanismof pattern control. One can imagine partial-pupae as resulting from deformation of the pattern mechanism. But when the pattern-forming mechanism is still a mystery speculations along such lines are hardly useful. Clearly, more work remains to be done in order to elucidate the induction mechanism of G. melt’onella partial-pupae. ACKNOWLEDGMEKTS
To ProfessorA. Glenn Richards,Departmentof Entomology,Fisheries,and Wildlife, University of Minnesota,I expressmy appreciationfor pointing out the problemand for his carefulreadingof the manuscript. REFERENCES
BALDWIN, W. F., AND SALTHOUSE, T. N. 1959. Derma1 glandsand mucinin the molting cycle of Rhodnius prolizus Stol. 1. Insect Physiol., 3, 345348. ELFTMAN, H. 1959. Aldehyde-fuchsin for pituitary cytochemistry.J. Histochem. Cytochem., ‘7, 9% 100. HALMI, N. S. 1952. Differentiationof two types
of basophils in the adenohypophysis of the rat andthe mouse.Stain Technol., 27, 61-64. LEES, B. 1950. “The Microtomist’sVade-Mecum” (J. B. Gatenby and H. W. Beams,eds.),11th ed., 753 pp. Blakiston, Philadelphia,Pennsylvania. RICHARDS, A. G. 1958. The ecdysialmembrane of the moth, Ephestia kiihniella Z. Z. Natwforsch., 13b, 811-812. TAYLOR, R. L., AND RICHARDS, A. G. 1963. The subimaginal cuticle of the mayfly Callibaetis sp. (Ephemeroptera). Ann. Entonzol. Sot. Am., 56, 418-426. TAYLOR, R. L., AND RICHARDS, A. G. 1965. Integumentary changes during moulting of arthropods with special reference to the subcuticle and ecdysial membrane. J. Morphol., 116,1-22. WII.LIAMS, G., AND JACKSON, D. S. 1956. Two organic fixatives for acid mucopolysaccharides. Stain. Technol., 31, 189-191.