The morphology and development of Nosema carpocapsae, a microsporidian pathogen of the codling moth, Cydia pomonella (Lepidoptera: Tortricidae) in New Zealand

JOURNAL

OF INVERTEBRATE

PATHOLOGY

38, 315-329 (1981)

The Morphology and Development of Nosema carpocapsae, a Microsporidian Pathogen of the Codling Moth, Cydia pomonella (Lepidoptera: Tortricidae) in New Zealand L. A. MALONE’ Department

of Zoology,

of Auckland,

University

Neic* Zealand

AND

P. J. WlGLEY Entomology

Division,

Department

of Scientific Auckland.

and Industrial New Zealand

Research.

Mt.

Albert.

Received May 20, 1980 The morphology of Nosema carpocapsae and its development in experimentally infected codling moth larvae are described. Spherical &nucleate meronts were the first stages. Nuclear division produced binucleate meronts which were the most abundant vegetative stage, although additional uninucleate and a few tetranucleate meronts were also observed at this time. All meronts were spherical and ranged from 2.8 to 5.8 pm in diameter. Uninucleate and binucleate fusiform sporonts then appeared followed by some tetranucleate and dividing forms. Oval sporoblasts developed after these and did not divide before maturing into spores. Sporonts were approximately 5.0 to 7.9 x 2.4 to 3.0 pm. Spores developed in all host tissues except the nervous tissue. The binucleate spores showed considerable variation in spore size, 2.4 to 3.9 x 1.3 to 3.1 pm (alcohol fixed, Giemsa stained). The polar filament was usually coiled 11 times (range 9 to 13) at an angle of 53” to the long axis of the spore. Its maximum observed length was 75 pm. KEY WORDS: Cydia pomonella; codling moth; Microsporida: Nosema carpocapsae, life cycle, generation time, spore size, spore ultrastructure.

INTRODUCTION

Poland. N. carpocapsae infections have since been recorded in Notocellia uddmanniana in Poland (Lipa, 1977) and in Carpocapsa pyrivoru in the Soviet Union (Issi and Lipa, 1968). Other recent papers (Nordin and Maddox, 1974; Watanabe, 1976) mention Nosema carpocapsae in attempts to identify other microsporidia but do not add to its description. This paper describes the life cycle and histopathology of N. carpocapsae in codling moth as shown by a time-course experiment. Changes in the relative proportions of the various life stages during the course of infection were examined and this information was combined with their sequence of appearance to suggest a possible life cycle for N. carpocupsae. The results of an electron microscope study of the spores are presented.

is a common pathogen of codling moth in New Zealand (Malone and Wigley, 1980). It is widespread in Europe (Huger, pers. commun.) and was first reported from codling moth collected at Saint-Genis-Lava1 in France by Paillot (1938, 1939), who described details of its life cycie and histopathology, and noted that it was transovarially transmitted. Weiser (1961) listed N. carpocapsae in his monograph, noting the morphology of developmental stages and histopathological details and suggested that transovum transmission may also occur. Lipa (1963) redescribed the life cycle and histopathology, and investigated its epizootiology in Nosema

carpocapsae

’ Present address: DSIR Entomology Division, F’rivate Bag, Auckland, New Zealand. 315

