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Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae)
Journal of
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 87 (2004) 51–58 www.elsevier.com/locate/yjipa
Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae) ´ lvarez* E. Quesada-Moraga, R. Santos-Quiro´s, P. Valverde-Garcı´a, C. Santiago-A Departamento de Ciencias y Recursos Agrı´colas y Forestales, ETSIAM, Universidad de Co´rdoba, Apartado 3048, 14080 Co´rdoba, Spain Received 25 March 2004; accepted 22 July 2004 Available online 12 September 2004
Abstract Virulence of Metarhizium anisopliae (Metschnikoff) Sorokin strain EAMa 01/121-Su against the German Cockroach, Blatella germanica (L.), was determined using four concentrations ranging from 4.2 · 106 to 4.2 · 109 spores per milliliter. The LD50 value was 1.4 · 107 spores per milliliter (56,000 spores per cockroach) and LT50 values were 14.8 days and 5.3 days for 4.2 · 108 and 4.2 · 109 spores per milliliter, respectively. An experiment was conducted to evaluate whether a fungal transmission could exist among infected and healthy cockroaches. Percentage mortality at a ratio of 1:10 of infected to unexposed cockroaches was 87.5% and LT50 was 12.2 days, which indicated the potential of this strain to be horizontally transmitted and to rapidly spread the infection in the insect population. The effect of a sublethal dose (ca. LD60) of M. anisopliae EAMa 01/121-Su strain, applied topically on German cockroaches, was studied by reciprocal crossing. Othecal production, oothecal hatchability, and nymphal production declined upon exposure to M. anisopliae EAMa 01/121-Su strain. The mean number of oothecae laid by female was progressively and significantly reduced by fungal treatment from second oviposition period onwards. Oothecal hatch of fungally challenged females was reduced by 46–49%, oothecal viability by 48–85%, and nymphal production by 22–35%. Only treated females showed an effect on oothecal production, oothecal hatch, and nymphal production, although oothecal hatch was also governed by treated males at a higher significance level. Our results on virulence and horizontal transmission of fungal conidia of M. anisopliae EAMa 01/121-Su strain and its sublethal reproductive effects on German cockroach females are discussed in terms of its potential to decrease the pest status of B. germanica in the short and long terms. 2004 Elsevier Inc. All rights reserved. Keywords: Blatella germanica; German cockroach; Blattodea; Metarhizium anisopliae; Entomopathogenic fungi; Virulence; Horizontal transmission; Reproductive effects; Sublethal effects; Biological control
1. Introduction The German cockroach, Blatella germanica, is a worldwide distributed species surviving well in association with any human habitation that provides warmth, moisture, and food in places such as apartments, homes and food-handling facilities (Cochran, 2003). German *
Corresponding author. Fax: +34 957218440. ´ lvarez). E-mail address: [email protected] (C. Santiago-A
0022-2011/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.07.002
cockroaches are primarily controlled through the use of synthetic organic insecticides (organophosphates, pyrethroids, and carbamates), but many factors including insecticide resistance, concerns about human and environmental safety, and increased developmental cost of new insecticides have intensified the search for new control methods. One potential alternative is the use of biologically based insecticides, such as those containing entomopathogenic fungi (Kaakeh et al., 1996, 1997; Murali Mohan et al., 1999).
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The entomopathogenic fungus, Metarhizium anisopliae, is a common fungal pathogen showing a wide insect host range including B. germanica (Zimmermann, 1993). Despite considerable research on the use of Metarhizium sp. for crop insect pest control, there are few reports on the application of this fungus to control B. germanica. From them, it can be concluded that although M. anisopliae has the potential to be an effective control agent, its slow action has limited effective use of this fungus under field conditions, as reported for the Bio-Path Cockroach Control Chamber (Pachamuthu et al., 1999). Therefore, efforts have been made to enhance the biological activity of M. anisopliae by integrating it with sublethal doses of chemical insecticides (Pachamuthu and Kamble, 2000; Zurek et al., 2002). To date, a majority of the work has focused on mortality of the target pest caused directly by the fungus. This was due to the urgent need for more active fungal strains. In addition, mortality due to initial fungus application may be followed by horizontal transmission within the target population (Kaakeh et al., 1996), a phenomenon that may be attributable to the gregarious behavior of cockroaches. Horizontal transmission may contribute greatly to the spread of infection and thus should be studied as part of a commercial development effort. However, to make an accurate estimation of the influence that a fungal pathogen has on a cockroach population is necessary to consider all effects, other than mortality on target individuals (Arthurs and Thomas, 2000; Blanford and Thomas, 2001; Fargues et al., 1991; Mulock and Chandler, 2001). It has been demonstrated that sublethal effects of fungal infection can have important implications for the population dynamics of the host, which ultimately contribute to the status of the target insect as pest (Arthurs and Thomas, 2000; Blanford and Thomas, 2001). There is some documentation on the effects of different chemical insecticides on oothecae carried by German cockroach at application time (Abd-Elghafar et al., 1991; Appel and Abd-Elghafar, 1990; Harmon and Ross, 1987), and on the sublethal effects of azadirachtin on reproduction of Periplaneta americana (L.) (Richter et al., 1997), and of deltamethrin and propoxur on longevity and reproduction of B. germanica (Lee et al., 1998), but to our knowledge, no information is available on the sublethal effects of entomopathogenic fungi on cockroach reproduction. From our collection of strains of M. anisopliae, we have selected strain EAMa 01/121-Su for its pathogenicity to B. germanica. This research was undertaken to evaluate the virulence of M. anisopliae strain EAMa01/ 121-Su against B. germanica, to study whether it may be transmitted from infected to healthy cockroaches and to assess the sublethal effects of fungal infection on German cockroach reproduction.
