Seasonal progress of Nosema pyrausta in the European corn borer, Ostrinia nubilalis

JOURNAL

OF INVERTEBRATE

PATHOLOGY

52,

130-136 (1988)

Seasonal Progress of Nosema pyrausta in the European Corn Borer, Ostrinia nubilalis J.P. SIEGEL, J. V. MADDOX, AND W. G. RUESINK Illinois

Natural

History

Survey

and Illinois

Agricultural

Experiment

Station,

Champaign,

Illinois

61820

Received May 25, 1987; accepted December 2, 1987 A 4-year study of the epizootiology of Nosema pyrausta in the European corn borer, Ostrinia was conducted in Woodford County, Illinois, during 1980-1983. N. pyrausta infections in the first-generation larvae resulted primarily from transovarial infection. Second-generation N. pyrausta larval infections resulted from both transovarial infection and horizontal transmission. There was a significant relationship between larval density and percentage infection of N. pyrausta for the second generation, whereas no such relationship could be demonstrated for the first generation. We attempted to determine empirically a critical density for horizontal transmission in the second generation and concluded that no single threshold value existed. o 1988Academic press, IIIC. KEY WORDS: Nosema pyrausta; Ostrinia nubilalis; epizootiology; transmission; population dynamics . nubilalis,

INTRODUCTION

The microsporidium Nosema pyruustu, widely distributed throughout central Illinois, is an important biological mortality factor of the European corn borer, Ostrinia nubilafis, (Kramer, 1959; Decker, 1960). The pathogen is transmitted horizontally in the field through ingestion of spores (Lewis, 1978) and transovarially, both within the egg and on the chorion (Kramer, 1959). N. pyruusta infections of laboratory populations of the European corn borer reduce adult life span and fecundity as well as increase larval mortality (Zimmack and Brindley, 1957; Kramer, 1959; Van Denburgh and Burbutis, 1962; Windels et al., 1976; Siegel et al., 1986). Horizontal transmission of N. pyruustu is densitydependent and infections reduce host population levels (Hill and Gary, 1979; Andreadis, 1984). The European corn borer was introduced into the United States between 1909 and 1914 and first appeared in northern Illinois in 1939. There are two complete generations of 0. nubihlis in central Illinois today. The borer overwinters as a diapausing Sth-instar larva in corn stubble and pupation begins in May. Most of the adults

emerge over a 2-week period in early June (first moth flight). Mating occurs in the evening in moist patches of grass and weeds at the field margins, after which the female flies into the corn field to oviposit. An averge egg mass contains 15-30 eggs and a female lays 15-20 egg masses during her lifetime. There are five instars; firstgeneration adult emergence (second moth flight) begins in August and continues for 4 to 5 weeks (Anonymous, 1980). In order for level of infection to increase within a population, the population must exceed a critical density or host threshold. Factors determining this threshold include pathogen transmission efficiency, natural mortality, pathogen-induced mortality, and host recovery (Anderson and May, 1980). To avoid extinction, virulent pathogens require a higher host threshold density than do nonvirulent pathogens, and pathogens with a high transmission efficiency, such as the Microsporida (transovarial transmission) may have relatively low threshold densities (Anderson and May, 1980). If a host threshold can be calculated for a pathogen, it is possible to predict future infection levels of the pathogen based on current host densities. For exampb, Nordin et al. (1983), working with the alfalfa weevil,

130 0022-2011188 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

SEASONAL

PROGRESS

and the entomopathogenic phytonomi, reported a threshold value of 1.7 weevils per alfalfa stem. Fungal infections are complicated by their dependence on atmospheric moisture for conidial and resting spore gemineration, and relative humidity may be as important as host density in determining the occurrence of an epizootic. Because protozoan infections do not depend on relative humidity, it should be easier, in theory, to calculate their host thresholds. The objectives of our study, conducted in Woodford County, Illinois, from 1980 through 1983, were to describe the epizootiology of N. pyruustu and assessits impact on 0. nubilalis populations infesting field corn. In this paper we present our data on the seasonal progress of the disease and use the concept of host threshold density to explain the observed fluctuations in N. pyruustu infection levels between generations. Hyperu postica, fungus Erynia

MATERIALS

AND METHODS

Field selection. For a period of 4 years, 1980-1983, a 3.2-km2 area of Woodford County, Illinois, was sampled at weekly intervals, beginning with moth flight in June and ending after corn harvest in September. In 1980, eight cornfields within the study area were chosen for intensive sampling and a portion of these fields was subdivided into three replicates. In the following years some of these fields could not be used due to crop rotation, and new fields were chosen near the original sites. Six fields were chosen for intensive sampling in 1981, four in 1982, and only one large field was available within the study area in 1983. All fields were divided into three replicate plots as in 1980. There were two complete generations of 0. nubilalis each year during the course of the study. Sampling. Adults were captured using black-light traps from 1980 through 1982; adults were not sampled in 1983. Firstgeneration larvae were sampled in each replicate by selecting a row of corn at ran-