0022-2011/81/060315-15$01.00/O Copyright All rights

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

316

MALONE

AND

WIGLEY

MATERIALS AND METHODS nucleate, binucleate, and tetranucleate merHealthy codling moth larvae were ob- onts, sporonts, and mature spores. Every 24 hr two additional larvae were tained from our stock colony, reared on arfixed in Bouin’s fluid, embedded in 54°C tificial diet (Brinton et al., 1969) at 28°C melting point wax (Paraplast), sectioned at with 60% RH, in a 16:8-hr 1ight:dark regimen. A spore suspension of 1 x lo8 5 pm, and stained in Giemsa stain. Infected spores/ml was prepared by macerating in- adults of each sex were also prepared for fected larvae in distilled water in a glass histological study in this way. The nuclei of the spores were revealed by Giesma stainDounce tissue grinder. The homogenate ing after hydrolysis in 1 N HCl at 60°C for 10 was filtered through nylon gauze to remove min. Polar filaments were extruded by fragments of cuticle and spore concentramechanical pressure applied with a small tion was determined by a stained dry film vice and measured from Polaroid prints. technique (Wigley, 1980). Spores and vegetative stages were meaThird-instar larvae were infected by sured with eyepiece micrometer. Spores feeding them individually in plastic tubes (7.5 x 1.0 cm) on cylindrical plugs (0.5 x were measured from wet mounts of Giesma0.7 cm) of artificial diet (Singh, 1977) to stained preparations; all other stages were which 25 ~1 of the spore suspension had measured stained only. For electron mibeen applied. After 30 hr, fresh plugs were croscopy, pellets of spores set in small agar substituted for the spore-contaminated diet. blocks were fixed in buffered (0.02 M phosphate, pH 6.9) 5% glutaraldehyde and postLarvae were infected and subsequently fixed in similarly buffered 1% 0~0,. The reared in constant light at 21°C and 50% RH. spores were dehydrated in ethanol or aceLarvae were killed at 6, 24, 30, and 36 hr tone and embedded in Spurr’s embedding and every 12 hr thereafter until 276 hr from medium (Spurr, 1969); a JEOL JEM 1008 the initial exposure. Three larvae were reelectron microscope was used at an accelmoved at each time and smears of pieces of erating voltage of 80 kV. the fat body, midgut, and silk gland of each larva were made on three areas of a slide. RESULTS These were fixed in ethanol for 2 min, immersed in picric acid at 40°C for 2 min, and The results are presented in three parts. then stained in 2% (v/v) Giemsa stain The first deals with the life cycle which was (Gurr’s Improved R66) in 0.02 M phosphate examined both qualitatively and quantitabuffer, pH 6.9, overnight. tively, the second with the histopathology, Each smear was examined at 250x and and the third part describes features relat900~ magnification to study the morpholing to the spore, in particular the polar filaogy of life stages and to determine the time ment, spore size, and spore ultrastructure. at which each first appeared. Control smears of 100 uninfected third- and The L$e Cycle fourth-instar larvae were also examined at Qualitative description. Stages of the life the end of the experiment. Changes in the cycle are illustrated in Figures 1 to 38. With relative numbers of life stages were deter- Giemsa’s stain, the nuclei of the vegetative mined by a series of sample counts on stages stained red and the cytoplasm smears from an infected larva from each stained blue. In the midgut the first stages time point. At each count, the number of observed were two densely stained uninueach stage seen in a randomly chosen field cleate cells, 3.70 and 3.86 pm in diameter of view at 900X magnification was re- (Fig. l), which appeared 30 hr after infeccorded. Fifteen counts were made for each tion. At 48 hr, two protozoan cells with individual, five from each organ as above. twin-lobed nuclei (Fig. 2) were seen in one Stages identified and recorded were uni- specimen and a uninucleate cell and a dis-

Nosema

6

16

7

8

carpocapsae

IN

9

17

20

0f! 21

22

23

24

25

-

26 -1

FIGS. I-26.

Appearance of developmental stages of in air-dried, alcohol-fixed, and Giemsa-stained smears. Fig. 1. Uninucleate meront. Fig. 2. Uninucleate meront undergoing nuclear division. Figs. 3 and 4. Binucleate meronts. Fig. 5. Elongate binucleate meront. Fig. 6. Two uninucleate meronts. Fig. 7. Binucleate meront undergoing nuclear division. Figs. 8 and 9. Tetranucleate meronts. Fig. 10. Tetranucleate meront undergoing cytoplasmic cleavage. Fig. 11. Two binucleate meronts. Fig. 12. Very elongate binucleate meront. Figs. 13 and 14. Binucleate fusiform sporonts. Fig. 15. Binucleate fusiform sporont undergoing cytoplasmic cleavage. Fig. 16. Uninucleate fusiform sporont. Fig. 17. Uninucleate fusiform sporont undergoing nuclear division. Fig. 18. Tetranucleate fusiform sporont. Fig. 19. Tetranucleate fusiform sporont undergoing cytoplasmic cleavage. Fig. 20. Uninucleate oval sporoblast. Fig. 21. Uninucleate oval sporoblast undergoing nuclear division. Fig. 22. Binucleate oval sporoblast. Fig. 23. Binucleate, vacuolated sporoblast. Figs. 24 and 25. Immature spores. Fig. 26. Mature spore. Noserna

carpocapsae

tinctly binucleate cell in another. By 60 hr, binucleate (Figs. 3-5, 11, 12, 28, 29), uninucleate (Figs. 6, 27), and tetranucleate meronts (Figs. 8, 9, 30) were seen, not as