2. Materials and methods 2.1. Insects The insects used in this study came from a stock colony established in our laboratory. The colony was reared at 27 ± 2 C, 60 ± 10% RH, and photoperiod of 12:12 h light:dark cycle and fed with Purina dog chow and water provided in glass tubes with cotton stoppers. Each plastic cage (30 · 20 · 20 cm) was provided with a paper egg carton shelter. 2.2. Conidial production The M. anisopliae strain used was originally isolated from a cotton field soil at Marchena, Sevilla (Spain) in 2001. This strain is deposited in the fungal collection of CRAF Department of the Universidad de Co´rdoba with the access number EAMa 01/121-Su. Conidia were produced on Malt Agar (Panreac No. 413781) following the instructions of the fabricant. After autoclaving at 121 C for 20 min, approximately 15 ml of media was poured into sterile petri dishes (10 · 15 cm). Ten microliter of the spore suspension prepared in 0.05% Triton X-100 was placed in the center of each Petri dish containing the media and sealed with parafilm. Spore cultures in sealed Petri dishes were incubated at constant temperature (27 ± 2 C) in darkness for 21 days. Conidia were harvested in sterile water containing 0.05% Triton X-100. Conidia from each plate were scraped with a sterile spatula, and the spore suspension was filtered through an eight-layered cheese cloth, centrifuged (4000g for 15 min at 4 C), and resuspended in sterile water containing 0.05% Triton X-100. The spore concentration was determined using a hemocytometer. Germination of conidia was over 95%. 2.3. Bioassay to determine the biological activity of M. anisopliae strain EAMa 01/121-Su Four spore concentrations (4.2 · 106, 4.2 · 107, 4.2 · 108, and 4.2 · 109 spores per milliliter) were prepared from the stock suspension, based on the results of a preliminary experiment. Then, by means of a micropipette, 4 ll from each spore dilution was applied topically on the first ventral abdominal segment of each cool immobilized adult German cockroach. Controls were treated with 4 ll of sterile distiller water plus 0.05% Triton X-100. The treated cockroaches were placed in plastic containers (12 · 12 · 6 cm) conditioned as described above. Cockroaches were fed with Purina dog chow and water provided in glass tubes with cotton stoppers. The bioassay was conducted at 27 ± 2 C and 75 ± 10% RH. Mortality was monitored daily for 31 days, and dead cockroaches were removed daily. Each treatment was replicated four times with 10 adult cock-
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roaches per treatment, and the entire experiment was conducted twice. Cadavers were immediately removed and placed into sterile Petri dishes. Afterwards, they were surface sterilized with 70% ethanol and placed on sterile wet filter paper in sterile Petri dishes that were then sealed with parafilm and kept at room temperature. The emergence of hyphae was monitored for 10 days. The mortality data were pooled and LD50 calculated by probit analysis using SPSS (Statistical Package for Social Sciences in personal computers) 8.0 for Windows (SPSS, 1997). The LT50 values were determined using the probit analysis method for correlation data (Throne et al., 1995). 2.4. Transmission of M. anisopliae from sporulating adult cadavers to healthy adults Single EAMa 01/121-Su-killed adult cockroach with profuse mycosis was placed at the center of plastic containers (12 · 12 · 6 cm) with 10 newly emerged healthy adult cockroaches (one container per replication) and incubated at 27 ± 2 C and 75 ± 10% RH. Mortality was monitored daily for 31 days, and dead cockroaches were removed daily. The treatment was replicated four times and the entire experiment was conducted twice. Kaplan–Meier survival analysis was performed using SPSS (Statistical Package for Social Sciences in personal computers) 8.0 for Windows (SPSS, 1997). The LT50 values were determined using the probit analysis method for correlation data (Throne et al., 1995). 