Occurrence and effects of Nosema fumiferanae infections on adult spruce budworm caught above and within the forest canopy

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1 Agricultural and Forest Entomology (2007), 9, DOI: /j x Occurrence and effects of Nosema fumiferanae infections on adult spruce budworm caught above and within the forest canopy Eldon S. Eveleigh *, Christopher J. Lucarotti *, Peter C. McCarthy *, Benoit Morin *, Tomo Royama * and Anthony W. Thomas * * Natural Resources Canada, Canadian Forest Service Atlantic Forestry Centre, PO Box 4000, Fredericton, New Brunswick E3B 5P7, Canada and Population Ecology Group, Faculty of Forestry and Environmental Management, University of New Brunswick, Fredericton, New Brunswick E3B 6C2, Canada Abstract 1 Nosema fumiferanae infections in populations of both sexes of spruce budworm Choristoneura fumiferana moths, collected live above the forest canopy (canopy moths), within the tree crown (crown moths) and in drop trays (dead moths), were examined over a 5-year period in New Brunswick, Canada. 2 The incidence of infection and of moderate heavy infections in canopy and crown moths of both sexes increased concomitantly with moth eclosion, indicating that N. fumiferanae retards larval/pupal development, with infected moths, particularly those having higher disease loads, emerging later in the season. 3 Infection rates differed among canopy, crown, and dead female, but not male, moths. Canopy (i.e. emigrating) females had a lower incidence of infection, lower incidence of moderate heavy infections, and had longer forewings and higher dry weights, than crown females. These results suggest that N. fumiferanae infections negatively affect aspects of female, but not male, flight performance. Regardless of infection, forewing length and dry weight of both canopy and crown females declined over the moth flight period, but infected females in both moth types were smaller than their uninfected counterparts. Forewing lengths and dry weights of moderately heavily infected females were most severely affected. 4 Despite high annual infection rates in parents, only a small percentage of offspring (second-instar larvae) that established feeding sites each spring were infected, indicating that high rates of horizontal transmission occurred annually throughout the larval period. 5 The present study indicates that whether N. fumiferanae infections are a debilitating sublethal factor in spruce budworm populations depends more on the disease load than on the overall incidence of infection. The potential importance of N. fumiferanae infections on various fitness parameters related to host dispersal is discussed. Keywords Choristoneura fumiferana, dispersal, entomopathogen transmission, Nosema fumiferanae, spruce budworm. Introduction Nosema fumiferanae (Thompson) (hereafter Nosema ) is the most prevalent entomopathogen of the spruce budworm, Correspondence: Eldon S. Eveleigh, Natural Resources Canada, Canadian Forest Service Atlantic Forestry Centre, PO Box 4000, Fredericton, New Brunswick E3B 5P7, Canada. Tel.: ; fax: ; eeveleig@nrcan.gc.ca Choristoneura fumiferana (Clem.), with incidences of over 80% being reported in natural populations ( Thomson, 1960 ). It has been observed in all stages of the host ( Percy, 1973 ), and is efficiently transmitted both horizontally (perorally) and vertically (transovarially but not venereally) ( Wilson, 1982 ). Consequently, the incidence of Nosema has been reported to increase within ( Wilson, 1977 ) and across host generations ( Thomson, 1960; Wilson, 1977 ). Journal compilation 2007 The Royal Entomological Society

2 248 E. S. Eveleigh et al. The effects of Nosema on the host depend on the disease load (incidence of moderate heavy infections). High disease loads can cause host mortality ( Wilson, 1981 ), with progeny mortality being positively correlated with final maternal disease load ( Bauer & Nordin, 1989 ). By contrast, low disease loads (sublethal infections) tend to prolong development of immature stages ( Wilson, 1983; Bauer & Nordin, 1988a ), reduce pupal (and hence moth) weight and shorten moth longevity ( Wilson, 1981 ). These detrimental effects may be more pronounced in females than males ( Wilson, 1981 ). Nosema does not affect mating success or egg fertility but it does reduce fecundity and total egg complement of females ( Bauer & Nordin, 1989 ). Despite the reported importance of Nosema as either a mortality or debilitating factor in spruce budworm populations, most studies have been laboratory based. Little is known about the ecology of this entomopathogen in field populations of host larvae and pupae, and no detailed field studies have been conducted on Nosema in spruce budworm moths. As part of a larger field study on the population ecology of spruce budworm in New Brunswick, Canada, Nosema infections