CODLING

MOTH

317

densely stained as the early uninucleate stages. Some binucleate cells appeared to be undergoing nuclear division and others cytoplasmic cleavage (Figs. 5, 6). Cleaving tetranucleate stages (Figs. 9, 10) and uninuclear division and cytoplasmic cleavage (Fig. 2) were also observed at this time. The first sporonts appeared at 72 hr. These were uninucleate or binucleate spindle-shaped cells, usually faintly staining and sometimes vacuolated (Figs. 13, 14, 16, 31-33). Fusiform sporonts in various stages of nuclear division and cytoplasmic cleavage (Figs. 15, 17-19, 34) were seen at 84 hr together with faintly stained oval sporoblasts with one or two nuclei (Figs 20-22, 36, 37). Some binucleate oval sporoblasts had a well-defined vacuole at one end (Fig. 23). Immature spores with blue cytoplasm and two red nuclei (Figs. 24, 25) and thick-walled mature spores (Figs. 26, 38) were first detected at this time. Mature spores had two nuclei. Developmental stages appeared in a similar sequence in the fat body smears, although the first stages were not observed until 72 hr after infection. By contrast, no stages were observed in the silk glands until 96 hr when all stages and mature spores were present. Satisfactory smears of the silk glands were difficult to obtain and this may explain the apparent absence of early stages in this tissue. None of the stages described above were found in the control smears. The dimensions of these life cycle stages are given in Table 1. Quanfitafit~e aspects. The proportions of stages progressively changed as the proportion of each type of meront or sporont reached a peak and then declined (Fig. 39). Spores also built up gradually until they accounted for nearly all of the stages seen. Binucleate meronts were the first to appear in detectable numbers, followed by uninucleate meronts, and later by tetranucleate meronts. Two methods were used to measure the minimum generation time. The

MALONE

AND WIGLEY

28

FIGS. 27-38. Smears of codling moth larvae infected with Nosema carpocapsae (air dried, alcohol fixed, and Giemsa stained). Fig. 27. Uninucleate meront. Figs. 28 and 29. Binucleate meronts. Fig. 30. Tetranucleate and binucleate meronts. Fig. 31. Uninucleate fusiform sporont. Figs. 32 and 33. Binucleate fusiform sporonts. Fig. 34. Cleaving fusiform sporont. Fig. 35. Tetranucfeate fusiform sporont. Fig. 36. Uninucleate oval sporoblast. Fig. 37. Binucleate oval sporoblast. Fig. 38. Mature spores.

Nosema

carpocapsae

IN

TABLE MEASUREMENTS

(pm) OF GIEMSA-STAINED

CODLING

319

MOTH

1

DEVELOPMENTAL

STAGESOF

N

Uninucleate meront Binucleate meront Tetranucleate meront Uninucleate sporont (fusiform) Binucleate sporont (fusiform) Uninucleate sporoblast (oval) Binucleate sporoblast (oval) Binucleate sporoblast (oval. vacuolated) Spores

M ? SD

Uninucleote

Range

N

M -+ SD

Range

30 30 30

3.20 k 0.06 3.66 z!z0.09 5.42 t 0.18

2.50-3.78 2.90-4.79 4.47-7.17

30 30 30

2.87 t 0.06 3.43 ? 0.08 4.82 + 0.13

2.09-3.62 2.74-4.75 4.03-6.92

25

6.79 t 0.23

5.15-9.26

25

2.81 + 0.07

1.69-3.30

30

7.46 zi 0.23

5.07- 10.31

30

2.92 i 0.04

2..54-3.62

25

5.70 2 0.01

4.11-7.25

25

2.55 2 0.06

1.97-3.06

25

5.37 + 0.19

4.11-7.41

25

2.53 k 0.07

2.13-3.18

30 200

5.96 k 0.15 3.13 c 0.02

4.95-7.65 2.42-3.75

30 200

2.62 -t 0.05 1.88 2 0.01

2.01-3.02 1.29-2.25

time taken for development from spore to spore (Kramer, 1965) was 84 hr at 21°C and the interval elapsing before 50% of the stages seen in a sample were spores (Milner, 1972a) ranged from 144 to 180 hr (6 to 7% days).

