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Biology of Nosema plodiae sp. n., a microsporidian pathogen of the Indian-meal moth, Plodia interpunctella (Hübner), (Lepidoptera: Phycitidae)
JOURNAL
OF INVERTEBRATE
PATHOLOGY
11,
1od-lll
1968)
(
Biology of Nosemu plodiue sp. n., a Microsporidian Pathogen of the IndianMeal Moth, Plodia inteqmwtella (Hiibner ), (Lepidoptera:Phycitidae) WILLIAM
R. KJZLLEN AND JAMES E. LINDEGRJZN
Stored-Product Insects Research Branch, Market Quality Research Division, Agricultural Research Service, U.S. Department of Agriculture, Fresco, California 93727 Received July 18, 1967 The morphology and developmental cycle of a previously undescribed species of Nosema are described from the Indian-meal moth, Plodia interpunctellu. The pathogen invades a variety of tissues and organs, including fat bodies, muscles, the gut wall, Malpighian tubes, and the salivary and silk glands. In laboratory tests larvae were readily infected by feeding them fresh spores. Larvae with acute infections showed signs and symptoms of disease 6 days after initial feeding; such larvae were stunted, and usually succumbed before pupation. Larvae with subacute infections survived to the adult stage. Pathogens invaded developing ova, and transovarian transmission was observed. Larvae of the almond moth, Cadra cautellu (Walker), the tobacco moth, Ephestia elutella (Hiibner), and the dried fruit beetle. Carpophilus hemipterus (Linnaeus), did not become infected when fed spores; however, larvae of the greater wax moth, GalleTiu melkmda (Linnaeus) acquired subacute infections.
INTR~Du(;TION
The Indian-meal moth, Plodif interpunctella, is one of the most widely distributed and economically important lepidopterous pests known to infest stored products. Efforts to control this moth have received worldwide attention, and although it has been the subject of many biological and ecological investigations, very little is known about its associated protozoan pathogens. The only record of a microsporidian pathogen of P. interpunctellu was published by Steinhaus and Marsh (1962) in a report of diagnoses of diseased insects. They noted having examined a diseased larva which was received from J. Weiser in 1953; the specimen was from a laboratory culture that had been maintained in Prague, Czechoslovakia. The larva was diagnosed as being infected with Nosema; however, descriptions of the spores or of the affected tissues
were not included in the report. From other geographical sources they reported the presence of the schizogregarine Matte& dispora, and earlier Steinhaus (1947) reported the finding of the coccidian Ad&m mesnili in the insect. The original slides or subsequent additional preserved material of the Prague Nosew are not available for comparative study; however, Dr. Weiser has kindly examined spores of the Nosemu which we isolated from diseased adults of P. interpunctella collected in Fresno, and he has compared our material with his original laboratory notes. Weiser (1966) has indicated that the California species is not similar to the Nosemu that he had observed in Prague. We have, therefore, concluded from studies of the morphology, life cycle, and hostpathogen relationships that the Nosema from Fresno has not been previously re104
MIC33OSPOIUDIAN
PATHOGEN
ported from P. interpunctella, and that it represents an undescribed species. MATERIALS
AND
METHODS
All stages of P. interpunctella were collected from infested dried figs in a warehouse in Fresno, California, during the summer of 1966. Larvae and adults were returned to the laboratory in plastic bags, and dissected within 24 hr in 0.65% saline solution Squash preparations of fresh tissues were examined with a phase-contrast microscope at a magnification of 1250 diameters. Smears of infected tissues were fixed with methyl alcohol and stained with Giemsa’s solution. Whole bodies of laboratory-infected larvae and adults were fixed in Duboscq-Brasil solution for 24 hr, embedded in paraffin (mp 60°C ) , and sectioned at 6~. Serial sections were stained with Heidenhain’s hematoxylin or Sharp’s ( 1914) modification of Mallory’s triple stain. Spores were measured with an A. E. I. Vickers image-splitting eyepiece at a magnification of 1006 diameters. The mean value and standard error of the mean were determined from measurements of 50 spores. The sizes of typical stages in the schizogonic cycle of the pathogen were also determined. Infectivity studies were conducted with laboratory strains of Plodia interpunctella, Ephestiu elutellu, Cadra cautella, Gall&a mellonella, and Carpophilus hemipterus. Cultures of the moths had been maintained in the insectary for at least 5 years. Larvae were reared in l-quart Mason jars, and were provided a rearing medium consisting of 16 parts chicken mash, 1 part honey, and 1 part glycerol. Calcium propionate was added to the mixture to retard fungus growth. SYSTEMATICS
The holotype and 10 paratype slides of the new species have been deposited in the
OF
THE
INDIAN-MEAL
105
MOTH
type collection of the U. S. National Museum. Additional paratype slides have been deposited in the collections of J. J. Lipa, Laboratory of Biological Control, Institute of Plant Protection, Poznan, Poland; J. Weiser, Department of Insect Pathology, Institute of Biology, Czechoslovakian Academy of Sciences, Prague; J. P. Kramer, Department of Entomology and Limnology, Cornell University, Ithaca, New York; and in the collection of the authors. Nosema plodiae sp. n. (Figs. l-50) Derivation Plodia.
