Quantitative studies on the pathogenicity of Nosema carpocapsae, a microsporidian pathogen of the codling moth, Cydia pomonella, in New Zealand

JOURNAL

OF INVERTEBRATE

PATHOLOGY

38, 330-334

(1981)

Quantitative Studies on the Pathogenicity of Nosema carpocapsae, a Microsporidian Pathogen of the Codling Moth, Cydia pomonella, in New Zealand Department

of Zoology,

L. A. MALONE’ University c>fAuckland,

New Zealand

AND

P. J. Entomology

Division,

Department

WIGLEY

of Scientific Auckland, New

and Industrial Zealand

Received

20, 1980

May

Research,

Mt.

Albert,

The pathogenicity of Nosema carpocapsae for codling moth was studied using dose-infectivity experiments. The IC,, (median infective concentration) was similar for the five larval instars (range 4.0 x IOP to 6.7 x 104 spores/ml). Spore loads in moths ranged from 6.0 x 1W to 7.1 x 10’ spores per moth and varied with dose and with larval age at infection. The infection does not cause mortality but does reduce the fecundity and fertility of infected moths. Nosema carpocapsae is transmitted transovarially as well as horizontally. Infected eggs were not produced by healthy females mated with infected males, although such pairs generally produced fewer eggs than healthy pairs. KEY WORDS: Cydia pomonella; codling moth: Microsporida; Nosema carpocapsae; infectivity; codling moth fertility, fecundity.

INTRODUCTION

MATERIALS AND METHODS The source of host insects and the method of rearing have been described previously (Malone and Wigley, 1981). An ICjO was determined for each of the larval instars. Decimal dilutions were made from a stock spore suspension of 1.2 x lOa spores/ml to give seven suspensions ranging from 1.2 x 108 to 1.2 x lo2 spores/ml. Fifty larvae of each instar were treated with each dose. An eighth, more concentrated spore suspension (5.2 x 108 spores/ml) was prepared by centrifugation of the stock suspension. Doses were administered by allowing larvae to feed individually for 24 hr on plugs (1.0 x 0.7 cm) of insect diet (Singh, 1977) to which 25 ~1 of spore suspension had been applied. Larvae were then transferred to clean tubes containing uncontaminated diet and incubated at 21”C, 50% RH, in constant light. After 2 months, Giemsa-stained smears were made of the dead adults and pupae

Nosema carpocapsae is a common pathogen of codling moth in New Zealand (Malone and Wigley, 1980). It has previously been described from codling moth in Europe (Paillot, 1938, 1939; Lipa, 1963) and its effects on natural codling moth populations have been studied in Poland (Lipa, 1963). However, there have been no laboratory studies quantifying its pathogenicity for codling moth larvae or its effects on codling moth adults. The morphology and development of N. carpocapsae are described in detail in an earlier paper (Malone and Wigley, 1981). This paper describes the pathogenicity of this microsporidian for codling moth larvae and adults as shown by a dose-infectivity study.

’ Resent

address:

DSIR,

Entomology

Division,

Pri-

vate Bag, Auckland, New Zealand. 330

EFFECTS

OF

N. cnrpocapsa~

and overwintering larvae. These were examined for the presence of microsporidian spores or schizonts. Does-infectivity responses for each instar were analyzed by the method of Finney (1952). Spore loads of infected adults from two larval dose groups were quantified by a direct microscopical counting technique (Wigley, 1980) using whole insect homogenates of a known volume. To determine the mode of N. carpocapsae transmission and to assess its effects on fecundity and fertility, a population consisting of both infected and uninfected moths was obtained by exposing fifth-instar larvae to a range of spore doses. Uninfected moths were those which failed to become infected at low spore doses. Five decimal dilutions from 1 x lo8 to 1 x 104 spores/ml were used, with 40 larvae being fed at each dose level. Infection and rearing methods were as described above except larvae were only removed to fresh artificial diet (Brinton et al., 1969) when the contaminated diet plug had been completely consumed. Emerging moths were sexed and then paired according to the dose they received as larvae to maximize the chance of obtaining all combinations of infected and healthy moths. Paired moths were left in individual oviposition containers at 22°C in constant darkness until they died. The eggs produced were surface sterilized for 20 min in 2% formaldehyde. Most were left to hatch at 22°C in a l&14 hr 1ight:dark regimen and stained smears were made of the remainder. Smears were also prepared from the dead parent moths, the progeny larvae, and any unhatched eggs, and examined for the presence of N. carpocupsae. Results were classified according to whether each parent moth was infected or not.

