Horizontal and vertical transmission of a Nosema sp. (Microsporidia) from Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

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Journal of Invertebrate Pathology 95 (2007) 9–16 www.elsevier.com/locate/yjipa

Horizontal and vertical transmission of a Nosema sp. (Microsporidia) from Lymantria dispar (L.) (Lepidoptera: Lymantriidae) Dörte Goertz a,c,¤, Leellen F. Solter b, Andreas Linde c a

Department of Forest and Soil Sciences, BOKU – University of Natural Resources and Applied Life Sciences, Hasenauer Straße 38, A-1190 Vienna, Austria b Illinois Natural History Survey, 1816 S. Oak St., Champaign, IL 61820, USA c Fachhochschule Eberswalde, Alfred-Möller-Str. 1, 16225 Eberswalde, Germany Received 2 June 2006; accepted 7 November 2006 Available online 23 January 2007

Abstract The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae), a serious defoliator of deciduous trees, is an economically important pest when population densities are high. Outbreaking populations are, however, subject to some moderating inXuences in the form of entomopathogens, including several species of microsporidia. In this study, we conducted laboratory experiments to investigate the transmission of an unusual Nosema sp. isolated from L. dispar in Schweinfurt, Germany; this isolate infects only the silk glands and, to a lesser extent, Malpighian tubules of the larval host. The latent period ended between 8 and 15 days after oral inoculation and spores were continuously released in the feces of infected larvae until pupation. Exclusion of feces from the rearing cages resulted in a 58% decrease in horizontal transmission. The silk of only 2 of 25 infected larvae contained microsporidian spores. When larvae were exposed to silk that was artiWcially contaminated with Nosema sp., 5% became infected. No evidence was found for venereal or transovum (including transovarial) transmission of this parasite. © 2006 Elsevier Inc. All rights reserved. Keywords: Transmission; Lymantria dispar; Nosema sp.; Microsporidia; Latent period

1. Introduction Transmission is a key factor in pathogen–host interactions that can inXuence the population dynamics of the host (Anderson and May, 1981; McCallum et al., 2001). There are several potential pathways by which pathogens are transmitted within a host population; the most common are vertical transmission, the direct transfer of infection from parent to progeny (Fine, 1975; Becnel and Andreadis, 1999), and horizontal transmission, the transmission of the pathogens from one individual to another of the same generation (Steinhaus and Martignoni, 1970). Microsporidia, obligate pathogens that are commonly found infecting insects, may be transmitted vertically, hori-


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zontally, or by both means, depending on species-speciWc microsporidium–host interactions. Transovarial transmission, a form of vertical transmission, is known for a wide range of microsporidian species (Becnel and Andreadis, 1999) and is deWned as transmission within the egg yolk or embryo. Transovum transmission, a broader term that encompasses transovarial transmission, also includes infection that occurs when hatching neonate larvae feed on egg chorion contaminated with spores (Becnel and Andreadis, 1999). Only a few cases of venereal transmission via the male host have been documented and even fewer unequivocally (Solter, 2006). Microsporidia are horizontally transmitted when a susceptible host ingests spores that are released into the environment in the feces of infected individuals or via decomposed cadavers (Becnel and Andreadis, 1999). Spores of enterogastric microsporidia are released in the feces of the host insect throughout the larval stage (Weiser, 1961) and are also found in the meconium of newly


