Nosema whitei, a microsporidan pathogen of some species of Tribolium

JOLXh-AL

OF INVERTEBRATE

Nosema

PATHOLOGY

whitei,

19. 231-238

a Microsporidan Species

I. Morphology,

Life

of Agricvltural

Pathogen

of Some

of Tri bolium Cycle,

R. J. Department

(1972)

Zoology,

and

Generation

Time’s2

MILXER~

University

Received August

of :Vewcastle-upon-Tyne,

England

16, 1971

The morphology of Nosema whitei is described from 4 host beet,les, Tribolium castaneum, T. conjusum, T. anaphe, and Oryzaephilus surinamensis. The effect of host species on the sizes of the various stages w&s small. The predominant schizogonic stages were mononuclear (26yG) and binuclear (73yc) although schieonts with up to 5 nuclei were seen. In stained preparations the schizonts were approximat,ely 2.7-7.0 pin diameter. The sporonts, which do not divide, were elongate (5.6 X 3.1 pj, and had 1 or 2 nuclei. Both the sporoblast (4.2 X 2.2~) and the spores (3.5 X 2.0 p) were binucleate. Fresh spores averaged 4.6 X 2.9 1. The polar filament length ranged from 75 to 135 p (mean = 112 p). The only tissue found infected was the fat body. Host species, dose, and temperature were all found to affect the generation time, which ranged’from 8 to 17 dais. INTREDUCTION

A recent paper (Lipa, 1968) clarified t,he generic status of Nosema ,whitei, which had previously been in doubt (Thomson, 1960b), and also confirmed the suggestion by Weiser (1961) that N. whitei is a senior synonym of Nosema buckleyi Dissanaike, 1955. Another microsporidan, Nosema oryzaephili, has been described from 0 yzaephilus surinamensis and will also infect Tribolium castaneum (Burges et al., 1971). The differences between N. oyzaephili and N. whitei have already been discussed (Burges et al., 1971). Other studies on N. whitei have included the biology (West, 1960) and some aspects of t.he host-pathogen relationship (Fisher and Sanborn, 1962a, b, 1964). ,4 study of the morphology and life cycle has been made with T. castaneum as the host 1 This work is based on a thesis submitted for a degree of Ph.D. at the University of Newcastleupon-Tyne. 2 Financed by a Science Research Council (U.K.) Studentship. a Present address: C.S.I.R.O., Division of Entomology, Armidale, N.S.W., 2350, Australia.

species. Three other host beet,les, Tribolium anaphe, T. con&sum., and Oryzaephilus surinamen.sis were used as comparisons. Finally, some dat.a on the generation time of N. whitei are presented. MATERIALS

0 1972 by Academia Press, Inc.

METHODS

Healt,hy cultures of the host beetles were obtained from the Pest Infestation Laboratory, Slough, England. The insects were cultured in a 12: 1 plain flour to dried yeast medium at. 30°C. The flour was autoclaved at 15 psi for 90 min. Young larvae were easily infected by exposure to the rearing medium mixed with W. ,whitei spores. Disease-killed insects were sieved out, and stored at &4”C until required. The dry disease-killed cadavers were ground to a spore powder in a mortar. A weighed sample of this powder was homogenized wilth aqueous potassium hydroxide solution, and the number of spores was estimated using a hemacytometer. The powder contained bet,ween 1 X lOlo and 3 X lOlo spores per gram. Finally, the powder was diluted with the rearing medium to give the 231