of
name:
The
Nosema
of
Host: All stages of Plodia interpunctellu (Hiibner). Invades many tissues and glands, including silk and salivary glands, Malpighian tubes, gut wall, ovaries, oviduct, testes, fat bodies, muscle, and nervous tissue (Figs. 53 and 54). Morphology: Fresh material. Mature spores, 4.09p + 0.24~ X 1.89, & 0.03~; polar filament, SO,; immature spores with distinct vacuoles, 5.11~ k 0.31~ X 2.58~ 4 0.15~. Diameter of mononucleate schizonts, 2.6-3.6~; quadrinucleate schizonts, 5.0-5.4~; diplokarya, 3.6-4.0~. Stained material fixed with methyl alcohol. Mature spores, 3.43~ 2 0.04~ X 1.61~ t 0.03~; diameter of planont, about 1.0~; mature mononucleate schizonts, 2.84.0/.&; quadrinucleate schizonts, 3.5-4.5~; diplokarya, 2.5-2.8~; sporoblasts, 4.0-4.5~ x 2.3-4.0j.b. Locality fornia.
Record:
Fresno County,
Cali-
Type Material: Holotype slide, VIII-1766, Fresno County, California; Paratype slides VIII-17-66, and 1X-7-66, Fresno County, California. LIFE
CYCLE
Figures l-50 are in a sequence which represents the developmental cycle of N.
106
KELLEN
AND
LJNDEGREN
18
23
24
25
38
48 FIGS. l-50. Developmental stages of Nosemu pbdiae sp. n. Giemsa-stained: 29, 30. Sporoblasts. 31-36. 1627. Second schizogony. 28. Diplokaryon. 39. Mature spore spore. 38. Mature spore stained by Feulgen’s method. hematoxylin. 40-44. Young spores in saline mount. 45-49. Fresh mature extruded with pressure.
1-13. First schizogony. Young spores. 37. Mature stained with Heidenhain’s spores.
50.
Polar
filament
MICROSPORIDIAN
PATHOGEN
OF
THE
INDIAN-MEAL
FIG. 51. Larvae of Plodia intmpuncte2Zu (Hiibner), showing acute infection with N. plodiue. Healthy larvae of comparable FIG. 52. Mature fresh spores of N. pZodiue in saline mount.
107
MOTH
stunted development after age at left. Phase-contrast microscopy,
10 days
of
FIG. 53. invasion
FIG. plodiue spores.
Transverse section through thoracic tergosternal muscles by N. plodiae and typical deposits of spores within muscle 54. Transverse section through optic lobe of P. interpondella, and resulting displacement of tissues: Ch, chiasmata; Ep, Both sections cut at 6 /A and treated with Sharp’s modification
108
of adult P. interpuncteZkz, showing bundles (arrows). showing typical invasion by N. epiopticon; Pe, periopticon; Sp, of Mallory’s triple stain.