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eclosed adults that survive larval infection (Inglis et al., 2003). When microsporidian infection is primarily restricted to the insect fat body, release via decomposing cadavers may be the most important means of transmission (e.g. Vairimorpha disparis, Vavra et al., 2006). The latent period, the time interval between the initial infection of the host and the beginning of the infectious period (Anderson and May, 1992), is an important factor in the horizontal transmission of a pathogen within its host population, resulting in a time delay in the spread of the pathogen that can inXuence the population dynamics of the host (Dwyer et al., 2000). The life history and biology of several microsporidian pathogens of Lymantria dispar (L.) have been intensively studied to elucidate interactions with the host and to facilitate decisions regarding use in biological control programs (Maddox et al., 1998; McManus and Solter, 2003; Goertz et al., 2004b). Isolates in the L. dispar Vairimorpha–Nosema complex, despite close genetic relationships, infect diVerent tissues, and means of transmission appear to vary. Unlike Vairimorpha disparis, which is primarily a fat body parasite, several of the Nosema isolates also infect the silk glands and gonads, and have been shown to infect susceptible larvae via spores isolated from feces (Solter; Goertz and Hoch, unpublished data) as well as transovarially (Bauer, unpublished data). JeVords et al. (1987) suggested that spore-containing silk is an important horizontal transmission route for the L. dispar microsporidium Nosema portugal. Silk is widely distributed and gypsy moth larvae spin silk mats for resting and during molts. They follow their own silk threads or those of other larvae when they wander between feeding and resting sites (Campbell, 1975; Leonard, 1967). The infection of silk glands with microsporidia is regarded as a prerequisite for the transmission of microsporidia by oral uptake of spores from silk strands, and spores of Nosema portugal were found regularly in silk threads of gypsy moth larvae (JeVords et al., 1987). A Nosema-type microsporidium was isolated from the silk glands of a L. dispar larva collected in 1999 near Schweinfurt, Bavaria, Germany. The isolate was identiWed as a Nosema species and is listed as Nosema sp. [Schweinfurt], Accession No. 1999-A in the collection of the Illinois Natural History Survey, Urbana, Illinois and the University of Applied Sciences at Eberswalde, Germany. This isolate primarily infects the silk glands and, to a lesser extent, the Malpighian tubules, in contrast to several other Nosema isolates that also heavily infect the fat body tissues. These characteristics were of particular interest when considering transmission of the L. dispar microsporidian complex. The aim of the present study was to investigate the role of silk and feces as possible horizontal transmission pathways, the potential for vertical transmission and the latent period during infection of Nosema sp. [Schweinfurt]. 2. Material and methods Lymantria dispar, New Jersey Standard Strain (NJSS) egg masses were provided by the USDA-APHIS Otis

Method Development Center, Cape Cod, Massachusetts, USA. The larvae were reared on meridic diet (Bell et al., 1981) in climate chambers with a photoperiod of 16 L:8 D and temperature of 24 § 1 °C day: 18 § 1 °C night. Fresh spores of Nosema sp. [Schweinfurt] were produced for the experiments in L. dispar larvae following the procedure described in Goertz et al. (2004b). The spore suspension was stored as a 1:1 distilled water:glycerin suspension in liquid nitrogen until experimental use. 2.1. Inoculation of L. dispar larvae with Nosema sp. [Schweinfurt] Newly molted third-instar Lymantriae dispar larvae were starved for 24 h. Diet cubes, 2 mm3, were placed into 24-well plates and were inoculated with 1-l spore suspensions of 2 £ 104 spores. Larvae were placed individually into the wells. Only larvae that consumed the entire diet cube within 24 h were used in the experiments. 2.2. Horizontal transmission studies 2.2.1. Latent period and time period of transmission The latent period was determined using the following experimental design. Nine experimentally inoculated L. dispar larvae were reared together in a 250-ml diet cup, from day 3 after molt to the third stadium until pupation. Beginning 1 day post inoculation (dpi), an uninfected L. dispar larva ( D test larva) of the same age and from the same egg mass was randomly selected from the colony, marked dorsally between the 5th and 7th abdominal segments with correction Xuid (TippEx) and placed into the cup. After 24 h, the exposed larva was removed from the cup and was replaced with a new uninfected, marked larva. The diet was not changed until fresh diet was needed at 7 dpi, as it was not known when the Wrst infective spores would be released in the feces. Following the 24 h exposure periods, the test larvae were reared individually in 30-ml diet cups until death or adult eclosion. This procedure was continued until death or pupation of all experimentally inoculated larvae in the cup or until test larvae of the same age began to pupate. The experiment was repeated 10 times for a total of 198 test larvae. At the end of the experiment, inoculated and test larvae were examined under phase contrast microscopy for microsporidian infection. 2.2.2. Occurrence of spores with silk and feces To investigate the release of Nosema sp. spores in silk or feces, 25 experimentally inoculated L. dispar larvae were reared individually in 30-ml diet cups, changing to 100-ml cups when body weight exceeded 1 g. All diet cups were replaced on a daily basis until pupation of inoculated larvae and were checked for the release of feces and silk. If silk or feces were found, one randomly selected fecal pellet and all of the recovered silk were microscopically examined for the presence of spores.