Binucleate

carpocapsur

Width

Length Stage

Nosema

meronts

meronts

Histopathology N. carpocupsae produced no obvious external symptoms in larvae, although milky white thickenings on the silk glands were visible upon dissection. The tissues in which spores developed and the sequence in which they became infected are presented in Table 2. Spores appeared in the midgut epithelium at first (Fig. 40) and eventually occurred in most larval tissues (Figs. 41-4.5). Adult tissues were similarly infected and spores were also found in the pericardial cells and the reproductive organs. In the female, the oocytes, the epithelium of the ovarioles and oviducts, and the bursa copulatrix (Fig. 46) were infected. In the male, spores were observed in the epithelia of the testis and the reproductive ducts and also among the spermatozoa within (Fig. 47). No spores were observed in the nervous tissue of infected larvae or adults. The Spore

0 30

70 HOURS

110 AFTER

150

190

INFECTION

FIG. 39. Changes in the proportions of life stages of during Nosema carpocapsae development. The proportion of each stage present at a particular time after the initial exposure to spores is given as a percentage of the total number of stages seen at that time.

Polar filament. Polar filaments were difficult to extrude. Considerable mechanical pressure was necessary to force extrusion as other commonly used techniques (Vavra and Maddox, 1976) were unsuccessful. At the completion of the process, stained polar filaments with an empty spore at one end

320

MALONE

TABLE 2 SEQUENCE OF INFECTION OF TWXJES OF CODLING MOTH LARVAE WITH Nosema carpocapsae Time

point

(hr after Infected tissues

infection)

96

Midgut

144

Midgut

192

Midgut, silk glands, fat body, hemolymph Malpighian tubules, tracheal epithelium, gonad epithelium

240

Midgut, silk glands, fat body, hemolymph Malpighian tubules, tracheal epithelium, gonad epithelium

408

Midgut, silk glands, fat body, hemolymph, Malpighian tubules, tracheal epithelium, gonad epithelium, epidermis, muscle

AND

WIGLEY

with normal spores, in diseased larvae from Havelock North (Table 4). Their stained appearance was similar to that of normal spores and although of similar width, they were considerably longer and oval or spindle shaped. Spore ultrastructure. The spores possess two nuclei (Fig. 48). Longitudinal and cross-sectional views show that the polar filament occurs in the spore as a single coil of 9 to 13 turns. In cross section, the polar filament is almost circular. Two peripheral layers, the outer electron lucent and the inner electron dense, enclose an electronlucent area with a dense central core which appears as a tube in some sections. These layers correspond to the outer and inner polar tubes. Only one section was suitable for measuring the anterior angle of coiling, which was 53”. The exospore appeared as an electrondense rippled layer with no clear evidence of layering within. The endospore is a thick electron-lucent layer, thinning considerably at the polar filament insertion point (Fig. 49). The anterior third of the mature spore contains a lamellar polaroplast (Figs. 48, 49). The polar filament is attached to the anterior endospore by an anchoring disc. An electron-dense area at the anterior pole of some immature spores (Fig. 50) was interpreted as part of the anchoring disc since fully developed polar filaments were present. Longitudinal and oblique sections of immature spores generally revealed a vacuolated area in the anterior third of the spore (Fig. 50) sometimes containing scattered lamellae (Fig. 51). Cytoplasm occupies the remaining space in the spore. In some mature spores this is packed with free ribosomes (Fig. 48). A scanning electron micrograph of mature spores shows the finely wrinkled exospore suggested by ultrathin sections (Fig. 52).