OF Nosema

IN

Ostrinia

131

dom and no row was sampled twice during the summer. Within a row, 100 consecutive corn plants were examined for infestation, indicated by leaf feeding damage. Ten plants showing evidence of feeding were selected for detailed examination, cut off at the base, and taken to the laboratory for dissection. Early-ins&n- larvae are readily dislodged when the corn is handled and beginning in 1982,the plants were dissected in the field to maximize the recovery of earlyinstar larvae; the larvae were placed individually into cups to minimize horizontal transmission. These data were converted to the number of borers per 100 cornstalks by multiplying the average number of borers per infested stalk by the number of infested stalks/100 plants examined. Two different methods were used for sampling second-generation larvae over the 4-year period. In 1980 and 1981, 10 corn plants within a row were randomly selected, cut off at the base, and taken to the laboratory for dissection. The number of borers per 100cornstalks was calculated by multiplying the total number of borers recovered in the 10 cornstalks by 10. In 1982 and 1983, 50 plants within a row were examined for the presence of fresh frass by stripping the leaves to recover more second-generation borers. Ten infested comstalks out of the 50 were dissected in the field and the larvae removed. The number of borers per 100cornstalks was calculated by multiplying the average number of borers per infested stalk by percentage infestation by 100. The change in sampling procedure did not increase the collection of early-instar larvae but minimized the loss of 3rd-instar larvae, which are not necessarily established inside the corn stalk. Diagnosis. N. pyrausta infections in corn borer larvae were determined in the laboratory by removing Malpighian tubules and examining these in wet mount preparations for the presence of spores. N. pyruusta infections in adults were diagnosed by examining tissue from the abdomen. All slides (larva and adults) were examined using

132

SIEGEL,

MADDOX,

phase-contrast microscopy at 400x ; at least five spores had to be present within the microscope field of view for a positive diagnosis. If infection was questionable, additional slides were made and examined. Analysis. In both generations of the corn borer, the relationship between larval density and the prevalence of N. pyrausta was investigated through regression analysis, and the slopes were compared using the Student’s t procedure of Zar (1974). Because first-generation fields were homogeneous with regard to larval density and infection levels, the data from all folds were pooled. Second-generation fields were not homogeneous because larval density varied, and we felt that by pooling data from all of the fields, specific information regarding host density and infection would be lost. Using two criteria, density within the field and the relationship between infection level and density, we divided the secondgeneration fields into two groups for regression analysis. First, fields were categorized as high density if their population exceeded 100borers per 100 cornstalks at some point during the growing season, and as lowdensity fields if the count was below this cutoff. All the data were then pooled to make high- and low-density groups, and the relationship between density and infection was then evaluated for each one. As an alternative method of analysis, we utilized the relationship between larval density and infection level within each field to form two categories of fields that were then pooled into two groups, one consisting of fields lacking a significant relationship between larval density and infection and the other group consisting of fields with a significant relationship between the two. The relationship between larval density and infection levels was then examined. RESULTS

was enzootic in the first generation and there was no significant relationship between larval density and the prevalence of N. pyruustu. First-generation N. pyrausta

AND

RUESINK

larval infection levels were similar to infection levels of the first motMight (Tables 1, 2). Larval density peaked at 14, 14,2.6, and 80 borers per 100 cornstalks, and larval N. pyrausta infections peaked at 55, 27, 25, and 27% for the 4 years reported. In the second generation, the prevalence of N. pyruustu increased from 1980through 1983, and N. pyrausta was epizootic in 1982 and 1983, peaking at 72 and 75%, respectively (Table 3). Second-generation larval density also increased over the 4-year period, with peaks of 85,76, 193, and 697 borers per 100 cornstalks; second-generation larval infection levels surpassed those observed for the second moth flight (Table 4). The second-generation regression between the natural log of density and the natural log of the prevalence of N. pyruustu was significant (P < 0.01) for the pooled high-density fields (Fig. 1); no such relationship existed in the pooled low-density fields. Among the individual fields in the second generation, 8 out of 17 had sign& cant regressions (P < 0.05) between density and infection and in an additional three TABLE