MICROSPORIDIAN
PATHOGEN
plodiae in P. interpunctelh. The sequence of stages is based on our interpretation of the various forms observed in tissue smears; these preparations were made from larvae at frequent intervals following initial infections. Relatively small mono- and binucleate trophozoites measuring about 1-2 ,J in diameter were the earliest stages observed (Figs. 1 and 2). They occurred infrequently in our preparations and stained very dark with Giemsa’s solution. They were interpreted as being the immediate result of planont development. We obtained no evidence to indicate that newly emerged planonts had twin nuclei when forceably extruded from spores. There are two types of schizogony in the life cycle of N. plodiae. The first is characterized by relatively large forms that do not develop beyond the binucleate stage (Figs. 3-13). Nuclei of these schizonts were typically large and dense; however, forms with relatively distinct chromosome formation were frequently present (Figs. 69). Our observations indicated that many of the binucleate forms of the first schizogony developed directly into diplokarya and sporoblasts. The second schizogony was characterized by the formation of quadrinucleate forms having relatively small compact ringlike nuclei (Figs. 14-22). As development of this stage progressed, the nuclei became associated in pairs, and the vesicular appearance became less evident ( Fig. 23). Division of the quadrinucleate schizont gave rise to daughter diplokarya (Figs. 2%27), each of which developed into a binucleate sporoblast (Figs. 29 and 30). Although a schizont with six twin nucIei was observed in one instance (Fig 24), the second schizogony typically did not produce chains of diplokarya; the formation of twin daughter diplokarya is characteristic of this species,
OF
THE
INDIAN-MEAL
MOTH
109
The binucleate condition of the sporoblast persisted in young spores (Figs. 3134), but only a single dense nucleus was evident in mature spores stained with Giemsa’s solution ( Figs. 35-37). However, treatment with Feulgen stain clearly showed twin nuclei in mature spores (Fig. 38). Staining of the various developmental stages with Heidenhain’s hematoxylin did not disclose additional information conceming nuclear transformation (Fig. 39). Fresh smears of infected tissues frequently contained many stages of spore development. Young spores were relatively large; they had a conspicuous vacuole at one end, and usually appeared dark when viewed with phase-contrast microscopy (Figs. 40-44). Mature spores varied slightly in size and shape and were highly refractile (Figs. 4549, 52). Polar filaments were about 80 p long and were easily extruded under pressure (Fig. 50). Our conclusion that N. plodiae represents a previously undescribed species is based on the morphological characteristics of mature spores and schizogonic stages, and on the absence of tissue specificity. In the key to the known species of Microsporidia from Lepidoptera that was devised by Weiser ( 1961), N. plodiae is accommodated in the couplet including N. bombycis Nageli, N. lotmaris Weiser, and N. phryganidiae Lipa and Martignoni. Differences in spore morphology easily segregate N. plodiae from these other species. BIOLOGY
P. interpunctellu is an important pest of stored products, including a variety of dried fruits, nuts, grains, and cereal products. Biological studies of this common pest have been reported by Hamlin et al. (1931), Richards and Thomson (1932), and Tzanakakis ( 1959). Although many authors state that P. interpunctella is a native of the Old World, Heinrich (1956) indicates that
110
KEXLEN AND LlNDl3GREN
the genus Plodia is definitely of American origin. The only other known congeneric species, P. d~lorosu Dyar, and its nearest relatives, the genera Ribua, Caudellia, and Bath&u, are all confined to the New World. Larvae of P. interpunctetla are omnivorous, and cannibalism is common under crowded rearing conditions in laboratory cultures. Larvae overwinter in cold areas, but breeding may continue all year in warmer regions. The life cycle usually requires from 4 to 6 weeks, and in Fresno, California there may be four to six generations a year.