and a sporoplasm at the other were not found. It therefore remains uncertain whether the maximum extruded length has been recorded. The longest polar filament was 75 ,um. Spore size. The mean spore size of IV. carpocapsae in New Zealand is 3.16 x 1.7 km. A study of the geographical variability of this character was made. The dimensions of spores from different regions of New Zealand, together with those of spores, originally from Te Atatu, Auckland, after their passage through four generations of laboratory-reared codling moth, are given in Table 3. Spores from Te Atatu differed significantly (P < 0.05) in length from those from Havelock North but not Taranaki or Otago. The widths of spores from Te Atatu differed significantly (P < 0.001) from those from Taranaki, Havelock North, or Otago. After passaging through four generations of laboratory hosts, Te Atatu spores differed in length (P < 0.05) and width (P < 0.001) DISCUSSION from their original condition. The ranges of lengths and widths overlap for spores from This study confirms the findings of Paillot all localities. (1938, 1939) and Lipa (1963) on the develSome macrospores were found, together opmental stages of N. carpocapsae and

Nosema

carpocapsae

IN

CODLING

MOTH

321

FIGS. 40-43. Giemsa-stained sections of infected larval codling moth tissues showing Noset~u c-arpocapsae spores. Fig. 40. Midgut cells. Note spores being expelled into the gut lumen. Fig. 41. Fat body cells. Fig. 42. Epidermal cells. Fig. 43. Tracheal epithelium.

provides quantitative evidence for their sequence in the life cycle. Paillot (1938, 1939) also noted the long, spindle-shaped tetranucleate sporonts which are unusual for Nosema spp. Lipa (1963) described the

sporonts of N. carpocapsae as-elongate but drew them as oval cells. However, in his report of N. carpocapsae from Notocrllia uddmanniana (Lipa, 1977), the sporonts are described as “spindle-shaped binucfeate

322

MALONE

AND WIGLEY

FIGS. 44-47. Giemsa-stained sections of infected larval and adult codling moth tissues showing Nosema carpocupsae spores. Fig. 44. Malpighian tubule. Fig. 45. Muscle block. Fig. 46. Bursa copulatrix of infected adult female. Fig. 47. Spores among the spermatozoa of an infected adult male.

bodies.” Neither Paillot nor Lipa followed the life cycle of N. carpocapsae with a time-course experiment but merely assembled the stages into a likely sequence.

Time-course experimentation has obvious advantages over this method. The proposed life cycle for N. carpocapsae presented in Figure 53 was made on the

Nosema carpocapsue IN CODLING

MEASUREMENTS (Fm) OF Nosema

323

MOTH

TABLE 3 SPORES FROM DIFFERENT NEWZEALAND

carpocapsae

Width

Length Origin of spores

N

M f SD

Te Atatu, Auckland Te Atatu. after four passages through cultured hosts Taranaki Havelock North Otago

341

3.23 + 0.02

200 100 100 100

3.13 3.15 3.11 3.16

2 ? 2 ?

0.02 0.03 0.03 0.02

LOCALITIES

Range

N

M t_ SD

A0

2.50-4.25

341

2.00 k 0.01

A

Range -1.41-3.13

B A, B B A, B

2.42-3.75 2.70-3.78 2.58-3.86 2.62-3.86

200 100 100 100

1.88 5 1.70 z 1.75 c 1.69 +

B c c C

1.29-2.25 1.29-2.17 1.25-2.21 1.37-2.21

0.01 0.02 0.02 0.02

Nofe. All spores were alcohol fixed and Giemsa stained. ‘I Values without a letter in common differ significantly at P = 0.05.

basis of qualitative and quantitative observations. Binucleate meronts and binucleate sporonts were the most abundant life stages seen and these are shown as the foci of the meront and sporont replicative cycles. Two uninucleate meronts observed at 30 hr were the first stages seen after sporoplasm penetration, but these did not appear in numbers large enough to be detected by the sample counts (Fig. 39). An initial cleavage of the sporoplasm is proposed to produce these early uninucleate stages. Binucleate meronts appear in large numbers at 48 hr (Fig. 39) and probably have arisen from nucelar division of the early uninucleate meronts. Large numbers of uninucleate meronts first appear at 60 hr. The most likely source of such large numbers of uninucleate meronts is the cleavage of binucleate meronts. Subsequent nuclear division of the uninucleate meronts produces further binucleate merants. Tetranucleate meronts first appeared TABLE 4 MEASUREMENTS (pm) OF MACROSFQRESOF