were investigated in immature and adult stages from 1983 to 1987, a period during which spruce budworm populations reached high densities and then declined to low densities. The present study addresses Nosema infections in moths and the incidence of vertically transmitted infection to F 1 progeny (second-instar larvae; L 2 ) that established feeding sites in the spring of each year. The incidence of Nosema infections in other larval instars, life stages in which horizontal transmission occurs, will be reported elsewhere. Entomopathogens have been shown to affect the flight performance of some insects ( Bradley & Altizer, 2005 ), possibly by influencing their energetic resources ( Schiefer et al., 1977; Marden & Cobb, 2004; Bradley & Altizer, 2005 ). Because many female and male spruce budworm moths emigrate, sometimes traveling several hundred kilometers from their natal sites (for details of moth flight and dispersal, see Greenbank et al., 1980 ), the present study specifically attempted to examine emigrating and non-emigrating moths to determine: (i) whether the incidence and levels of Nosema infection differ between emigrating and non-emigrating moths, and years, and (ii) if certain morphological characteristics associated with flight (e.g. forewing length and body weight) differ with the incidence of infection between emigrating and non-emigrating female moths. Materials and methods Study site This field study was carried out at the Acadia Research Forest (66 25 W, N) near Fredericton, New Brunswick, Canada, in a site consisting primarily (98% of basal area) of 25- to 30-year-old balsam fir ( Abies balsamea (L.) Mill.) averaging 9 15 m in height. Red spruce ( Picea rubens Sarg.) and red maple ( Acer rubrum L.) accounted for 1% each. The site has been described in detail by Lethiecq & Régnière (1988) and MacLean et al. (1996). Information on defoliation of balsam fir and budworm population density in the site is provided in MacLean et al. (1996). Life history The spruce budworm has a univoltine life cycle. In the study site, adults begin emerging in late June to early July. Most females are mated on the day of eclosion ( Greenbank et al., 1980 ). Eggs are laid in masses, averaging approximately 20 eggs/mass, on the underside of host needles, and larvae emerge approximately 10 days later. First instars disperse to find sheltered sites to build hibernacula, within which they molt into second instars and spend the winter. After emergence in late April of the next year, second instars disperse to find and establish feeding sites. Larvae continue to feed throughout a total of six instars, pupating on the foliage in approximately mid-june. Pupation lasts approximately 10 days, after which adults emerge and the cycle is repeated. Trapping methods Initially, the objective was to examine infections in emigrating and non-emigrating moths throughout the moth flight period each year by capturing live moths above the forest canopy (emigrants) and dead moths in the tree crown (non-emigrants). However, after collecting 2 years of data, it was found that: (i) catches of dead moths were very low, particularly for females, relative to catches of emigrating moths above the forest canopy, and (ii) local moth populations could be supplemented periodically with immigrant moths from both nearby and distant sites. Thus, in subsequent years, infections in moths captured in the tree crown were also examined. Canopy trap catches. Square-based pyramidal traps (canopy traps) were installed above the forest canopy to collect, each day, moths that were emigrating from the site (hereafter canopy moths). The bottom of the traps was 1 m 2 and open, so moths could enter through the bottom and crawl up the screened sides to the apex, where they would enter a collecting jar. A piece of Vapona (2,2-dichlorovinyl dimethyl phosphate) (Scotts Canada Ltd, Canada ) was suspended in each collecting jar to ensure that trapped moths died quickly. Traps were attached to the top of scaffold towers by steel pipes and suspended approximately 1 m above the tops of trees near the towers. The traps were drawn toward the t owers using ropes and pulleys to examine the collecting jars. Six traps were deployed, two from each of three towers, from 1983 to The towers were placed across the study site at distances of approximately 100 m apart. Crown trap catches. Moths flying around and between trees at time of capture (hereafter crown moths) were collected daily using modified, 1-m 3 Malaise traps ( Nyrop & Simmons, 1982 ). From 1985 to 1987, the traps were placed, with the open sides perpendicular to the tree trunk, at the top, middle and lower crown levels on a single tree located approximately mid-way between two of the canopy trap towers. As explained in the Appendix, these crown catches were probably a mixture of both emigrant and non-emigrant moths.