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2.2.3. Silk and feces as a source for infections To determine whether spores in silk or fecal pellets of infected larvae are possible sources for microsporidian infections, the following experimental design was chosen: On day 1 after the inoculation procedure, groups of two inoculated and eight uninfected test larvae were placed in 250-ml diet cups and reared together until pupation. The cups were arranged in three diVerent ways to test possible sources of new infections and/or transmission pathways: Standard rearing conditions (Treatment A). The diet was placed at the bottom of the cup so that fecal pellets of the infected larvae remained directly on the diet. Feces separated from diet (Treatment B). The diet was placed at the top of the cup (the cups were incubated upside down). In this way, the fecal pellets did not fall on the diet surface, reducing contamination of the diet with Nosema spores. The fecal pellets, however, were a possible source for new infections with Nosema sp. as they remained in the rearing cup. Feces removed from rearing container (Treatment C). As described for Treatment B, the diet was placed at the top of the diet cup (cups upside down). Additionally, the bottom of the diet cup was replaced by a gauze net with a mesh width of 1 or 3 mm, depending on the age and size of larvae, so that the fecal pellets of the infected larvae fell through the net and were removed from the rearing environment. There was no direct contact of larvae with sporecontaining fecal pellets. Each of these assays were performed four times with six repetitions each for a total of 24 cups per treatment. At the end of the experiment, after pupation of larvae, all individuals were examined for the presence of spores. Cups with less than two infected L. dispar larvae were excluded from further data analysis. Three of 122 individuals died in the larval stage during the experiments and were immediately removed. The diet was not changed until fresh diet was needed at 7 dpi. 2.2.4. Experimentally contaminated silk as a source for infections To quantify horizontal transmission of microsporidia via silk, this larval product was experimentally contaminated with Nosema sp. spores. Beginning with the molt to the third larval stadium, 10 uninfected L. dispar larvae were selected daily from the colony and placed individually into 30-ml diet cups in two groups of Wve cups. The larvae were held in the diet cups for 24 h in order to produce silk and then were treated as follows: Group (A). The silk deposited by each larva at the side of the cup was inoculated with 1-l spore suspension of 1 £ 103 spores. All larvae were held for another 24 h in the same cup.

11 Group (B). The silk deposited by each larva at the side of the cup was inoculated with 1-l spore suspension of 1 £ 103 spores; the larvae were rotated between cups; and each larva was held for an additional 24 h in the cup with inoculated silk produced by a diVerent larva. The following day, all larvae were placed individually in fresh diet cups until death or adult eclosion. This procedure was continued until the pupation of the test insects, when they were no longer available for initiation of infection with Nosema sp. At the end of the experiment, all individuals were examined for the presence of spores. 2.3. Vertical transmission 2.3.1. Infection of F1-Generation with Nosema sp. [Schweinfurt] To determine if Nosema sp. is vertically transmitted, the following experiment was performed. Third-instar larvae experimentally inoculated with Nosema sp. were reared on diet until adult eclosion. The adult L. dispar males and females were paired in separate breeding cages for mating. The breeding cages consisted of 250 ml plastic cups containing Wlter paper as a substrate for the deposition of eggs. After oviposition, all parental adults were examined for infection. Egg masses oviposited by the infected female moths were stored for 6 weeks in a climate chamber with a photoperiod of 16 L:8 D and temperature of 24 § 1 °C day: 18 § 1 °C night, then allowed to diapause for 3 months in a moist chamber (lidded 20 £ 10 £ 5 cm plastic box; water for humidity supplied in an open Petri dish) in a refrigerator at 6 °C. After the diapause period, the egg masses were placed on fresh diet in a climate chamber, 16 L:8 D and temperature of 24 § 1 °C day: 18 § 1 °C night, until hatch. Fifteen newly emerged larvae from each egg mass (n D 10) were reared individually on diet until molt to third instar, then dissected and microscopically examined for infection. 2.3.2. Examination of the gonads of Lymantria dispar, infected with Nosema sp. [Schweinfurt] Twenty L. dispar individuals experimentally inoculated in third instar, 10 females and 10 males, were dissected at diVerent developmental stages. The gonads of Wve late Wfthinstar male and Wve late sixth-instar female larvae were removed 26 and 33 dpi, respectively, and the gonads of Wve male and Wve female pupae were removed 41 dpi. Silk glands were examined routinely for the presence of spores before the removal of the gonads. The gonads were Wxed in Bouin for 24 h (Heinrich, 1957), followed by rinses in 70% ethanol until the ethanol remained clear and without an acidic odor. The samples were dehydrated in ethanol and embedded in HistoResin (Reichert-Jung, commercial embedding kit). Tissue sections were cut with a microtome (Leica Jung M2055), C-knife, angle of 0° and 5 m thickness. Every Wfth section was placed on a slide, Wxed and stained using Richardson’s method (60 °C, 20 s; Richardson et al., 1960) and micro-