1

PREVALENCE OF Nosema pyrausta IN FEMALE EUROPEAN CORN BORERS CAPTURED WITH BLACK-LIGHT TRAPS IN W~ODFORD COUNTY, ILLINOIS, DURING THE FIRST MOTH FLIGHT, 1980-1982

Julian date

No. captured

No. examined

Percentage infection

1980

154 157 161 168 171 175

21 111 243 119 35 144

20 30 20 20 20 20

50 23 20 45 50 30

1981

152 159 167 175

53 264 9 6

20 20 9 6

25 40 22 83

1982

153 159 166 174 181

26 148 217 52 11

11 20 20 20 10

9 20 5 15 40

Year

SEASONAL TABLE

2

PREVALENCE OF Nosema pyrausta IN FIRST-GENERATION EUROPEAN CORN BORER LARVAE (DATA COLLECTED IN WOODFORD COUNTY, ILLINOIS,’ 198&1983) Year 1980

1981

1982

1983

JIllian date

No. examined

Borers per 100 stalks

182 189 1% 203 210 217

166 133 79 81 32 21

10 14 6 7 4 4

167 173 182 189 197

22 70 47 20 14

3 14 6 7 4

182 189 1% 202 210

14 9 11 5 4

181 187 194 199

55 41 34 21

Year

27 17 23 20 0

YW

Julian date

No. examined

Borers per 100 stalks

BORER COUNTY,

Percentage infection

1980

233 240 248 255

19 141 130 124

20 63 80 85

19 32 37 56

1981

238 246 252 259

30 67 86 70

40 76 76 60

34 42 47 42

1982

231 237 253 258 267

18 62 ti 48

112 120 193 158 164

16 31 65 72 54

229 235 243 251 258

49 107 268 % 83

160 570 697 550 500

49 60 69 75 75

1983

No. captured

No. examined

COUNTY, MOTH

Percentage infection

21 310 20 40 540 219

20 40 20 30 10 30

25 15 10 27 30 27

1981

198 202 208 212 215 219 224 232 238

39 71 127 104 180 69 54 5 72

20 20 20 20 20 20 20 5 20

15 15 15 5 30 30 20 40 45

1982

202 210 216 224 231 237

73 303 499 1326 33 371

20 20 20 30 10 20

5 25 10 10 30 40

27 12 15 12

IN

TRAPS IN WOODFORD DURING THE SECOND FLIGHT, 1980-1982

202 209 212 214 217 220

2:

fields, the relationship between density and infection was of borderline significance (0.10 > P > 0.05). When the fields were divided into significant and nonsignificant

Julian date

TABLE 4 Nosema pyrausta IN FEMALE CORN BORERS CAPTURED WITH OF

1980

20

80 60

PREVALENCE SECOND-GENERATION EUROPEAN CORN LARVAE (DATA COLLECTED IN WOODFORD ILLINOIS, 1980-1983)

PREVALENCE EUROPEAN BLACK-LIGHT ILLINOIS,

Percentage infection

1.4 2.1 1.6 1 2.6

TABLE 3 OF Nosema pyrausta

133

PROGRESS OF Nosema IN Ostrinia

groups based on this relationship and then pooled, the regression of the natural log of density and the natural log of prevalence of N. pyrausta was significant in both groups (P C 0.05) (Table 5). DISCUSSION

First-generation

larvae acquired N.

pyrausta infections primarily as a result of

transovarial transmission, with the possible exception of 1980, where infection rose from 22 to 55% (Table 2). However, infection did not increase in the larvae 3 out of the other 4 years, but remained at the level observed in the females (Tables 1, 2). If substantial horizontal transmission had occurred, we should have observed higher levels of infection in the larvae than in the eggs. In 1980the average egg infection level was 37.3%, whereas the average larval infection level was 31%. In 1983 the average

134

SIEGEL.

MADDOX.