3 Entomopathogen infection and host dispersal 249 Drop-tray catches. Dead moths (non-emigrants) were collected daily in drop trays consisting of a square wooden frame, 0.25 m 2 in area, with fine cloth mesh attached to the bottom. To prevent invertebrate and vertebrate predators or scavengers from removing cadavers, the upper edges of the frame were coated with petroleum jelly, and the top was covered with removable hardware cloth (mesh = 1.61 cm 2 ). One tray was placed at the top crown level, and two trays, placed side by side, at both the middle and lower crown levels. Traps were placed in three trees, each near one of the three canopy trap towers, and were operated from 1983 to Trap/tray monitoring All traps/trays were in operation 24 h/day from just before the start of local moth eclosion until no moths were caught for several consecutive days after local moth eclosion ceased. Traps were emptied between 07:30 h and 09:30 h each day and all specimens were stored frozen ( 20 C) until processed. On each of 3 days in 1985, moths were collected from canopy and crown traps before and after normal emigration flight occurred [i.e. approximately 17:00 h Atlantic Daylight time (ADT) at the study site]. Data are shown in the Appendix. Wing length and dry weight Forewing length (distance measured between thoracic attachment point to distal tip of a detached wing using a binocular microscope) and dry weight less a forewing (48 h at 65 C) were determined for canopy and crown females collected daily throughout the entire 1986 moth flight period. In a few cases, the forewings or dry weight could not be measured on the same individual because of damage or loss, resulting in unequal sample sizes in these measurements on certain days. The forewing length of subsamples of female moths caught in canopy traps from 1983 to 1987, inclusive, were also measured. Moth eclosion and sampling of offspring surviving to feed as second instars (L 2 ) in spring Each year, spruce budworm development was monitored daily at the site using samples collected from whole, midcrown balsam fir branches. A weather station was placed at the site and maximum and minimum daily temperatures were used to calculate degree-day (DD) accumulation as described by Baskerville & Emin (1969). Percentage moth eclosion was determined daily from pupae and pupal exuviae collected as indicated above. Starting in early spring, spruce budworm larvae were regularly sampled nearly every weekday from mid-crown balsam fir branches cut from ten to 20 preselected trees at the study site. On each sample day, 50 living larvae were picked randomly from one or more of these branches depending on the number of larvae per branch, placed in gelatin capsules and frozen for subsequent examination. Nosema diagnosis and measure of level of infections (disease load) All male and female moths caught in each trap/tray were counted daily and all, or a random subsample ( Table 1 ), of each sex were examined for the presence of Nosema spores. A portion of the abdomen was macerated in distilled water and the homogenate examined under a compound microscope at 400 magnification. All L 2 sampled daily were examined in a similar manner, except that the entire body of each specimen was macerated and examined. If other entomopathogens were present, they were also noted. Level of infection (disease load) was scored as either light or moderate heavy based on the number of spores observed in each moth sample. Samples of females were oven dried overnight (60 C), cooled in a dessicator, weighed, and homogenized in 130 L of sterile distilled water in 1.5-mL microcentrifuge tubes. The tissue in each tube was resuspended by vortexing for 5 s followed by quickly inverting the tube. A 10- L aliquot was placed on a clean slide and covered with a mm coverslip. Nosema spores were counted in five fields of view in a compound microscope using the 40 objective to obtain an average number of spores. The levels of infection were set at light, an average in five fields of ten spores or less, and moderate heavy as ³ 11 spores. Once the infection level of each abdomen had been determined, the number of spores per mg dry weight of tissue was determined by diluting a 10- L aliquot of each sample in 90 L of sterile distilled water. The suspension was vortexed as above and 10 L was touched to the edge of a coverslip over a Neubauer Table 1 Total number of canopy, crown and dead spruce budworm moths of each sex caught each year Canopy moths Crown moths Dead moths Year Females ( n ) a Males ( n ) Females ( n ) Males ( n ) Females ( n ) Males ( n ) (719) 6979 (440) b 158 (150) 440 (356) (962) 4169 (505) 250 (107) 566 (234) (786) 4064 (420) 2902 (837) 4555 (815) 263 (225) 541 (405) (561) 1594 (350) 668 (634) 842 (282) 101 (101) 165 (165) (34) 47 (41) 18 (18) 26 (26) 0 0 Total (3062) (1756) 3588 (1489) 5423 (1123) 772 (583) 1712 (1160) a The number of each sex examined for the presence of Nosema spores is shown in parentheses. b Crown moths not collected because crown trapping did not commence until 1985.