infected areas of the Malpighian tubes and the silk and salivary glands were hypertrophied by the multiplication of schixogonic stages and formation of young spores. At 144 hr after infection, larvae showed signs of stunted development, and large areas of spore deposition were present in many tissues. After about 10 days larvae became sluggish; they usually succumbed shortly thereafter (Fig. 51). Such larvae quickly became dehydrated and were sources of highly infective spores. Larvae with subacute infections survived to the adult stage. N. plodiae was capable of invading and partially destroying the reWe have examined samples of P. interproductive organs of adult moths; howpunctelhz from several areas in California, ever, eggs harboring pathogens were frebut only one population from Fresno was quently laid. In laboratory tests, about 50% infected with N. plodiae. Three out of 45 of the progenies of surviving females acadults harbored the pathogen, and an inquired infections transovarially. fected laboratory culture was subsequently The host specificity of N. pZodiue was established. An undescribed species of tested with young larvae from laboratory Thelohania frequently accompanied N. plocolonies. Larvae were maintained on 2.5 X diae in dual infections, and a morphologic10” spores/g of rearing medium for 10 days, ally similar species of Glugea was observed and histological sections were then prein larvae that were collected in Sacramento, pared and examined for pathogens. Larvae California. of P. interpunctella were also exposed to In the laboratory N. plodiae was readily the material to verify the infectivity of transmitted perorally to P. interpunctella spores. In this test larvae of P. interby adding spores to the rearing medium. punctella showed signs and symptoms of Moreover, transmission was effected rouinfection after 6 days, and most tissues tinely by contaminating the mouth parts of harbored dense accumulations of spores. young larvae with dense concentrations of Larvae of G. mellonella acquired inapparent fresh spores. infections of N. plodiue, and limited inTissue smears and serial sections of larvae vasion of the silk and salivary glands and were made at certain intervals following Malpighian tubes was evident in serial secinitial exposure to spores in order to de- tions. Data indicated that N. pZodiae was termine the course of invasion. First signs considerably less pathogenic to G. melloof infection were usually evident in the wall nella than it was to its normal host, P. of the ventriculus at about 72 hr after in- interpunctella. There was no evidence of fection; such small loci were composed infection in larvae of C. cautella, E. elutella, primarily of schizogonic stages, however, a and C. hemipterus. We concluded that N. few diplokarya and young spores were presplodiue has a limited host range, and that ent. After 96 hr, infected areas measuring maximum development is probably attained about 40 p in diameter were evident in the in P. interpunctella. Further study is reMalpighian tubes, silk glands, abdominal quired to determine the relative pathogenicity expressed in G. mellonella. muscles, and visceral fat. By 120 hr ihe
MICROSPORIDIAN
PATHOGEN
The infectivity of spores was determined after storage for various periods at three different temperatures. The criterion for infectivity was the proportion of young P. interpunctella larvae acquiring infections after 10 days of exposure to 6.4 X 10’ spores/g of rearing medium. In our tests there was no decrease in the infectivity of spores that were maintained in aqueous suspensions for up to 9 months at 20” and 6°C. Similar suspensions stored at 30°C showed a 50% reduction in infectivity after 60 days, and were not infective after 90 days. Spores that were maintained in dried frass from infected laboratory cultures of P. interpunctella were not infective after storage at 20” and 30°C for 90 days. The infectivity of dried spores kept at 6°C was reduced by 75% after 90 days; these spores were not infective after 120 days. The effect of lyophilization on spores of N. plodiae has not been determined. Further studies are under way to determine the comparative host-pathogen relationships of N. plodiae and a more virulent species of Ghgea which was isolated from a sample of a larval population of P. interpunctella collected in Sacramento, California. The feasibility of manipulating such pathogens for the regulation of natural
OF
THE
INDIAN-MEAL
111
MOTH
populations of P. interpunctella consideration.
is under
REFERENCES HAMLIN, J. C., REED, W. D., AND PHILLIPS, M. E. 1931. Biology of the Indian-meal moth on dried fruits in California. U. S. Dept. Agr. Tech. Bull. No. 242, 26 pp. HEINRICH, C. 1956. American moths of the subfamily Phycitinae. U.S. Nat. Museum Bull. No. 207, 581 pp. RICHARDS, 0. W., AND THOMSON, W. S. 1932. A contribution to the study of the genera Ephestiu, Gn. (including Strymax, Dyar), and Pbdiu, Gn. (Lepidoptera, Phycitidae), with notes on parasites of the larvae. Trans. En-
tomol. Sot. London,
80, 169-250.
SHARP, R. G. 1914. Diplodinium ecaudatum with an account of its neuromotor apparatus. Uniu. Calif. Publ. Zool., 13, 43-122. STEINHAUS, E. A. 1947. A coccidian parasite of Ephestiu kiihniella Zeller and of Plodiu interpunctella ( Hbn. ) (Lepidoptera, Phycitidae) .