ON

CODLING

331

MOTH

Probit analyses of dose -infectivity responses for all instars are shown in Table 1. The IC$,s for different larval instars range from 4.0 x 103 to 6.7 x lo* spores/ml. The relatively narrow range of I& values and the absence of significant differences at the 5% level between the probit line slopes indicate that for any given spore concentration there is a similar probability that a larva will become infected regardless of its age. The mean spore loads of adults developing from larvae infected at different stages and with different concentrations of spores are given in Table 2. Adult spore loads varied according to the age at which the larvae were originally infected; those infected at an early age contained more spores as adults than those infected later (Spearman rank correlation: rs = 0.9, P < 0.05). The effect of larval dose was not as clear. There is a trend for decreased spore load- with decreased dose over the first four instars but the mean load for fifth-instar larvae infected at 1 x loj spores/ml was anomalously high. Fecundity

and Fertility

Two principal effects were observed. First, nearly half of all the moths tested, both infected and healthy, laid no eggs at all during the experiment. Failure to lay eggs was not related to infection as 48% of infected females and 41% of healthy females laid none (x2 = 0.21, P > 0.5). Second, of those moths laying eggs, infected females laid significantly fewer (P < 0.05, Witcoxon

PROBET

ANALYSIS

TABLE 1 IHFECT~OH OF CODLING LARVAL IN~TAR~ ~0%

MOTH

RESULlS Dosage Infectivity Study No significant mortality occurred in codling moth larvae orally infected with N. carpocapsae, even in those fed spores at a concentration of 5.2 x 108 spores/ml.

1st 2nd 3rd 4th 5th

233 112 230 218 263

6.7 l.i 4.7 4.4 4.0

x x x x x

10’ 1w 1w IF IQ

3.7 x 4.8 x 4.9 x 2.3 x 2.1xIW

IQ IB 1W IW

1.2 x 2.7 x 4.6 x 8.5 x 7.3x18

1Q 10’ 10s IW

332

MALONE TABLE

MEAN SPORE LOADS ORAL INFECTION

Dose (spores/ml) 1 x 108 1 x 101

1st 6.9 (4) 4.1 (3)

2

(x 107 IN ADULTS OF THE DIFFERENT

Instar infected

AND WIGLEY

FOLLOWING

150

-

125

-

100

-

75

-

50

-

.

INSTARS

(and number tested)

2nd

3rd

4th

5th

7.1 (4) 3.7 (4)

2.9(4) 1.4 (1)

2.9(3) 0.8 (3)

0.6(4) 1.6 (4)

0

Q s ti? IA-

0

%

. 0

l

. .

2

rank iurn test) (Table 3) eggs than healthy females. There was no clear relationship between weight and fecundity in either group (Fig. 1). However, infected females did not show as wide a range of fecundities as healthy females. Transovarian transmission of N. carpocapsae was suggested by microscopical examination of infected adult tissues. Histological examination of infected female moths revealed developing spores in the ovarioles, and smears of eggs contained spores and vegetative stages. The mating experiments showed that pairs in which both the male and the female were infected produced a considerable proportion of infected eggs (Table 3). However, when only the male was infected, or both moths were healthy, only healthy eggs were produced. Infected eggs gave rise to infected larvae even when the eggs had been surface sterilized. This demonstrates that true transovarial transmission occurs, although transovum transmission may also occur. Egg hatch varied according to the presence or absence of both egg and adult in-

I3 .