AND

RUESINK

cause the egg infection level presumably mirrored the female infection level. The increased second-generation population density facilitated horizontal transmission, and the host threshold level was surpassed. Consequently, second-generation N. 10~ 200 400 600 800 1000 1200 pyrausta infections have both a transovariBorers Per 100 Cornstalks al and horizontal component. FIG. 1. The relationship between the natural log of Choosing the infestation level of the second-generation larval density of the European corn study fields in order to determine the borer and percentage infection with Nosema pyrausta threshold population level for horizontal in the high-density group. Data were collected in transmission of N. pyrausta is problematic. Woodford County, Illinois, 1980-1983. Y = -0.2891 In their study of second-generation N. + 0.1579 In X; rZ = 0.49; P < 0.01. pyruusta infections in Nebraska, Hill and Gary (1979) used only fields with 100% inlarval infection level was 16.5% and the av- festation, whereas our study area included erage egg infection level was 16%. There- fields with a variety of infestation levels. It fore, the corn borer population density was is important to note that although epizootbelow the host threshold, so infection lev- its occurred in our study area during the 2 els for the first generation were controlled years with the highest population levels, inprimarily by the female input. Andreadis fection levels also rose when the average (1986), working with 0. nubihlis infesta- density was below 100 borers per 100 comtions of sweet corn in Connecticut, also stalks (Table 5). Therefore, we believe that found that horizontal transmission is lim- infestation of 100% of the corn plants is not ited in the first generation because of low a prerequisite for horizontal transmission larval densities. and excluding fields below that level may Second-generation larvae had infection result in an overestimate of the critical denlevels surpassing those of the second moth sity for horizontal transmission. flight. This rise in prevalence of N. Two factors complicate secondpyraustu above the female level provides generation threshold determination: the evidence of horizontal transmission, be- rate of horizontal transmission and the TABLE 5 SUMMARY OF THE AVERAGE WEEKLY PREVALENCE OF Nosema pyrausta IN THE SECOND-GENERATION EUROPEAN CORN BORER (DATA COLLECTED IN W~~DFORD COUNTY, ILLINOIS, 19804983). Significant group0

Nonsignificant group

Week

Borers per 100 stalks”

Percentage infection

1 2 3 4 5

72 192 241. 202 332

21 33 52 56 67

Week

Borers per 100 stalksc

Percentage infection

1 2 3 4

30 59 93 93

20 38 47 50

LIIndividual fields with a statistically significant (P c O:tif relationshlpbetwee~larval density and percentage infection. b Regression of density and infection: ln Y = In 0.0079 + 0.7862 In X, P < 0.05, iL = 0.86. c Regression of defisity and infection: h Y = ln 0.0148 + 0.7749 hi X, P < 0.01, 3 = 0.98.

SEASONAL

PROGRESS OF Nosema IN Ostrinia

presence of infectious cornstalks from the first generation. Horizontal transmission dynamics are not instantaneous because there is a time lag between infection with N. pyruustu and the appearance of spores in the frass of infected larvae. Spores did not appear in the frass of transovarially infected larvae until they reached the 3rd instar, and 3rd-instar larvae infected and held at 24.E did not produce spores for at least 6 days (Siegel, unpubl.). Therefore, infection levels observed in the field for one date may have resulted from conditions occurring 1to 2 weeks earlier. Unfortunately, we did not take enough samples to enable analysis of time lag effects; sampling the larvae at least twice weekly is recommended for future studies. The issue of infectious or spore contaminated corn stalks is also important because infected first-generation larvae contaminate the cornstalks they inhabit with N. pyrausfa spores and their infectious frass may be blown onto uninfested cornstalks, making these stalks infectious too. Depending on first-generation larval density and infection level, a variable percentage of the cornstalks may be infectious prior to the emergence of the second generation. These contaminated cornstalks are an. additional source of N. pyrausta, and may result in higher levels of infection than would have occurred if secondgeneration larval density solely determined infection. We calculated our empirical threshold by analyzing the fields according to two criteria, larval density within each field and the relationship between larval density and percentage infection within each field. When the fields were divided into high- and lowdensity groups, the low-density group had corn borer population levels similar to those observed in the first generation. As with the first generation, there was no significant relationship between larval density and infection in the low-density group, while there was a significant regression of density and infection in the high density

135

group (Fig. 1). The curve is steepest in the region below 100borers per 100cornstalks; we believe that this is the area of interest for threshold determination. Our second method of categorizing the fields utilized the relationship between larval density and infection in the individual fields, and the significant group included many of our high-density fields. Statistical analysis of both groups of pooled fields (significant and nonsignificant) resulted in a significant regression of average weekly density and average weekly infection for both groups. Although the initial density of the significant group was twice that of the nonsignificant group, the initial levels of N. pyruusta were the same and the infection rates were similar. Because infection increased in the lowdensity group, the host threshold lies within its range, i.e., 30 to 93 borers per 100 stalks. Using host threshold parameters of Anderson and May (1980), we calculated the rate of acquisition of infection by multiplying the pathogen transmission efficiency by the number of infected and susceptible individuals present. Because the rate of acquisition depends on both the initial number of infected individuals and the total population, the rate should increase as the number of infected individuals rises and it should vary between years as the corn borer population fluctuates. We feel that the inoculum of N. pyrausta present within the stalks also plays an important role in transmission; one cannot calculate the corn borer host threshold solely on the basis of larval density. Furthermore, it is misleading to search for a single threshold because both the pathogen and population inputs vary for the second generation. We prefer to think in terms of a “floating threshold,” determined by both larval density and the initial prevalence of N. pyruustu (female input plus contaminated cornstalks). For example, if the initial inoculun is high, a much lower larval density would be required to initiate horizontal transmission than if the initial level of N. pyruustu was low; con-