4 250 E. S. Eveleigh et al. haemocytometer ( Cantwell, 1970 ). Spore counts were made over ten grids and three separate counts were made for each sample. Light infections had 0.53 ± spores per mg dry weight of tissue (mean ± S E ; n = 14) and moderate heavy infections had ± spores per mg dry weight of tissue ( n = 30). Data analyses Polynomial logistic regressions (proc genmod) were used to determine if the overall infection rate and the moderate heavy infection rate (both treated as binomial response variables) in females and males differed between moth types (i.e. canopy, crown and dead moths) and year. In all analyses, DD was treated as a continuous covariate and its inclusion in quadratic form consistently provided a better model fit. The effects of infection on the forewing length and dry weights of canopy and crown female moths were examined using analysis of covariance with DD as a covariate (proc glm). The Student Newman Keuls multiple range test was used to compare the means of dry weight, and the means of forewing length, among infection levels. All statistical analyses were performed using SAS statistical software (SAS Institute Inc., 1988). Results Trap catches Canopy ( Fig. 1a d ) and crown ( Fig. 1e,f ) catches started each year at approximately 530 DD and ended at 800 DD. Over the study period, more females than males were caught in canopy traps, whereas more males than females were caught in crown traps ( Table 1 ). Subsequent to the decline in spruce budworm populations after 1985, both canopy and crown trap catches were low in 1986 and 1987 ( Table 1 ). Details on flight activity before and after emigration are presented in the Appendix. Each year, except 1987 when no dead moths were found ( Table 1 ), dead moths appeared in drop trays shortly after moths first appeared in canopy and crown traps ( Fig. 1g j ). As with crown catches, more males than females were present in drop trays each year ( Table 1 ). Figure 1 Percentage of moth eclosion, percentage of total trap catch for male and female spruce budworm moths and incidence of overall Nosema infections in male and female moths in relation to degree-day accumulation for canopy moths in (a) 1983, (b) 1984, (c) 1985 and (d) 1986; for crown moths in (e) 1985 and (f) 1986; and for dead moths in (g) 1983, (h) 1984, (i) 1985 and (j) 1986 (n.b. trapping for crown moths did not commence until Incidences of infection in catches containing fewer than ten moths were not used in graphs, but were used in all data analyses. Data for 1987 were not graphed because of very low daily trap catches).

5 Entomopathogen infection and host dispersal 251 Overall infection rate Few of the early emerging canopy ( Fig. 1a d ) and crown ( Fig. 1e,f ) males and females were infected each year. As DD accumulated and more moths entered the population, the overall infection rate increased, reaching a peak or plateau near 100% moth eclosion in all years except In that year, moth eclosion increased at a slower rate than in previous years and the overall infection rate reached a plateau at approximately 40 50% moth eclosion. Due to low trap counts in certain years, overall infection rates among dead female and male moths ( Fig. 1g j ) were not as readily discernible as they were among canopy and crown moths. However, it appears that the daily overall infection rate among dead moths of both sexes tended to increase with moth eclosion. The overall infection rate differed significantly among canopy, crown and dead females, but not among canopy, crown and dead males ( Table 2 ). In females, the overall infection rate was lowest among canopy moths and highest among dead moths, with crown moths having intermediate infection rates ( Fig. 2 ). The overall infection rate also differed significantly between years ( Table 2 ). Moderate heavy infection rate The moderate heavy infection rate for both sexes of canopy ( Fig. 3a d ), crown ( Fig. 3e,f ) and dead ( Fig. 3g j ) moths exhibited the same general patterns in relation to moth eclosion as those observed each year for the overall infection rate. The moderate heavy infection rate differed significantly among canopy, crown and dead females, but not among canopy, crown and dead males ( Table 3 ). In females ( Fig. 4a ), the lowest moderate heavy infection rate occurred in canopy moths, followed by crown and dead moths. As seen in the overall infection rate, more canopy males than females were moderately heavily infected, but fewer dead males than females were moderately heavily infected ( Fig. 4a,b ). The moderate heavy infection rate varied significantly between years in females, but not in males; however, in both sexes, Table 2 Summary of logistic regression analysis relating the incidence of Nosema infection in female and male canopy, crown and dead (moth type) spruce budworm moths to degree-day accumulation and year Source d.f. 2 P Females Degree-day < Degree-day < Year < Moth type < Year Moth type Males Degree-day < Degree-day < Year < Moth type Year Moth type Figure 2 Mean ± SE percentage of canopy, crown and dead male and female spruce budworm moths infected each year with Nosema. there was a significant interaction between the infection rate of moth types and