Copyright

Am

232

MILNER

required doses. The mixture was kept dry as a proportion of spores are known to hatch on contact with water. The stages of the life cycle were studied both in fresh smears, observed with phase contrast optics, and in methanol-Giemsa stained smears; approximately 1,000 stained preparations were studied. The smears were prepared by teasing the larvae, with needles, on a coverslip. Other smears were fixed in either> Carnoy’s fluid, or Schaudinn’s, or acetic-alcohol, while other stains included the Feulgen reaction, the periodic acidSch8 (PAS) reaction, Toluidine blue, and Heidenhain’s hematoxylin. The nuclei of the spores were revealed by Giemsa staining after hydrolysis in N HCl at 60°C for 5 min. For sectioning, portions of larvae were fixed in Carnoy le Brun, embedded in 60°C melting point wax (B.D.H., Poole, England): and sectioned at 2-5 p. These sections were stained with Giemsa’s, Feulgen’s, Heidenhain’s hematoxylin, or Gunther’s (1957) stain. The most effective technique for extruding the polar filament was the wet-dry-wet method of Kramer (1960). Another successful technique was to allow a smear of spores, in water, to dry out on a warm hot plate. Mechanical pressure was not effective. Only complete filaments, i.e., those with an empty spore at one end and the emerged sporoplasm at the other, were measured. The fresh spores and the polar filaments were both measured from an enlarged photographic image. The other stages were measured with a filar micrometer eyepiece from stained smears. To study the generation time, newly hatched larvae were infected by exposure to flour mixed with spores. The dose was varied both by altering the time of exposure and/or the number of spores per gram. The larvae were then placed with spore-free medium in individual tubes and incubated at the appropriate temperature. The control larvae were placed directly into the spore-free

medium. At roughly 24 hr-intervals the fat bodies of three larvae were dissected out, smeared, and stained. Each smear was searched for the stages of the N. whit& and at least 100 parasites, from a total of 5 fields of view, were assigned to a stage of development and counted. RESULTS

Morphology and Life Cycle The following account is based on the stages seen in infected T. castaneum larvae reared at 30°C. The principal stages are illustrated in Figs. l-11. In fresh preparations t,he schizonts were approximately spherical, with the cytoplasm full of refractile globules, and dark, discrete, spherical nuclei. Schizonts were not observed to move. In smears treated with methanolGiemsa, the schizonts were oval or spherical with blue-staining cytoplasm and red nuclei (Figs. l-3). The large schizonts stained less densely, with unstained areas in the cytoplasm giving the appearance of vacuoles, but ultrastructural studies showed that these were probably artefacts (Milner, 1970). Schizonts were Eeen with up to 5 nuclei. A series of counts totaling 1,865 schizonts showed that mononuclear schizonts comprised on average 26% of the schizont population while binucleate schizonts made up 73%, and schizonts with more than 2 nuclei comprised 1% of the population. Chains of schizonts were not seen either with the light microscope or with the electron microscope (Milner, 1970). The only dividing stage seen in the life cycle was a dividing schizont with karyokinesis complete and cytokinesis in progress. The first stage of this division was an unusually elongate schizont with a nucleus at either end. Subsequently, a constriction developed and the typical bilobed dividing form was produced (Fig. 4). In fresh preparations this stage was only recognized once, and little of the internal structure was

FIGS. 1-11. The stages of Nosen~a zuhitei, all from methanol-Giemsa treated smears except 10 and 11 which are fresh preparations. The scale on Fig. 2 refers t.o Figs. l-10. FIG. 1. Mononuclear schiaont. FIG. 2. Two binuclear schizonts. FIG. 3. Quadrinuclear schizont. FIG. 4. Schizont dividing into two. FIG. 5. Schizont dividing into three. FIG. 6. Sporont. FIG. 7. Sporoblast. FIG. 8. Immature spore showing the developing polar filament. FIG. 9. Mature spore. FIG. 10. Fresh spores. FIG. 11. Empty spore (c) with extruded polar filament (b) and sporoplasm (a).

for Figs.