D. Goertz et al. / Journal of Invertebrate Pathology 95 (2007) 9–16

Fig. 1. Latent period of L. dispar larvae inoculated with Nosema sp. [Schweinfurt] and time periods of horizontal transmission: 䊐 infection in test insects, 䊏 no infection in test insects.

scopically checked for the presence of merogonic stages, primary spores and environmental spores. 2.4. Statistical analysis of the experiments All data were analyzed with the software program SPSS 11.0 (SPSS Inc. 1989–2001). The Kolmogorov–SmirnoVtest was used to test the data sets for normal distribution. The medians of more than two not normally distributed data sets were tested for signiWcant diVerences using the Kruskal–Wallis-H-test. Multiple post hoc comparisons between two groups were made using the Tukey–Kramertest. For the analysis of frequency data the 2-test was used. 3. Results 3.1. Horizontal transmission 3.1.1. Latent period and time period of horizontal transmission Experimental inoculation with Nosema sp. produced infection in 100% of the treated larvae. The experiment was terminated by the pupation of uninfected test larvae, at 20 § 1 dpi (Fig. 1). Of the 198 test larvae, 31 (15.7%) became infected with Nosema sp. The transmission rate varied between 0.0 (replication no. 5) and 33.3% (replication no. 6) per diet cup. The average duration of the latent period was 11 § 3 days and varied between cups. The Wrst transmission of Nosema sp. occurred at 8 dpi; the longest latent period was 15 days. No transmission occurred later than 18 dpi; the time period for horizontal transmission ended on average 5 § 2 days prior pupation. 3.1.2. Occurrence of spores with silk and feces Feces. One single larva released fecal pellets with spores 3 dpi (Fig. 2). No L. dispar larvae released sporecontaining feces between 4 and 7 dpi. The shortest time period between the inoculation and the onset of continuous spore release in fecal pellets was 8 days. The longest time period between the inoculation of a larva and the Wrst