year ( Table 3 ). Female dry weight and forewing length The mean size (dry weights ( Fig. 5a,b ) and forewing lengths ( Fig. 5c,d ) of both uninfected and infected canopy and crown females declined over the 1986 eclosion period ( Table 4 ). Canopy moths (8.12 ± 0.16 mg (mean ± SE; n = 535) were significantly heavier than crown moths (6.79 ± 0.12 mg; n = 626) ( Table 4 ), regardless of infection. However, infection negatively affected dry weight; infected canopy and crown females weighed significantly less (6.81 ± 0.15 mg; n = 394) than infected females in both moth types (7.70 ± 0.13 mg; n = 766) ( Table 4, Fig. 5a,b ). Dry weights of moderately heavily infected females (6.51 ± 0.18 mg; n = 240) were most severely affected, differing significantly from both uninfected (7.69 ± 0.13 mg; n = 768) and lightly infected (7.21 ± 0.25 mg; n = 158) females, which did not differ (Student Newman Keuls). Independent of infection, canopy moths (65.89 ± 0.21 units; n = 559) had longer forewings than crown moths (64.60 ± 0.19 units; n = 626) ( Table 4, Fig. 5c,d ). Nevertheless, infected canopy and crown females (63.28 ± 0.25 units; n = 404) had significantly shorter forewings than uninfected females in both moth types (66.20 ± 0.16 units; n = 780) ( Table 4, Fig. 5c,d ). As with dry weights, females with moderate heavy infec tions were most severely affected, having significantly shorter forewings (62.43 ± 0.16 units; n = 244) than lightly infected (64.60 ± 0.38 units; n = 164) females, which, in turn, had significantly shorter forewings than un infected females (66.19 ± 0.16 units; n = 782) (Student Newman Keuls).

6 252 E. S. Eveleigh et al. Figure 3 Percentage of moth eclosion, percentage of total trap catch for male and female spruce budworm moths and incidence of moderate heavy Nosema infections in male and female moths in relation to degree-day accumulation for canopy moths in (a) 1983, (b) 1984, (c) 1985 and (d) 1986; for crown moths in (e) 1985 and (f) 1986; and for dead moths in (g) 1983, (h) 1984, (i) 1985 and (j) 1986 (n.b. trapping for crown moths did not commence until Incidences of moderate heavy infections in catches containing fewer than ten moths were not used in graphs, but were used in all data analyses. Data for 1987 were not graphed because of very low daily trap catches). The forewing lengths of canopy females varied considerably between years ( Fig. 6a ), with infected individuals having significantly shorter forewings than uninfected individuals each year ( > P < 0.046; data for 1988 generation year Table 3 Summary of logistic regression analysis relating the incidence of moderate heavy Nosema infections in female and male canopy, crown and dead (moth type) spruce budworm moths to degree-day accumulation and year Source d.f. 2 P Females Degree-day < Degree-day < Year < Moth type < Year Moth type Males Degree-day < Degree-day < Year Moth type Year Moth type were not analyzed because of small sample sizes). However, because canopy females were, on average, larger than crown females, regardless of infection (see above) and, in addition, only 24 39% of the canopy females were infected annually, whereas 40 68% of crown and dead females were infected annually ( Fig. 6b ), canopy females consisted mainly of the larger individuals in the local population each year. Overall infection rate in L 2 progeny in spring Despite high annual infection rates in crown and dead females ( Fig. 6b ), only a small percentage of their L 2 offspring that established feeding sites in the spring of each year were infected (6.30 ± 2.23; mean ± SE) ( Fig. 6c ). Discussion Infection rate within seasons Among the first male and female moths to eclose each year, only a small percentage were infected with Nosema, the only

7 Entomopathogen infection and host dispersal 253 Figure 4 Percentage of moderate heavy Nosema infections in (a) female and (b) male canopy, crown and dead spruce budworm moths caught each year. entomopathogen found in moths in the present field study. Thereafter, as the eclosion period progressed, overall and moderate heavy infection rates in both sexes increased more or less steadily to a peak at or near 100% moth eclosion. Because horizontal transmission does not occur in this life stage, these increasing trends in infections throughout the moth eclosion period indicate that Nosema retards the development of the larval/pupal stages such that more of the un- healthy, infected moths in the population, having higher disease loads, emerge later in the season. After local moth eclosion ceased, infections tended to remain at high levels until the late-emerging moths completed their lifespan. These observations indicate that assessment of annual incidences of overall and moderate heavy infections in moths requires that sampling cover the entire eclosion period regardless of the sampling method employed. Figure 5 Mean ± SE dry weight (mg) of uninfected, and Nosema- infected (a) canopy and (b) crown spruce budworm female moths caught each day, and mean ± SE forewing lengths (ocular units) of uninfected, and Nosema- infected (c) canopy and (d) crown spruce budworm female moths caught each day in relation to degree-day accumulation and moth eclosion in 1986.