234

MlLNEEt

visible under phase. In Giemsa-stained smears it was often difficult to determine the exact number of nuclei in either lobe; however, ultrastructural studies suggested that either “lobe” could be mononucleate or binucleate (Milner, 1970). Finally two, probably binucleate, stages were produced. The dividing form measured about 7 X 4 p. Occasionally atypical forms dividing into three (Fig. 5)’ and/or with one lobe anucleate, were seen. The initial stage of sporogony was the sporont. This stage (Fig. 6) was rarely seen in fresh preparations as it was diflicult t,o recognize when fresh and never constituted more than 13 % of the total number of stages seen in stained preparations. The sporonts were elongate, oval cells which stained relatively faintly wit.h Giemsa. The cytoplasm was normally homogeneous, but a developing posterior “vacuole” was sometimes seen in older sporonts. The nuclear material was variable in appearance; probably the sporont has an initial, single, large nucleus which divides into the two smaller nuclei of the sporoblasts. The nuclear material is normally situated toward one end of the cell and stains faintly red. The binucleate sporont developed directly into the sporoblast (Fig. 7), which was shorter and narrower. Viewed under phase, the sporoblast was normally dark, although a light, posterior “vacuole” was present in older forms. In Giemsa-stained preparations the “vacuole” did not stain but the cytoplasm stained intensely blue. In young forms two intensely red-staining areas were seen, situated peripherally at one end; these were interpreted as being nuclei. The immature spore, which was the same shape and size as the mature spore, stained similarly to the sporoblast but no nuclei were ever visible. As the spore formed, a developing polar filament could be seen as a diagonal, densely staining, band (Fig. 8). The mature spore (Fig. 10) showed no internal structure under phase. After Giemsa

staining, the polar filament was an intense blue and the thick wall st)ained a lighter blue (Fig. 9). Hydrolyzed spores revealed two discrete, central nuclei, often very close together, giving a bilobed appearance. The spore had a subapical PAS-positive cap. The sizes of the stages measured from methanol-Giemsa treated smears are given in Table 1. To avoid variation due to temperature, all the measurements were from insects reared at 30°C. The life cycle of the pathogen was similar in all 4 host species, and no consistent size differences were found. The differences between fixed spores from different species (Table 1) were also not consistent with those of fresh spores (Table 2). The fresh spores measured from 3 different T. caslaneum larvae did not vary significantly (Table 3). A preliminary t,est on the effect of rearing temperature on spore size, as measured with fresh spores in distilled water, indicated that spore size was not significantly different at 25 and 30°C (P > 0.05), but spores were significantly smaller (P < 0.01) at 35°C. Maddox and Luckmann (1966) suggested that the spores of a Nosema sp. from Hypera post&a were larger when formed at high temperatures. It is interesting that temperature has the opposite effect on the spores of N. whitei, i.e., they are smaller when formed at high temperatures. A sample of 50 polar filaments (Fig. 11) were measured from spores from T. castaneum larvae. The filaments ranged in length from 75 to 135 p, with a mean f standard error of 112 f 6.5 p. Site of Infection In sectioned material, N. whitei was confined to the fat body, also no host reaction was detected. The report by West (1960), that nervous tissue is also infected, was not confirmed. Gtmration Time The generation time is the time taken after infection for the first spore to develop

SIZES

4.70f0.94 (3.5-6.7

T. anaphe

Oryzaephi~ussuri4.97f0.76 namensis n=U) (3.5-7.0 n SD = standard deviation b n = number meltsured.

n-20

n = 20

4.42f0.69 (3.14.4

(2.84.8

-

of the mean.

2.7-7.0)

(4.2-6.1

x

X 2.9-5.5)

(3.5-7.0

X 4.25f1.145.36f0.47

X 4.25f0.665.67f0.50 X 3.1-5.4) (4.74.6

X 4.59ztO.735.73f0.53 X 3.2-5.5) (4.44.2

(4.G7.5

4.15&0.625.31&0.51

X 2.8-5.7)

x

1

SDa X width

MEASURED

-

f

~

(3.14.8

X 2.6-3.6)

(3.3-4.5

X 3.14f0.273.76f0.30

X 2.94zt0.274.48f0.49 (3.7-5.6 X 2.5-3.6)

TREATED

(2.9-3.8

X 2.0-2.9)

(2.7-3.7

X 2.30f0.213.31~t.~0.35

x 2.87f0.393.41zk0.28 (3.0-3.9 X 2.1-3.9)

X 2.94f0.353.6110.25 (3.14.1 X 2.3-3.5)

X 1.63.1)