observation of spores in the feces was 15 days. On average, infected L. dispar larvae began to release spore-laden feces at 11 § 3 dpi. Once infected larvae had begun to release Nosema sp. spores in their feces, 52% of the larvae continued to release spores with feces until pupation, 24% of L. dispar larvae interrupted the release of spores for one day and 24% of the infected individuals did not produce feces with spores for a time period of 2 to 9 days. After 8 dpi, the probability that an infected larva produced fecal pellets on a daily basis was 82% and the probability that the released feces contained observable spores was 66.3%. Silk. An average of 60% (range: 24–100%) of all infected L. dispar larvae released silk until day 8 dpi. By 9 dpi, the percentage of larvae that released silk decreased signiWcantly to 3.9% (2 D 222.4;  < 0.001). Two individual larvae released silk containing spores; one larva at 19 dpi and the other at 21 dpi. Therefore, the probability that silk was produced by a larva and that this silk contained spores was 0.6% after dpi 9. 3.1.3. Silk and feces as source for infection with Nosema sp. [Schweinfurt] Standard rearing conditions (Treatment A). An average of 46.6% of susceptible test larvae reared under standard conditions was infected at the end of the experiment (Fig. 3). The average proportion of new infections of test larvae varied between trials, between 0.0 and 85.5% of the test larvae were infected at the end of the experimental periods. Feces separated from diet (Treatment B). When the feces did not fall onto the diet, an average of 29.5% of test larvae developed infections, a decrease of 17.1% from that of standard rearing conditions. For all trials, between 0.0 and 56.3% of larvae became infected prior to pupation. Feces removed from rearing container (Treatment C). Removal of feces from the diet cups resulted in the lowest transmission of Nosema sp. The average percentage of new infections was 18.4% (2.5–27.1%), an overall average

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Fig. 2. Release of silk and feces, and occurrence of spores on silk and in feces of L. dispar larvae after an infection with Nosema sp. [Schweinfurt]. 䊐 no silk (A) or feces (B) present. silk (A) or feces (B) present; no spores observed. 䊏 silk (A) or feces (B) present; spores observed.

reduction of 28.2 and 11.1% compared to Treatments A and B, respectively. 3.1.4. Experimentally contaminated silk as a source for new infection with Nosema sp. [Schweinfurt] When silk produced by L. dispar larvae was contaminated artiWcially with 1 £ 103 spores of Nosema sp., 4.6% (n D 195) of test larvae became infected. When larvae were placed in diet cups with their own silk (Group A), 6.2% (n D 98) became infected. Successful infections occurred during exposure 2, 9, 10, 18 and 19 days after molt to the third instar, during the third, fourth and Wfth larval stadia. Of the larvae that were placed in diet cups containing silk produced by a diVerent larva (Group B), 3.1% (n D 97) of the larvae developed infections. Infections occurred during exposure 6, 12 and 19 days after molt to third larval instar, during the third, fourth and Wfth larval stadia. The percentage of successful infections of group A was not signiWcantly diVerent from that of group B (2 D 1.02;  < 0.05).

3.2.2. Occurrence of Nosema sp. [Schweinfurt] in the gonads of Lymantria dispar Infected male L. dispar larvae were in the Wfth larval stadium at 26 dpi. Microscopic examination showed infections of the silk glands and no infection of the gonads. No vegetative stages, primary spores or environmental spores were found. Infected female L. dispar larvae were in the ultimate larval stadium at 33 dpi. The connective tissue adjacent to the ovary of a single larva contained environmental spores of Nosema sp. No vegetative stages, primary or environmental spores were found inside the ovarioles of the larvae. Male and female pupae, dissected 41 dpi, contained diVerent microsporidian stages: meronts and spores of Nosema sp. were found in the peritoneal tissue of the testis in one male pupa. Neither the testicular epithelium nor the spermatocysts contained microsporidial stages. Environmental spores were found in the basal laminae covering the ovaries in Wve ovarioles of two female pupae. Vegetative stages were not found. Oocytes and the feeding cells of the dissected ovarioles did not contain developing stages of Nosema sp.

3.2. Vertical transmission 4. Discussion 3.2.1. Infection of F1-Generation with Nosema sp. [Schweinfurt] The progeny of infected adults of L. dispar were examined for the presence of spores after molt to the third instar. None of the 150 larvae examined were infected with Nosema sp.