3

=

25

0

-

00”.

0.0 O o Ino ’ 12

16

. 20

o I

24

28

0 .I 32

WEIGHT (mg) FIG. 1. Relationship between weight and fecundity in healthy (@) and infected (0) codling moths.

fection (Table 4). Not all of the eggs laid by infected female moths were infected. Uninfected eggs laid by infected female moths hatched at a rate similar to those laid by healthy pairs of moths. When only the male parent was infected, only uninfected eggs were produced, but these hatched at a rate significantly lower than normal (x2 = 15.6, P < 0.01). However, this can be attributed to the poor hatch of eggs from only one of the three pairs of moths in this category, so that it is difficult to judge the significance of this result without further experimentation. In general, infected eggs hatched at a lower rate (x2 = 12.88, P < 0.01) than uninfected eggs from the same parents. DISCUSSION

TABLE

3

AVERAGE NUMBER OF EGGS LAID AND PROFQRTION OF INFECTED EGGS PRODUCED BY HEALTHY AND INFECTED PARENTS

Parents

IF IF HF HF

x x x x

IMu (7 pairs) HM (5 pairs) IM (3 pairs) HM (7 pairs)

Average number of eggs laid by each female

9.2 10.4 17.7 22.3

Percentage wits

infected 73.8 59.6 0 0

a I, infected; H, healthy; F, female; M, male.

N. carpocapsae is highly infectious to codling moth larvae but not lethal to larvae or adults. Larvae became infected by spore suspensions containing only 100 spores/ml, yet even first-instar larvae were not killed by suspensions of 5.2 x 108 spores/ml. Experiments with N. melolonthae using fixed doses of spores for each larval instar showed that young larvae were infected by lower doses than old larvae (KharaziPakdel, 1968). Weiser (1976) attributed this effect to the different gut volumes of small

EFFECTS

OF N. carpocapsae ON CODLING TABLE

HATCHING

OF INFECTED

AND

UNINFECTED

4

EGGS

FROM

INFECTED

Uninfected eggs Parents

% hatching

Total number of eggs

333

MOTH

AND

HEALTHY

PARENTS

Infected eggs % hatching

Total number of eggs

Overall % hatch

IF x IM” (7 pairs)

53

17

13

48

23

IF

HM (5 pairs)

52

23

33

27

42

x

HF

x

IM (3 pairs)

38

56

None laid

0

38

HF

x

HM (7 pairs)

67

177

None laid

0

67

” I. infected; H. healthy: F, female; M, male.

and large larvae. In the present experiments with N. carpocapsae, the codling moth larvae were not given a fixed dose of spores, but fed with contaminated diet for 24 hr, so that each instar received a dose of spores proportional to its feeding rate and gut size. When measured in this way, the I& did not alter with host age to any marked degree. This method is more appropriate for the assessment of larval susceptibility to infection in the field, where the probability of larval infection depends not only on the concentration of spores encountered, but also on the rate of food consumption. As N. carpocapsae is not lethal to larvae, it was not surprising to find high spore loads (up to 7.1 x 10’) in adult moths. The size of these spore loads was directly related to the time available for microsporidian multiplication and to the concentration of the larval dose. Larvae infected at an early age, and with a high dose, developed into adults containing spore loads that were higher than those of moths infected at a later date and with a low dose. These results do not correspond to those reported by Hostounsky and Weiser (1972) and Weiser and Hostounsky (1973) who found that both Nosema plodiae and Nosema heterosporum multiplied in fourth-instar Mamestra hrassicae larvae only up to specific numeric limits that were relatively constant over a wide range of larval spore doses. However, both N. plodiae and N. heterosporum kill their host and infect only the fat body whereas N. carpocapsae in-