X 2.34f0.243.38f0.33

Sporoblasts

X 3.13f0.384.36ztO.46 (3.5-5.4 X 2.5-3.8)

X 2.6-3.6)

METHANOL-GIEMSA

SD (range)

FROM

X 2.96f0.223.86f0.36

Sporonts _-__

SPECIES,

TABLE 4 How

Mean size, length f

FROM

X 4.14f0.624.99f0.91

X 4.09f0.575.OOf0.84 X 3.1-5.8) (3.4-6.8

X 4.07f0.505.14f0.66 X 2.8-5.1) (3.94.2

X 2.6-5.9)

4.2811~0.80 X 3.73f0.764.72f0.88

(2.74.6

whitei

Binuclear schizonts ~~.-__-

OF Nosema

schizonts ___

STAGES

___ Mononuclear

(,A) OF TIIE

T. confusum

l’ribolium castaneum n = 25”

~-

Host species

THE

X 1.8-2.7)

X 2.15~1~0.27

X 1.88&0.17 x 1.8-2.7)

X 1.97fO.20 x 1.6-2.4)

X 1.7-2.7)

X 2.05x1=0.23

Spores

SMEARS

: g

2 $

8

i

P 3 z

8

5

0” 2

236

MILNER

TABLE 2 A COMPARISON OF TEE SIZES 6) OF FRESH SPORES OF Nosemu whitei FROM 3 HOST SPECIES

Tribolium

Mean width Z!ZSD (raw)

Host

Number sampled

castaneum

309

4.65 i 0.42 (3.8 to 5.9)

3.06 & 0.39 (2.6 to 3.6)

265

4.64 f 0.32 (4.0 to 6.0)

2.83 * 0.20 (2.3 to 3.5)

47

4.54 f 0.31 (4.0 to 5.2)

2.97 f 0.73 (2.5 to 3.6)

T. confusum

Oryzaephilus

surinamensis

a SD = standard deviation of the mean. Only the differences in width between T. castaneum and between T. confusum and 0. surinamensis are significant P < 0.01 (t test).

and

T. confusum,

TABLE 3 A COMPARISON OF SPORE SIZE (p) OF Nosema whitei FROM 3 Tribolium ca&aneum LARVAE Larva

NllUlFiPled

1 2 3

102 123 84

Mean length f SD0 (range)

Mean width zk SD b-w.)

4.68 * 0.33 (3.8-5.2)

3.04b f 0.20 (2.6-3.6)

4.63 f 0.53 (4.0-5.9)

2.97 f 0.34 (2.5-3.4)

4.70 * 0.30 (4.1-5.4)

2.90” f 0.21 (2.4-3.4)

a SD = standard deviation of the mean. b The only significant difference. P < 0.01 (t test).

(Kramer, 1965). It is difhcult to detect the presence of one spore in a host animal, and also there is considerable variation between insects. Thus it is probably more accurate to determine the average tune that 50% of the stages seen are mature spores. Another time, probably correlated, is the prepatent period. The effect of dose and temperature on these times have been studied for N. whitei. The results for the effect of dose are summarized in Table 4. The minimum generation time for first-inst,ar T. ca.stun4?um reared at 30°C was 8 days, and this time was increased at lower doses. At doses above 24 hr exposure to 2 X lOlo spores per gram, an increasing

TABLE

4

THE EFFECT OF DOSE ON GENERATION Nosema whitei

TIME

OF

Time in days Doses

24

4 1

‘Watent period

First spore

50% spores

8 8 12

15 15 17

6 6 7

0 Expressed as the time lOI spores per gram.

(hr) exposed to 2 X

TABLE 5 THE EFFECT OF TEMPERATURE ON GENERATION TIME OF Nosema whitei

Temperature (“C) 35 30 25

Time in days Prepatent period

First spore

50% spores

6 7 9

9 12 14-17”

14 17 20

= No spores present on day 13; spores present on day 17.

degree of mortality occurs before spore production has started. The LT~o for this dose (2 x lOlo spores per gram) was found to be 2-3 days for newly hatched larvae. The results for the effect of temperature are summarized in Table 5. The newly hatched larvae were infected by exposure to