Several aspects of vertical and horizontal transmission may inXuence the successful spread of a pathogen in its host population. While vertical transmission is an important factor for survival of microsporidia between non-over-


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Fig. 3. Percentage (median) of new microsporidian infections acquired by L. dispar test individuals in three diVerent treatments (two infected and eight uninfected larvae per cup) and for diVerent trials. 䊐 Treatment A: diet provided at the bottom of the rearing cup with accumulating larval feces (standard); Treatment B: diet provided at the top of the rearing cup, the fecal pellets fell to the bottom of the cup; Treatment C: diet provided at the top of the rearing cup, fecal pellets removed from cage via a gauze net at the bottom of the rearing cup. DiVerent letters above bars indicate signiWcant diVerences (**p < 0.01; *p < 0.05; n.s., not signiWcant).

lapping host generations, horizontal transmission allows the microsporidian parasite to infect new hosts of the same generation. Such microsporidian infections may occur upon ingestion of food contaminated with spore-laden feces or exposure to contaminated silk trails between the feeding and resting sites of host larvae. Lymantria dispar larvae that ingest microsporidian spores undergo a latent period, which constitutes a time delay in the transmission of a pathogen before they begin to release infective spores with feces or silk. The aim of the present study was to investigate aspects of horizontal and vertical transmission of a microsporidian isolate that was found in Germany in 1999 and is considered as a possible candidate for biological control. The latent period, the time period for horizontal transmission and the percentage of new infections were diVerent for Nosema sp. [Schweinfurt] than for two other Nosema isolates from Bulgaria (INHS Accession Nos. 1996-A and 1997-E) infecting L. dispar larvae (Goertz et al., 2004b, Pilarska et al., 1998). The two Bulgarian Nosema isolates were transmitted to a higher percentage of test larvae than Nosema sp. [Schweinfurt]. The latent period for Nosema sp. [Schweinfurt] was longer, averaging 11 days, than for the two Bulgarian isolates, which averaged 7–8 dpi (Goertz et al., 2004b). First transmission of infections occurred at 3 dpi for the Bulgarian isolates (Goertz et al., 2004b) and at 8 dpi for Nosema sp. [Schweinfurt]. Although transmission of the Bulgarian isolates occurred before the maturation of spores in the host and

is due to presence of ungerminated spores (inoculum) in the feces, the infections produced in susceptible larvae could enhance dispersal and horizontal transmission of some microsporidia. While there is reason to believe that transmission of ungerminated spores will also occur when larvae ingest Nosema sp. [Schweinfurt], the longer latent period of this isolate might be one factor in the lower horizontal transmission rate compared to other isolates. More than 40% of test larvae became infected with the Bulgarian Nosema isolates (Goertz et al., 2004b), while only 15% of all test larvae became infected with Nosema sp. [Schweinfurt] in horizontal transmission studies. The Bulgarian isolates may also have higher infectivity, or contain more infective spores in the feces; at least 70% of susceptible test larvae became infected after dpi 13 in contrast to Nosema sp. [Schweinfurt], which produced infection in, at most, 60%, of exposed larvae (Fig. 1). While the latent period following an infection with a microsporidian species results in a time delay in the transmission of a parasite, pupation of susceptible L. dispar larvae can limit successful transmission because they are no longer available to contract an infection by ingesting microsporidia. The variable duration of the entire larval stage explains diVerences in the number of larvae tested and the diVerences in time period available for transmission. Low transmission rates prior to pupation might be explained by lower susceptibility of late stage L. dispar larvae to Nosema sp. [Schweinfurt] spores for late stage gypsy moth larvae.