fections are not lethal and occur in all tissues except the nerves. Thus, N. carpocapsue has a much greater relative potential for multiplication in Cydiu pomonella and differences in the growth dynamics of these microsporidia are simply reflections of their different patterns of pathogenesis. N. carpocapsae reduced both the fecundity and fertility of codling moth under laboratory conditions. These results were expected as microsporidia are known to deplete the nutritive reserves normalfy used for reproduction, reducing fecundity (Smirnoff and Chu, 1968; Thomson, 1958; Veber and Jasic, 1961), fertility (Tanabe and Tamashiro, 1967), longevity (Gaugler and Brooks, 1975: Thomson, 1958), and mating success (Gaugler and Brooks, 1975). Infected eggs contain spores or vegetative stages and are less likely to hatch than uninfected eggs. This effect is probably due to a depletion of embryonic food reserves by the microsporidian. The effects of N. carpocapsae on natural populations of codling moth in New Zealand are unknown. The only large-scale study of codling moth population dynamics in New Zealand (Wearing, 1979) was carried out at Appleby Research Orchard where N. carpocapsue occurs only at a very low level. However, such low levels of infection are not typical of high-density codling moth populations. In a recent survey of the incidence of N. carpocupsae in codling moth populations on untended apple trees (Malone and Wigley, 1980), in-

334

MALONE

AND WIGLEY

fection rates exceeded 50% in 10 out of 15 sites surveyed and exceeded 75% in 5 of these sites. Similar infection levels were reported by Simchuk and Sikura (1978) from a survey of the incidence of N. carpocupsae in codling moth populations in tended and untended apple orchards in the USSR. The results of the present study thus suggest that N. carpocupsae causes little mortality and that its effects on natality are likely to significantly influence codling moth population dynamics only at the high population densities characteristic of untended apple trees. ACKNOWLEDGMENTS We thank Mr. G. Clare for the supply of codling moth larvae, Miss J. Hopkins for the statistical analysis, and Professor E. C. Young of the Department of Zoology, University of Auckland, for helpful discussions.

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13, 289-318.

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a microsporidian

pathogen

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57, 495-499.

SINGH, P. 1977. “Artificial Diets for Insects, Mites, and Spiders,” IFI/Plenum Data Co., New York. SMIRNOFF, W. A., AND CHU, W. H. 1968. Microsporidian infection and the reproductive capacity of the larch sawfly, Pristophora erichsonii. J. Invertebr. Pathol.. 12, 388-390. TANABE, A. M., AND TAMASHIRO, M. 1967. The biology and pathogenicity of a microsporidian (Nosema trichoplusiae sp. n.) of the cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae). J. Invertebr. Puthol., 9, 188- 195. THOMSON, H. M. 1958. The effect of a microsporidian parasite on the development, reproduction, and mortality of the spruce budworm, Choristoneura fimiferana (Clem.). Cunad. J. Zool., 36, 499-511. UNDEEN, A. H., AND ALGER, N. E. 1975. The effect of the microsporidan, Nosema algerae, on Anopheles

stephensi.

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19-24. VEBER, J., AND JASIC, J. 1961. Microsporidia as a factor reducing the fecundity in insects. J. Insect Pathol.. 3, 103-111. WEARING, C. H. 1979. Integrated control of apple pests in New Zealand. 10. Population dynamics of codling moth in Nelson. N.Z. J. Zool.. 6, 165- 199. WEISER, J. 1976. Microsporidia in invertebrates. Host-parasite relations at the organismal 1eveI. In “Comparative Pathobiology” (L. A. Bulla and T. C. Cheng, eds.), Vol. I, pp. 163-201. Plenum, New York. WEISER, J., AND HOSTOUNSKY, J. 1973. Production of spores of Nosema plodiae Kellen et Lindegren in Mamestra brussicae L. after different infective dosage. II. Comparison with Nosema heterosporum Kellen et Lindegren. Vest. Cs. Spol. Zool.. 37, 234- 237. WIGLEY, P. J. 1980. Quantitative methods. In “Microbial Control of Insect Pests” (J. Kalmakoff and J. F. Longworth, eds.), New Zealand Department of Scientific and Industrial Research, Bulletin 228.