8 254 E. S. Eveleigh et al. Table 4 Summary of analysis of covariance for the effect of Nosema infection (infected or uninfected) on dry weight and forewing length of canopy and crown (moth type) female spruce budworm moths in 1986 Source of variation d.f. Mean square F -value P Dry weight Moth type < Infection Degree-days < Moth type Infection Error Forewing length Moth type Infection < Degree-days < Moth type Infection Error Overall and moderate heavy infection rates varied considerably within years. A most likely source of this variation is immigrant moths which, on any given day, may be entering (and leaving) the site from nearby or distant areas ( Greenbank et al., 1980 ), possibly changing the overall levels of infection at the site for a period of time. Because it was impossible to distinguish between immigrant and local moths caught in the traps, the contribution of immigrant moths to the levels of infection observed throughout the local moth eclosion period could not be determined. However, after local moth eclosion ceases, immigrants from distant areas that are phenologically behind our Fredericton area study site ( Royama, 1984 ), being the only source of new moths, may be the cause of the decline in overall and moderate heavy infection rates within certain years. This is because immigrants tend to be healthier individuals that dispersed from other areas (see below). Effects of infection on adult dispersal activity, morphological characteristics and flight Newly-emerged moths spend their first few days in the natal site, mating and laying some of their egg complement (up to 50%; Greenbank et al., 1980 ). Some males and females then emigrate, and as males eclose before females, they are the first to leave the natal site each year. Nosema infections did not affect the tendency of males to emigrate because neither the overall, nor the moderate heavy infection rates differed among canopy, crown and dead moths. In contrast, infected females were less likely to emigrate each year; canopy females had both lower overall and moderate heavy infection rates than crown and dead females. Lower incidence and disease loads of the obligate protozoan parasite Ophryocystis elektroscirrha have also been found in migratory than in nonmigratory populations of the monarch butterfly Danaus plexippus ( Altizer et al., 2000 ). This tendency for more of the uninfected than infected spruce budworm females to emigrate resulted in a higher proportion of infected females being left behind in the site, whereas no such tendency existed in males. Thus, depending on whether canopy or crown moths are sampled, one could find gender-based differences in infection when, in fact, none may exist in the population as a whole. Nosema infections, particularly moderate heavy infections, resulted in smaller females. Because crown females were, on average, less healthy, had shorter forewings and lower body weights than canopy females, Nosema may have negative effects on flight activity, influencing dispersal ability and the distance flown. A possible association between Nosema disease loads, body size and flight performance of spruce budworm moths has not been studied. However, in monarch butterflies, adults infected with O. elektroscirrha were smaller ( Altizer & Oberhauser, 1999 ), flew shorter distances, had slower flight speeds and lost proportionally more body mass per km flown than uninfected monarchs ( Bradley & Altizer, 2005 ). These results suggest that diseases may negatively affect energy resources available for flight. Because the deleterious effects of infection are less severe on spruce budworm males than females ( Wilson, 1984 ), and the present results indicate that, by contrast to females, overall and moderate heavy infections in males were similar in canopy and crown individuals, it appears that male flight performance would not be as affected in infection as that of females. Sanders & Wilson (1990) found that male flight duration was not affected by Nosema infection; however, disease loads were not known and female moths were not examined. Given the potential importance of Nosema infections on flight behavior, experiments to determine the effects of disease load on aspects of the flight activity of female and male spruce budworm moths of various sizes need to be conducted. Regardless of infection, the forewing length and dry weight of both canopy and crown females declined throughout the moth flight period. As adult size in insects is determined by final larval size in response to the diet quality obtained during the larval period ( Davidowitz et al., 2003 ), this decline in size may be a result of later-developing larvae generally obtaining poorer quality nutrition than early developing larvae. Thus, later-eclosing females are smaller probably because of both the negative effects of Nosema infection on aspects of the nutritional physiology of heavily infected larvae ( Nolan & Clovis, 1985; Bauer & Nordin, 1988b ), and poor larval nutrition ( Davidowitz et al., 2003 ). Interestingly, at the end of the eclosion period in 1986, there was a sudden increase in