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The end of the latent period for Nosema sp. [Schweinfurt] corresponded with the Wrst observations of spores in the feces (Figs. 1 and 2). Silk produced by infected larvae appears to be of minor importance for the transmission of Nosema sp. [Schweinfurt], despite heavy infections observed in the silk glands. We observed reduced silk production after maturation of spores in the silk glands from approximately 12–14 dpi until pupation of larvae. The rare occurrence of spores in silk threads in only two larvae, one at 19 dpi and one at 21 dpi, corroborate the results of transmission studies. Additionally, the release of microsporidian spores with silk did not coincide with the occurrence of new infections of susceptible test larvae between 9 and 18 dpi, and only a very low proportion of test larvae became infected after being in contact with artiWcially contaminated silk. The higher transmission rates in diet cups when infected and uninfected larvae were reared together is not contradictory, because this experimental design did not exclude the spore transfer by individual contact of larvae or the contamination of diet surface by regurgitation or feces. Larval excuviae and cadavers can be excluded as infectious sources because the excuviae were removed regularly, and larval mortality was very low. The present study seems to contradict the results of JeVords et al. (1987). They found frequent occurrence of Nosema sp. (Nosema portugal; Maddox et al., 1999) spores in silk strands released by infected larvae (JeVords et al., 1987). JeVords et al. (1987) suggested, that spore-contaminated silk could be important for horizontal transmission of this L. dispar microsporidium. Our contrasting results and the rare number of publications (Kellen and Lindegreen, 1971; Thomson, 1958; JeVords et al., 1987) about the occurrence of spores in silk indicate that there are still questions about importance of silk as a horizontal transmission route. Our study shows that transmission of Nosema sp. [Schweinfurt] by feces containing spores is the primary transmission pathway. When feces of infected larvae did not fall onto the diet or were excluded from the rearing cups, the proportion of new infections of test larvae decreased by 17 or 28%, resp., but infections were not eliminated. These results indicate that the contamination of food by sporeladen feces is important for the transmission of microsporidia, even though direct feeding of L. dispar larvae on fecal pellets or the contamination of the gauze nets with spores cannot be completely excluded. We observed that feces of infected larvae regularly contained spores beginning 8 dpi. More than 50% of all infected larvae released spore-containing feces continuously without interruption after the latent period. The presence of spores in feces, probably a result of the infection of Malpighian tubules, and the initiation of infection of susceptible test larvae occurred during the same time period. Less than 50% of infected L. dispar larvae interrupted the release of spore-containing feces during molting. Our results indicate the importance of contaminated food for transmission of microsporidian spores and


of feces as a source for new infections even for species that are not primarily enterogastric. Transmission rates were lower in cages where feces were excluded. The variability of results among diVerent trials of the same experiment and treatment can be explained by diVerences in latent periods of individual larvae that ranged from dpi 8 to 15 and the unknown sex ratio of larvae at the beginning of the experiment. A longer larval stage for females compared to males may prolong the time period for transmission of microsporidia by and to female larvae (Goertz et al., 2004a; unpublished data). While uninfected male larvae pupate between 18 and 24 dpi, the larval stage of uninfected females may last up to 34 days (Goertz et al., 2004a). Therefore the infectious period for microsporidian transmission can last between 3 and 22 days, resulting in varying transmission rates or possibly no transmission for this Nosema isolate. While vertical transmission is considered to be common among microsporidia infecting insects (Solter, 2006), Nosema sp. [Schweinfurt] does not appear to be vertically transmitted. No infections were found in the F1 oVspring of infected parents. Mature microsporidian spores were found only rarely in the connective tissues of ovaries or peritoneal tissues of the male testes in otherwise heavily infected male and female larvae, and no spores or developmental stages were found in ovarian tissues or the testes. Our results suggest that horizontal transmission rates diVer among L. dispar Nosema isolates. We observed lower transmission rates for Nosema sp. [Schweinfurt] than for other isolates previously tested (Goertz et al., 2004b). Spore-laden feces is the major transmission pathway for Nosema sp. [Schweinfurt] but the latent period is longer and spore-containing silk appears to be of minor importance. Because horizontal transmission appears to be less eYcient for this isolate than for other Nosema biotypes isolated from L. dispar, and vertical transmission does not appear to occur, there remain questions about how Nosema sp. [Schweinfurt] is maintained in host populations. DiVerences in tissue speciWcity, latent period, and possibly infectivity, despite a close genetic relationship with other L. dispar Nosema isolates, are examples of variation in the microsporidia. Acknowledgments We thank G. Hoch for helpful comments on an earlier draft of the manuscript. The study was supported by the Fachhochschule Eberswalde, the Ministry of Science, Research and Culture of Brandenburg/Germany and the Illinois Natural History Survey. References Anderson, R.M., May, R.M., 1981. The population dynamics of microparasites and their invertebrate hosts. Philos. Trans. R. Soc. B 291, 451–524. Anderson, R.M., May, R.M., 1992. Infectious Diseases of Humans. Dynamics and Control. Oxford University Press, Oxford, New York, Tokyo.


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