MORPHOLOGY AND GENERATION

2 X lOlo spores per gram for 1 hr at 30°C. The larvae were then separated into three groups which were placed at 25, 30, and 35°C. The generation time was longer at the lower temperatures. In other experiments it was found that at higher doses the generation time was similar at 30 and 35”C, but the time was always increased at 25°C. Preliminary experiments have been carried out on the generation time of N. whitei in T. con&sum and 0. surinamensis. For T. con&sum, a dose of 24 hr at lo9 spores per gram resulted in a generation time of 14 days, with a prepatent period of 9 days, while for 0. surinamensis a lower dose of 15 hr at log spores per gram g resulted in a generation time of 8 days, with a prepatent period of 4 days. These results show that the generation time is longer in T. confusum than in 0. surinamensis. The doses are not strictly comparable t.o those used for T. castuneum, but it is suggested that the generation time is similar in T. castaneum and 0. surinamensis. DISCUSSION

Lom and Weiser (1969) have proposed that the genus Nosema is nynonomous with Glugea, since in both the type species, Nosema bombycis, and in another common species, Nosema apis, the sporont divides to give two sporoblasts. It is my opinion that the sporont of N. whitei does not divide; therefore, its place in t.he scheme of Lom and Weiser (1969) is dubious. Thus I propose that the genus Nosema should include all species in the family Kosematidae in which the sporont, if it divides, does not form more than two sporoblasts. However, in view of the obvious difficult,y of det,ermining this character, and the fact that in some species only a portion of the sporonts may divide (Ishihara, 1969; Lipa and Martignoni, 1960), a serious attempt should be made to find a more s&able basis for defining the genus Nosema. The effect of host species on the morphol-

TIME OF Nosemawhitei

237

ogy, including spore size, is imignificant. This supports the view of Thomson (1960a), who studied the spore size of Perezia fumierae and Nosema cerasivoranae in three host, species. This study of N. whitei suggests that the prespore stages are also moderately constant in size between different host species and thus may be of taxonomic significance. The generation time of N. whitei is affected by dose, temperature, and host species. The effect of dose was similar to that reported by Maddox (1968) for N. necatrix, and thus supports the statement that “a certain population density of vegetative forms must be attained before development will proceed to the spore stage” (Maddox, 1968). Predictably, increasing the temperature decreased the generation time. The differences between host species may be correlated with differences in LDso. Another factor which probably affects generation time is host age but attempts to study this were confounded by the greatly increased variation between insects. The minimum generation time, under precisely defined conditions in a particular host species, may be of taxonomic value though it might reveal strains of the same species of the pathogen with different generation times. Published times range from 24 hr for Glugea go&i (McLaughlin, 1969) to 20 days for Thelohania nana (Kellen and Lindegren, 1969). It is hoped that future records of generation time will be accompanied by details of such factors as dose, host age, and rearing temperature. ACKNOWLEDGMESTS Special thanks are due to Dr. B. J. Selman of the Department of Agricultural Zoology, Universit.y of Newcastle-upon-Tyne, for his interest, guidance, and supervision of this study, and for criticizing the manuscript. The author is greatly indebted to Dr. H. D. Burges of the Pest Infestation Control Laboratory, Slough, England for his interest in all phases of this work. Help in preparing this manuscript has also been given by Dr. T. Grace, Dr. R. J. Roberts and hlr. T. J. Ridsdill Smith, all of C.S.I.R.O., Division of Entomology.

MILNER

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microsporidian pathogen of Tribolium castane-um Herbst. Ph.D. Thesis, Univ. of Newcastle, 133 pp. THOMSON, H. M. 1969a. Variation of some characters used to distinguish between species of microsporidia. I. Spore size. J. Insect Pathol., 2, 147-151. THOMSON, H. M. 1969b. A list and brief description of the microsporidia infecting insects. J. Znsect Pathol., 2, 3S-385. WEISER, J. 1961. Die Mikrosporidien als Parasiten der Insekten. MonogT. Angew. Entumol.,

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