J. Parasitol., 33, 29-32. STFXNHAUS, E. A., AND MARSH, G. A. 1962. Report of diagnoses of diseased insects 19511961. Hilgardia, 33, 349490. TZANAKAKIS, M. E. 1959. An ecological study of the Indian-meal moth, Plodia interpunctella (Hiibner) with emphasis on diapause. Hil-
gardia, 29, 205-246. WEISER, JAROSLAV 1961. “Die Mikrosporidien Parasiten der Insekten,” Monograph. Angew Entomol., No. 17, 149 pp. WEISER, J. 1966. Personal communication.
aI.s zur
OF INVERTEBRATE
PATHOLOGY
11,
1od-lll
1968)
(
Biology of Nosemu plodiue sp. n., a Microsporidian Pathogen of the IndianMeal Moth, Plodia inteqmwtella (Hiibner ), (Lepidoptera:Phycitidae) WILLIAM
R. KJZLLEN AND JAMES E. LINDEGRJZN
Stored-Product Insects Research Branch, Market Quality Research Division, Agricultural Research Service, U.S. Department of Agriculture, Fresco, California 93727 Received July 18, 1967 The morphology and developmental cycle of a previously undescribed species of Nosema are described from the Indian-meal moth, Plodia interpunctellu. The pathogen invades a variety of tissues and organs, including fat bodies, muscles, the gut wall, Malpighian tubes, and the salivary and silk glands. In laboratory tests larvae were readily infected by feeding them fresh spores. Larvae with acute infections showed signs and symptoms of disease 6 days after initial feeding; such larvae were stunted, and usually succumbed before pupation. Larvae with subacute infections survived to the adult stage. Pathogens invaded developing ova, and transovarian transmission was observed. Larvae of the almond moth, Cadra cautellu (Walker), the tobacco moth, Ephestia elutella (Hiibner), and the dried fruit beetle. Carpophilus hemipterus (Linnaeus), did not become infected when fed spores; however, larvae of the greater wax moth, GalleTiu melkmda (Linnaeus) acquired subacute infections.
INTR~Du(;TION
The Indian-meal moth, Plodif interpunctella, is one of the most widely distributed and economically important lepidopterous pests known to infest stored products. Efforts to control this moth have received worldwide attention, and although it has been the subject of many biological and ecological investigations, very little is known about its associated protozoan pathogens. The only record of a microsporidian pathogen of P. interpunctellu was published by Steinhaus and Marsh (1962) in a report of diagnoses of diseased insects. They noted having examined a diseased larva which was received from J. Weiser in 1953; the specimen was from a laboratory culture that had been maintained in Prague, Czechoslovakia. The larva was diagnosed as being infected with Nosema; however, descriptions of the spores or of the affected tissues
were not included in the report. From other geographical sources they reported the presence of the schizogregarine Matte& dispora, and earlier Steinhaus (1947) reported the finding of the coccidian Ad&m mesnili in the insect. The original slides or subsequent additional preserved material of the Prague Nosew are not available for comparative study; however, Dr. Weiser has kindly examined spores of the Nosemu which we isolated from diseased adults of P. interpunctella collected in Fresno, and he has compared our material with his original laboratory notes. Weiser (1966) has indicated that the California species is not similar to the Nosemu that he had observed in Prague. We have, therefore, concluded from studies of the morphology, life cycle, and hostpathogen relationships that the Nosema from Fresno has not been previously re104
MIC33OSPOIUDIAN
PATHOGEN
ported from P. interpunctella, and that it represents an undescribed species. MATERIALS
AND
METHODS
All stages of P. interpunctella were collected from infested dried figs in a warehouse in Fresno, California, during the summer of 1966. Larvae and adults were returned to the laboratory in plastic bags, and dissected within 24 hr in 0.65% saline solution Squash preparations of fresh tissues were examined with a phase-contrast microscope at a magnification of 1250 diameters. Smears of infected tissues were fixed with methyl alcohol and stained with Giemsa’s solution. Whole bodies of laboratory-infected larvae and adults were fixed in Duboscq-Brasil solution for 24 hr, embedded in paraffin (mp 60°C ) , and sectioned at 6~. Serial sections were stained with Heidenhain’s hematoxylin or Sharp’s ( 1914) modification of Mallory’s triple stain. Spores were measured with an A. E. I. Vickers image-splitting eyepiece at a magnification of 1006 diameters. The mean value and standard error of the mean were determined from measurements of 50 spores. The sizes of typical stages in the schizogonic cycle of the pathogen were also determined. Infectivity studies were conducted with laboratory strains of Plodia interpunctella, Ephestiu elutellu, Cadra cautella, Gall&a mellonella, and Carpophilus hemipterus. Cultures of the moths had been maintained in the insectary for at least 5 years. Larvae were reared in l-quart Mason jars, and were provided a rearing medium consisting of 16 parts chicken mash, 1 part honey, and 1 part glycerol. Calcium propionate was added to the mixture to retard fungus growth. SYSTEMATICS
The holotype and 10 paratype slides of the new species have been deposited in the
OF
THE
INDIAN-MEAL
105
MOTH
type collection of the U. S. National Museum. Additional paratype slides have been deposited in the collections of J. J. Lipa, Laboratory of Biological Control, Institute of Plant Protection, Poznan, Poland; J. Weiser, Department of Insect Pathology, Institute of Biology, Czechoslovakian Academy of Sciences, Prague; J. P. Kramer, Department of Entomology and Limnology, Cornell University, Ithaca, New York; and in the collection of the authors. Nosema plodiae sp. n. (Figs. l-50) Derivation Plodia.