9 Entomopathogen infection and host dispersal 255 forewing length and dry weight of canopy moths coupled with an apparent decline in the incidence of infection. Given the arguments presented above, it is likely that these were not locally emerged moths, but rather small numbers of immigrant moths that were re-emigrating. It should be noted that the actual difference in infection rates between emigrating and non-emigrating female moths is likely to be greater than that reported in the present study. This is because the three trap types used here did not catch only genuine emigrants or genuine non-emigrants. As discussed in the Appendix, crown traps undoubtedly contained a mixture (proportion unknown) of emigrants and non-emigrants. The presence of emigrants, having lower infection rates than non-emigrants, would have diluted the observed infection rate in crown moths. Likewise, some small proportion (unknown) of the moths caught in canopy traps could be non-emigrants, causing the observed infection rate to be higher than if only emigrants were caught in these traps. As shown in Figs 2 and 4, this situation resulted in the infection rate in crown moths being intermediate between that of canopy (mostly emigrants) and dead moths (mostly non-emigrants). Thus, it is predicted that if it was technically possible to distinguish genuine non-emigrants, then the actual effects of Nosema infections on dispersal activity and morphological characteristics would be greater than that reported in the present study. Effects of infection on fecundity As infected females have lower fecundity than healthy females ( Wilson, 1982; Bauer & Nordin, 1989 ), and the potential fecundity of spruce budworm is positively correlated with body size ( Thomas et al., 1980 ), it is likely that the fecundity of a large proportion of the females in years with a high incidence of moderate heavy infections in a local population of relatively small individuals will be most adversely affected (e.g. generation years 1984 and 1986). However, it is difficult to assess the effects of entomopathogen infections on fecundity at the population (site) level because the number of eggs laid is also influenced by other factors that are largely independent of infections in individual moths, such as dispersal (emigration and immigration) ( Royama, 1992 ), male larval nutrition ( Delisle & Hardy, 1997 ), defoliation levels ( Nealis & Régnière, 2004 ), and age at time of mating ( Proshold, 1996; Fraser & Trimble, 2001; Evenden et al., 2006 ). The overall and moderate heavy infection rates in male moths were often equal to, or greater than, those in female moths. However, laboratory studies have shown that, in contrast to infections in females, infections in male moths do not contribute to infections in subsequent generations ( Wilson, 1982, 1984 ). Nor do infected males, when mated with healthy Figure 6 (a) Annual mean ± SE forewing length (ocular units) of uninfected, lightly infected and moderately heavily Nosemainfected canopy spruce budworm female moths in relation to generation year (i.e. year after moths were caught) (n.b. in the 1984 generation year, infections were not scored as light or moderate heavy and, in the 1988 generation year, the data for lightly and moderately infected moths were combined because of small sample sizes); (b) annual variations in the percentage of canopy, crown and dead females infected by Nosema ; and (c) annual incidence of Nosema infection in L 2 offspring that established feeding sites each spring.

10 256 E. S. Eveleigh et al. females, affect fecundity or percentage egg hatch ( Wilson, 1984 ). However, Neilson (1963) reported that fecundity was reduced when male only or male and female moths were infected, and suggested that infected males may have a greater effect on fecundity than infected females. Because the results of the present study suggest that the effect of Nosema infections on the host may depend more on the disease load than on the incidence of infection, these somewhat conflicting observations may be the result of differing disease loads in the males and females used in these laboratory studies. Indeed, paternal infections could influence fecundity in field populations where numerous biotic and abiotic factors are also involved, particularly when the disease load in both sexes is high. This warrants further investigation. Effects of infection on progeny The fact that healthier females tended to emigrate whereas infected females, particularly those with moderate heavy infections, tended to remain at the site, suggests that the latter would contribute most to maintaining local levels of infection from year to year, especially because transovarian (vertical) transmission efficiency of infected females to progeny is high (90%, Wilson, 1982 ; 100%, Bauer & Nordin, 1989 ). The percentage of the total eggs laid that was infected each year in the present study is not known, but it is expected that a large proportion of the eggs were infected in years in which parental infection rates were high, particularly in non-emigrating moths. However, because of high annual mortality from egg to feeding L 2 (unpublished data), only a small percentage of the F 1 offspring ( L 2 ) that survived to begin feeding each spring was infected. Subsequently, through horizontal transmission of new Nosema infections during the larval period within each generation, the annual incidence of infection in adults was always much higher than that in the initial (spring) population of feeding larvae. In a laboratory study of spruce budworm, Bauer & Nordin (1989) reported that progeny mortality is positively correlated with maternal disease load, and suggested that moth disease load may be a useful predictor of progeny mortality