136

SIEGEL,

MADDOX,

versely, a high initial larval density would require a lower inoculum for initiation of horizontal transmission. This system contrasts with that of Nordin et al. (1983), where the initial fungal inoculum is relatively unimportant and host density combined with the proper relative humidity determines the occurrence of an epizootic. In summary, transovarial transmission plays the dominant role in establishing firstgeneration N. pyrausta infections because larval densities generally are too low for a significant amount of horizontal transmission of the pathogen to occur. The initial input of N. pyrausta into the second generation depends on first-generation levels of infection and consists of infected egg masses and contaminated corn stalks. However, the greater larval density of the second generation facilitates horizontal transmission and, as a consequence, infection levels rise during the second generation. The initial level of N. pyrausta combined with larval density determined the rate of acquisition of infection; increased population levels facilitate horizontal transmission. The concept of a critical density or host threshold requires some modification for our system because of the variability in both the input of pathogen and host density, and there probably is no single host threshold level. ACKNOWLEDGMENTS Funding for the project came from the Illinois Natural History Survey and the Illinois Agricultural Experiment Station, College of Agriculture, University of Illinois at Urbana-Champaign, through Hatch Project 11-319, Biology and Impact of Entomophilic Microsporidia. We thank Jim Seiler and Terrie DePratt for their assistance in this study and also M. Berenbaum and T. Andreadis for reviewing earlier drafts of this manuscript.

REFERENCES ANDERSON, R. M., AND MAY, R. M. 1980. The pop-

AND RUESINK ulation dynamics of microparasites and their invertebrate hosts. Philos. Trans. R. Sot. London, 291, 451-524. ANDREADIS, T. G. 1984. Epizootiology of Nosema pyrausta in field populations of the European corn borer (Lepidoptera: Pyralidae). Environ. Entomol., 13, 882-887. ANDREADIS, T. G. 1986. Dissemination of Noserna pyrausta in feral populations of the European corn borers, Ostrinia nubilalis. J. Znvertebr. Pathol., 48, 335-343. ANONYMOUS. 1980. “Entomology Fact Sheet for Field Crop Insects.” University of Illinois Cooperative Extension Service and Illinois Natural History Survey. DECKER, G. 1960. Microbial insecticides and their future. Agric. Chem. 15, 30. HILL, R. E., AND GARY, W. G. 1970. Effects of the microsporidium Nosema pyrausta on field populations of the European corn borer in Nebraska. Environ. Entomol., 8, 91-95. KRAMER, J. P. 1959. Some relationships between Perezia pyruastae (Paillot) (Sporozoa, Nosematidae) and Pyrausta nubilalis (Hubner) (Lepidoptera, Pyralidae). J. Insect Pathol., 2, 25-33. LEWIS, L.C. 1978. Migration of larvae of Ostrinia nubilalis infected with Nosema pyrausta and subsequent dissemination of the microsporidium. Canad. Entomol. 220, 897-900. NORDIN, G. L., BROWN, G. C., AND MILLSTEIN, J. A. 1983. Epizootic phenology of Erynia disease of the alfalfa weevil, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) in central Kentucky. Environ. Entomol., 12, 1350-1355. SIEGEL, J. P., MADDOX, J. V., AND RUESINK, W. G. 1986. The lethal and sublethal effects of Nosema pyrausta (Protozoa: Microsporida) on the European corn borer (Ostrinia nubilalis) in Central Illinois. J. Znvertebr. Pathol., 48, 167-173. VAN DENBURGH, R. S., AND BURBUTIS, P. P. 1962. The host-parasite relationship of the European corn borer, Ostrinia nubilalis, and the protozoan parasite, Perezia pyrausta, in Delaware. J. Econ. Entomol., 55, 65-67. WINDELS, M. B., CHIANG, H. C., AND FURGALA, B. 1976. Effects of Nosema pyrausta on pupal and adult stages of the European corn borer, Ostrinia nubilalis. J. Znvertebr. Pathol., 27, 239-242. ZAR, J. H. 1974. “Biostatistical Analysis.” PrenticeHall, Englewood Cliffs, New Jersey. ZIMMACK, H. L., AND BRINDLEY, T. A. 1957. The effects of the protozoan parasite Perezia pyrausta Paillot on the European corn borer. J. Econ. Entomol. 50, 537-640.