of
name:
The
Nosema
of
Host: All stages of Plodia interpunctellu (Hiibner). Invades many tissues and glands, including silk and salivary glands, Malpighian tubes, gut wall, ovaries, oviduct, testes, fat bodies, muscle, and nervous tissue (Figs. 53 and 54). Morphology: Fresh material. Mature spores, 4.09p + 0.24~ X 1.89, & 0.03~; polar filament, SO,; immature spores with distinct vacuoles, 5.11~ k 0.31~ X 2.58~ 4 0.15~. Diameter of mononucleate schizonts, 2.6-3.6~; quadrinucleate schizonts, 5.0-5.4~; diplokarya, 3.6-4.0~. Stained material fixed with methyl alcohol. Mature spores, 3.43~ 2 0.04~ X 1.61~ t 0.03~; diameter of planont, about 1.0~; mature mononucleate schizonts, 2.84.0/.&; quadrinucleate schizonts, 3.5-4.5~; diplokarya, 2.5-2.8~; sporoblasts, 4.0-4.5~ x 2.3-4.0j.b. Locality fornia.
Record:
Fresno County,
Cali-
Type Material: Holotype slide, VIII-1766, Fresno County, California; Paratype slides VIII-17-66, and 1X-7-66, Fresno County, California. LIFE
CYCLE
Figures l-50 are in a sequence which represents the developmental cycle of N.
106
KELLEN
AND
LJNDEGREN
18
23
24
25
38
48 FIGS. l-50. Developmental stages of Nosemu pbdiae sp. n. Giemsa-stained: 29, 30. Sporoblasts. 31-36. 1627. Second schizogony. 28. Diplokaryon. 39. Mature spore spore. 38. Mature spore stained by Feulgen’s method. hematoxylin. 40-44. Young spores in saline mount. 45-49. Fresh mature extruded with pressure.
1-13. First schizogony. Young spores. 37. Mature stained with Heidenhain’s spores.
50.
Polar
filament
MICROSPORIDIAN
PATHOGEN
OF
THE
INDIAN-MEAL
FIG. 51. Larvae of Plodia intmpuncte2Zu (Hiibner), showing acute infection with N. plodiue. Healthy larvae of comparable FIG. 52. Mature fresh spores of N. pZodiue in saline mount.
107
MOTH
stunted development after age at left. Phase-contrast microscopy,
10 days
of
FIG. 53. invasion
FIG. plodiue spores.
Transverse section through thoracic tergosternal muscles by N. plodiae and typical deposits of spores within muscle 54. Transverse section through optic lobe of P. interpondella, and resulting displacement of tissues: Ch, chiasmata; Ep, Both sections cut at 6 /A and treated with Sharp’s modification
108
of adult P. interpuncteZkz, showing bundles (arrows). showing typical invasion by N. epiopticon; Pe, periopticon; Sp, of Mallory’s triple stain.