Length Width

Nosema

carpocapsae

N

M -t SD

Range

20 20

6.53 c 0.18 2.07 2 0.05

5.19-8.29 1.77-2.58

Note. All spores were alcohol-fixed stained.

and Giemsa

at 84 hr. They were uncommon and probably represent a number of binucleate meronts that have undergone nuclear division before cytoplasmic cleavage. A similar replication cycle explains the variety of spindle-shaped sporonts seen. Dividing and uninucleate forms were seen infrequently indicating that proliferation of sporonts is limited and that monosporoblastic sporonts predominate in sporogony . Reports of the histopathology of N. curpocapsae have minor variations but they generally agree that infection and replication occurs in all tissues except nervous tissue. The tissues infected in this study are the same as those reported by Paillot and Lipa. The time-course experiment shows that infection begins in the midgut and later spreads to other tissues. Weiser (1961), although noting the same pattern of tissue infection, stated that infection begins in the Malpighian tubules and silk glands, but does not give details of how this sequence was determined. N. carpocapsae has not been studied previously with the electron microscope. The number of coils of the polar filament varies widely between microsporidian species, ranging from 3 to 4 in EncephalitoZOOS cuniculi (see Petri and Schiodt, 1966) to 44 coils in Nosema apis (see Scholtyseck and Danneel, 1962). N. carpocapsae has

324

MALONE

AND

WIGLEY

FIG. 48. Oblique section of a mature spore of Nosema carpocapsae. The two nuclei (N) occupy a central position surrounded by a cytoplasm packed with free ribosomes (R), the coiled polar filament (PF), and the lamellar polaroplast (LPI.

about 11 coils, as do most Nosema species (Milner, 1972b). The anterior angle of coiling of N. carpocapsae, 53”, is well within the range of angles measured for other microsporidia, for example 35” in Nosema

to 80” in Glugea weissenbergi (Burges et al., 1974). New Zealand samples vary considerably in spore size but conform to that previously published for N. carpocapsae. Macrooryzaephili

Nosema

carpocapsae

IN

CODLING

MOTH

325

FIG. 49. Median longitudinal section of a mature spore of Nosema carpocopsa~.The lamellar polaroplast (LP) surrounds the descending polar filament. The endospore is considerably narrower a! the polar filament insertion point (IP). The two nuclei (N) are closely apposed.

spores occur in New Zealand as elsewhere. The published sizes of N. carpocapsae spores vary considerably. Paillot (1938, 1939) recorded the dimensions as 4 x 2 pm.

Lipa (1963) found that stained spores measured 3.1 to 4.1 x 2.1 to 2.7 pm. N. curpocapsae in New Zealand showed considerable variation in the size of spores from

326

MALONE

AND WIGLEY

FIG. 50. Oblique section of an immature spore of Nosema carpocapsae. The electron-dense area at the anterior pole of the spore (AD) was interpreted as being an anchoring disc for the polar filament. The developing polaroplast (DP) appears as a vacuolated area above the nucleus (NJ.

different localities and from a single location after four passages through laboratory-reared codling moth. Other workers have found that the spore size of a sin-

gle species may be altered by host age, the tissue in which the spores develop, the treatment of the tissue after removal from the host, and the medium in which the

Nosema

carpocnpsae IN CODLING

327

MOTH

FIG. 51. Anterior oblique section of an immature spore of Nosema carpocapsae. The scattered lamellae (L) of the developing polaroplast surround the descending part of the polar filament.

spores are measured (Blunck, 1954; Walters, 1958). Studies with N. carpocapsae show the importance of taking large samples from a variety of sites and support the conclusion reached by Nordin and Maddox (1974) that spore size is only useful for distinguishing species where the ranges of sizes do not overlap and in conjunction with other features such as the life cycle, his-

topathology, range.

spore ultrastructure,

and host

ACKNOWLEDGMENTS We thank Mr. G. Clare for the supply of codhng moth larvae, Dr. G. Dempsey for the transmission electron micrographs, Mrs. M. Fisher for histology, Mrs. H. Roberts for the scanning electron micrograph, and Professor E. C. Young of the Department of Zoology, University of Auckland, for helpful discussions.