from Nosema infections. Nosema infections are known to reduce egg hatch by 19% ( Wilson, 1982 ), but it is not known whether infections contribute to L 1 dispersal losses (disappearance) in late summer or to L 2 dispersal losses in the spring of the next year. Régnière & Duval (1998) reported that overall L 2 overwintering mortality in hibernacula, which amounted to 43.2%, was not related to Nosema infections in pupae of the previous generation but they did not examine the effects of maternal disease load. Given that Nosema infections affect the nutritional physiology of spruce budworm larvae, reducing nutrient assimilation needed for growth ( Nolan & Clovis, 1985; Bauer & Nordin, 1988b ), it is likely that infections, particularly moderate heavy infections, could not only affect the size of females as noted above, but also the energy reserves available to F 1 progeny. Thus, progeny of relatively small, infected females could be comparatively weaker and less able to survive L 1 dispersal, find suitable overwintering sites ( Régnière & Duval, 1998 ) and survive L 2 dispersal, than progeny of bigger, uninfected or lightly infected, females. This possible indirect effect of known disease loads of Nosema on the survival of early larval instars warrants further investigation. In summary, Nosema, a vertically and horizontally transmitted disease of low virulence, is not regarded as an important mortality factor of spruce budworm, although it can build to high incidences in the host population. Many debilitating and fitness effects of sublethal infections on the spruce budworm have been reported, but the potential effect of Nosema on adult dispersal has not been examined previously. The present study indicates that sublethal Nosema infections can have an important effect on adult, particularly female, dispersal activity, and perhaps on the distance travelled from natal sites, thus providing a preliminary understanding of the maintenance and spread of this entomopathogen in the host population over wide geographical areas. More research is now needed to explore the effects of Nosema disease loads on spruce budworm flight endurance (dispersal ability), and to determine the importance of adult dispersal as a disease dispersal mechanism in the host entomopathogen interaction ( White et al., 2000 ) Acknowledgements We thank George McDougall (retired), Laurie McNally, Carmen Cranley and numerous summer students for assistance with the conduct of this study. Earlier drafts of this manuscript were reviewed by Christina Campbell, Barry Cooke, Kees van Frankenhuyzen, Gaétan Moreau, Vince Nealis and Dan Quiring. This research was funded by the Canadian Forest Service. References Altizer, S.M. & Oberhauser, K.S. ( 1999 ) Effects of the protozoan parasite Ophryocystis elektroscirrha on the fitness of monarch butterflies ( Danaus plexippus ). Journal of Invertebrate Pathology, 74, Altizer, S.M., Oberhauser, K.S. & Brower, L.P. 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Proceedings of the Entomological Society of Ontario, 115, Accepted 16 February 2007 First published online 23 July 2007 Appendix Field observations in the present study indicated that flight activity of spruce budworm moths within the tree canopy during the day consists mostly of so-called buzzing activity around branches and short flights between trees. However, beginning in the early evening (approximately h ADT in the present study area), moths engage in pre-emigration flight activity, flying to the tops of trees before take-off. Emigration flight starts with an abrupt take-off from these trees ( Greenbank et al., 1980 ; present observations). Male and female moths were caught in canopy and crown traps during both observation periods, with most being caught after h ( Table A1 ). These observations confirm those of Greenbank et al. (1980) indicating that, if weather conditions permit, moth activity is greater during the evening and night than during the day. As in crown traps, some moths entered canopy traps during the morning and afternoon. Between h and h, up to 12% of the total daily female catch

12 258 E. S. Eveleigh et al. Table A1 Daily catch of moths (% of total catch), before and after h (Atlantic Daylight time), in canopy traps and crown traps on days 191, 193 and 196 in 1985 Day 191 Day 193 Day 196 Observation period Females Males Females Males Females Males Canopy traps h h Total catch Crown traps h h Total catch and up to 32% of the daily total male catch were caught in canopy traps ( Table A1 ). These daytime canopy trap catches raise two possibilities: (i) that moths may enter the traps accidentally during normal buzzing activity and/or (ii) some moths may actually emigrate during the day, which, incidentally, is a time when moth activity has received little direct field study. Direct observations of flight activity in the field (unpublished data) indicate, however, that most moths caught in canopy traps were genuine emigrants. On the other hand, crown traps undoubtedly caught a mixture of emigrating and non-emigrating moths (i.e. moths that were going to emigrate if not caught in crown traps, and those that were going to complete their lives in the site as genuine non-emigrants). Likewise, dead moths were also a mixture of non-emigrants and potential emigrants, although the proportion of emigrants was likely to be extremely low. Thus, drop-tray catches represented mostly genuine non-emigrants.