MICROSPORIDIAN
PATHOGEN
plodiae in P. interpunctelh. The sequence of stages is based on our interpretation of the various forms observed in tissue smears; these preparations were made from larvae at frequent intervals following initial infections. Relatively small mono- and binucleate trophozoites measuring about 1-2 ,J in diameter were the earliest stages observed (Figs. 1 and 2). They occurred infrequently in our preparations and stained very dark with Giemsa’s solution. They were interpreted as being the immediate result of planont development. We obtained no evidence to indicate that newly emerged planonts had twin nuclei when forceably extruded from spores. There are two types of schizogony in the life cycle of N. plodiae. The first is characterized by relatively large forms that do not develop beyond the binucleate stage (Figs. 3-13). Nuclei of these schizonts were typically large and dense; however, forms with relatively distinct chromosome formation were frequently present (Figs. 69). Our observations indicated that many of the binucleate forms of the first schizogony developed directly into diplokarya and sporoblasts. The second schizogony was characterized by the formation of quadrinucleate forms having relatively small compact ringlike nuclei (Figs. 14-22). As development of this stage progressed, the nuclei became associated in pairs, and the vesicular appearance became less evident ( Fig. 23). Division of the quadrinucleate schizont gave rise to daughter diplokarya (Figs. 2%27), each of which developed into a binucleate sporoblast (Figs. 29 and 30). Although a schizont with six twin nucIei was observed in one instance (Fig 24), the second schizogony typically did not produce chains of diplokarya; the formation of twin daughter diplokarya is characteristic of this species,
OF
THE
INDIAN-MEAL
MOTH
109
The binucleate condition of the sporoblast persisted in young spores (Figs. 3134), but only a single dense nucleus was evident in mature spores stained with Giemsa’s solution ( Figs. 35-37). However, treatment with Feulgen stain clearly showed twin nuclei in mature spores (Fig. 38). Staining of the various developmental stages with Heidenhain’s hematoxylin did not disclose additional information conceming nuclear transformation (Fig. 39). Fresh smears of infected tissues frequently contained many stages of spore development. Young spores were relatively large; they had a conspicuous vacuole at one end, and usually appeared dark when viewed with phase-contrast microscopy (Figs. 40-44). Mature spores varied slightly in size and shape and were highly refractile (Figs. 4549, 52). Polar filaments were about 80 p long and were easily extruded under pressure (Fig. 50). Our conclusion that N. plodiae represents a previously undescribed species is based on the morphological characteristics of mature spores and schizogonic stages, and on the absence of tissue specificity. In the key to the known species of Microsporidia from Lepidoptera that was devised by Weiser ( 1961), N. plodiae is accommodated in the couplet including N. bombycis Nageli, N. lotmaris Weiser, and N. phryganidiae Lipa and Martignoni. Differences in spore morphology easily segregate N. plodiae from these other species. BIOLOGY
P. interpunctellu is an important pest of stored products, including a variety of dried fruits, nuts, grains, and cereal products. Biological studies of this common pest have been reported by Hamlin et al. (1931), Richards and Thomson (1932), and Tzanakakis ( 1959). Although many authors state that P. interpunctella is a native of the Old World, Heinrich (1956) indicates that
110
KEXLEN AND LlNDl3GREN
the genus Plodia is definitely of American origin. The only other known congeneric species, P. d~lorosu Dyar, and its nearest relatives, the genera Ribua, Caudellia, and Bath&u, are all confined to the New World. Larvae of P. interpunctetla are omnivorous, and cannibalism is common under crowded rearing conditions in laboratory cultures. Larvae overwinter in cold areas, but breeding may continue all year in warmer regions. The life cycle usually requires from 4 to 6 weeks, and in Fresno, California there may be four to six generations a year.