328

MALONE

AND WIGLEY

FIG. 52. Scanning electron micrograph of Nosema carpocapsae

spores.

REFERENCES

FIG. 53. Proposed life cycle of Nosema carpocapsac based on qualitative and quantitative observations. The binucleate sporoplasm divides initially to produce a uninucleate meront. Merogony proceeds with the production of large numbers of binucleate meronts. There is also some prolife.ration of fusiform sporonts before spore formation.

BLUNCK, H. 1954. Mikrosporidien bei Pieris brussicae L. ihren Parasiten und Hyperparasiten. Z. Anger. Entomol., 36, 316-393. BRINTON, P. E., PROVERBS,M. D., AND CARTY, B. E. 1969. Artificial diet for mass production of the codling moth, Carpocapsa pomonella (Lepidoptera; Olethreutidae). Canad. Entomol., 101, 577-584. BURGES, H. D., CANNING, E. U., AND HULLS, I. K. 1974. Ultrastructure of Hosema oryzaephili and the taxonomic value of the polar filament. J. Invertebr. Pathol., 23, 135-139. ISSI, I. V.. AND LIPA, J. J. 1968. Report on the identification of Protozoa pathogenic for insects in the Soviet Union (1961- 1966), with descriptions of some new species. Acta Protozool., 6, 282-290. KRAMER, J. P. 1965. Generation time of the microsporidian Octosporea muscaedomesticae Flu in adult Phormia regina (Meigen) (Diptera, Calliphoridae). Z. Parasite&d.. 25, 309-313. LIPA, J. J. 1963. Studia inwazjologiczne i epizootiologiczne nad kilkoma gatunkami pierwotniakow z rzedu Microsporidia paszytujacymi w owadach. Pr. Nauk. Inst. Ochr. Rosl., 5, 103-165. LIPA, J. J. 1977. Infection of Notocellia uddmanniana L. (Lepidoptera:Tortricidae) by the microsporidian Nosema carporapsae Paillot. Acta Protozool., 16, 201-206. MALONE, L. A., AND WIGLEY, P. J. 1980. The distribution of Nosema carpocapsae. a protozoan pathogen of the codling moth, Cydia pomonella

Nosema carpocapsae IN CODLING (Lepidoptera:Tortricidae) in New Zealand. N.Z. Entomol. 7, 151- 153. MILNER. R. J. 1972a. Nosema whitei, a microsporidan pathogen of some species of Tribolium. I. Morphology, life cycle, and generation time. J. Invertebr. Parho/., 19, 231-238. MILNER. R. J. 197213.Nosemu whitei, a microsporidan pathogen of some species of Tribolium. II. Ultrastructure. J Im,ertebr. Parhol.. 19, 239-241. NORDIN, G. L., AND MADDOX, J. V. 1974. Microsporida of the fall webworm H.vphantria c~~nca. 1. Identification, distribution, and comparison of Noserna sp. with similar Nosema spp. from other Lepidoptera. J. Invertebr. Pathoi.. 24, I- 13. PAILLOT. A. 1938. Le cycle evolutif de Nosema carpocqsar. microsporidie nouvelle parasite du Carpocapse (Laspeyresia pomonella L.). C.R. Sot. Bid., 127, I 138- 1140. PAiLLor, A. 1939. Le Carpocapse dans la region Lyonnaise et les regions limitrophes. Ann. Epiphyr. Ph.wqyvzet., PETRI.

M..

5, 199-21 AND

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SCHIODT,

T.

1966.

On

the

ul-

trastructure of Nosema cuniculi in the cells of Yoshida rat ascites sarcoma. Acfa Pathd. Miuobiol.

&and.

I 66, 437-446.

MOTH

SCHOLTYSECK,

AND DANNEEL, R. 1962. Uber die der Spore von Nosema apis. Drur.

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