infected areas of the Malpighian tubes and the silk and salivary glands were hypertrophied by the multiplication of schixogonic stages and formation of young spores. At 144 hr after infection, larvae showed signs of stunted development, and large areas of spore deposition were present in many tissues. After about 10 days larvae became sluggish; they usually succumbed shortly thereafter (Fig. 51). Such larvae quickly became dehydrated and were sources of highly infective spores. Larvae with subacute infections survived to the adult stage. N. plodiae was capable of invading and partially destroying the reWe have examined samples of P. interproductive organs of adult moths; howpunctelhz from several areas in California, ever, eggs harboring pathogens were frebut only one population from Fresno was quently laid. In laboratory tests, about 50% infected with N. plodiae. Three out of 45 of the progenies of surviving females acadults harbored the pathogen, and an inquired infections transovarially. fected laboratory culture was subsequently The host specificity of N. pZodiue was established. An undescribed species of tested with young larvae from laboratory Thelohania frequently accompanied N. plocolonies. Larvae were maintained on 2.5 X diae in dual infections, and a morphologic10” spores/g of rearing medium for 10 days, ally similar species of Glugea was observed and histological sections were then prein larvae that were collected in Sacramento, pared and examined for pathogens. Larvae California. of P. interpunctella were also exposed to In the laboratory N. plodiae was readily the material to verify the infectivity of transmitted perorally to P. interpunctella spores. In this test larvae of P. interby adding spores to the rearing medium. punctella showed signs and symptoms of Moreover, transmission was effected rouinfection after 6 days, and most tissues tinely by contaminating the mouth parts of harbored dense accumulations of spores. young larvae with dense concentrations of Larvae of G. mellonella acquired inapparent fresh spores. infections of N. plodiue, and limited inTissue smears and serial sections of larvae vasion of the silk and salivary glands and were made at certain intervals following Malpighian tubes was evident in serial secinitial exposure to spores in order to de- tions. Data indicated that N. pZodiae was termine the course of invasion. First signs considerably less pathogenic to G. melloof infection were usually evident in the wall nella than it was to its normal host, P. of the ventriculus at about 72 hr after in- interpunctella. There was no evidence of fection; such small loci were composed infection in larvae of C. cautella, E. elutella, primarily of schizogonic stages, however, a and C. hemipterus. We concluded that N. few diplokarya and young spores were presplodiue has a limited host range, and that ent. After 96 hr, infected areas measuring maximum development is probably attained about 40 p in diameter were evident in the in P. interpunctella. Further study is reMalpighian tubes, silk glands, abdominal quired to determine the relative pathogenicity expressed in G. mellonella. muscles, and visceral fat. By 120 hr ihe
MICROSPORIDIAN
PATHOGEN
The infectivity of spores was determined after storage for various periods at three different temperatures. The criterion for infectivity was the proportion of young P. interpunctella larvae acquiring infections after 10 days of exposure to 6.4 X 10’ spores/g of rearing medium. In our tests there was no decrease in the infectivity of spores that were maintained in aqueous suspensions for up to 9 months at 20” and 6°C. Similar suspensions stored at 30°C showed a 50% reduction in infectivity after 60 days, and were not infective after 90 days. Spores that were maintained in dried frass from infected laboratory cultures of P. interpunctella were not infective after storage at 20” and 30°C for 90 days. The infectivity of dried spores kept at 6°C was reduced by 75% after 90 days; these spores were not infective after 120 days. The effect of lyophilization on spores of N. plodiae has not been determined. Further studies are under way to determine the comparative host-pathogen relationships of N. plodiae and a more virulent species of Ghgea which was isolated from a sample of a larval population of P. interpunctella collected in Sacramento, California. The feasibility of manipulating such pathogens for the regulation of natural
OF
THE
INDIAN-MEAL
111
MOTH
populations of P. interpunctella consideration.
is under
REFERENCES HAMLIN, J. C., REED, W. D., AND PHILLIPS, M. E. 1931. Biology of the Indian-meal moth on dried fruits in California. U. S. Dept. Agr. Tech. Bull. No. 242, 26 pp. HEINRICH, C. 1956. American moths of the subfamily Phycitinae. U.S. Nat. Museum Bull. No. 207, 581 pp. RICHARDS, 0. W., AND THOMSON, W. S. 1932. A contribution to the study of the genera Ephestiu, Gn. (including Strymax, Dyar), and Pbdiu, Gn. (Lepidoptera, Phycitidae), with notes on parasites of the larvae. Trans. En-
tomol. Sot. London,
80, 169-250.
SHARP, R. G. 1914. Diplodinium ecaudatum with an account of its neuromotor apparatus. Uniu. Calif. Publ. Zool., 13, 43-122. STEINHAUS, E. A. 1947. A coccidian parasite of Ephestiu kiihniella Zeller and of Plodiu interpunctella ( Hbn. ) (Lepidoptera, Phycitidae) .
J. Parasitol., 33, 29-32. STFXNHAUS, E. A., AND MARSH, G. A. 1962. Report of diagnoses of diseased insects 19511961. Hilgardia, 33, 349490. TZANAKAKIS, M. E. 1959. An ecological study of the Indian-meal moth, Plodia interpunctella (Hiibner) with emphasis on diapause. Hil-
gardia, 29, 205-246. WEISER, JAROSLAV 1961. “Die Mikrosporidien Parasiten der Insekten,” Monograph. Angew Entomol., No. 17, 149 pp. WEISER, J. 1966. Personal communication.
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