2.5. Bioassay to determine the sublethal effects of M. anisopliae strain EAMa 01/121-Su on B. germanica Based in the biological activity bioassay, we selected a spore concentration of 4.2 · 107 spores per milliliter (ca. LD60) to study the sublethal effects of M. anisopliae EAMa 01/121-Su strain on German cockroach reproduction. Newly emerged adult females and males were treated topically as above. As controls, newly emerged adult females and males were treated with 4 ll of sterile distilled water plus 0.05% Triton X-100. We established six mating combinations: Untreated females · untreated males, treated females · untreated males, untreated females · treated males, treated females · treated males, untreated virgin females, and treated virgin females. Couples were placed in plastic containers (12 · 12 · 6 cm), one couple per replication except for virgin females, conditioned as described above and incubated at 27 ± 2 C and 75 ± 10% RH. Each reproductive combination was replicated 14 times with one couple per treatment. Couples were checked daily for death and offspring for 100 days, and dead cockroaches were immediately removed and processed as above to evaluate fungal growth. Insects in the orthopteroid orders Blattodea, Mantodea, and Orthoptera secrete an egg case or pod
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surrounding the eggs, to give additional protection from desiccation and predation which it is commonly named as oothecae. The percentage of dropped oothecae from both live and dead females was determined. Comparisons of cumulative oothecae per surviving female over time were analyzed by a two-way repeated measures ANOVA for multi-observation data (Go´mez and Go´mez, 1984). Multiple comparisons between treatments for each sample date were performed by one-way ANOVA. Both analyses were made using SPSS (Statistical Package for Social Sciences in personal computers) 8.0 for Windows (SPSS, 1997). Surviving females with oothecae, dead females with oothecae, and dropped oothecae from live or dead females were held individually in 5 cm diameter plastic Petri dishes with a water wick and a piece of Purina dog chow. Cockroaches and oothecae were held at 27 ± 2 C and 75 ± 10% and photoperiod of 12:12 h light:dark cycle. We recorded the mean percentage of oothecal hatch and the mean carrying time until hatch. Nymph mortality was recorded for 20 days after hatching. Cadavers were immediately removed and processed as described before to evaluate fungal growth.
3. Results Based on initial mortality data, four tenfold concentrations from 4.2 · 106 to 4.2 · 109 spores per milliliter were selected for the biological activity bioassay. The average cockroach mortality ranged from 42.3 to 93.3% in the treated insects, whereas 13.6% mortality was observed in the untreated cockroaches. The dose– mortality response regression equation from assay of the four concentrations of EAMa 01/121-SV-strain against B. germanica newly emerged adults is shown in Fig. 1. The LD50 value was 1.4 · 107 spores per milliliter (56,000 spores per cockroach) [(95% CI: 6.0 · 106 spores
Fig. 1. Dose–mortality response of B. germanica newly emerged adults to four tenfold doses of M. anisopliae EAMa 01/121-Su strain (4.2 · 106–4.2 · 109 spores/ml). For each point N = 80 insects. Each insect was treated topically with 4 ll from each spore dilution.
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per milliliter (24,000 spores per cockroach)–2.7 · 107 spores per milliliter (108,000 spores per cockroach)]. The LT50 values were 14.8 days for 4.2 · 108 spores per milliliter [(95% CI: 10.8–20.6 days) (slope ± SE = 2.50 ± 0.38, v2 = 18.41)], and 5.3 days for 4.2 · 109 spores per milliliter [(95% CI: 3.3–7.3 days) (slope ± SE = 2.28 ± 0.40, v2 = 12.05)]. M. anisopliae grew from all cadavers in the 4.2 · 108 and 4.2 · 109 fungal treatments and from 20 to 60% of them in the 4.2 · 106 and 4.2 · 107 treatments. The fungus was horizontally transmitted from infected to healthy adult cockroaches. Mortality rates observed for unexposed and exposed cadavers were 10 and 87.5%, respectively (Fig. 2). Average survival time of healthy adults mixed with dead-infected cockroaches at a 1:10 ratio was 16.1 ± 0.96 days (means ± SE), which it is significantly lower than the one of 29.03 ± 0.96 days showed by unexposed cockroaches (log-rank statistic = 46.9, P < 0.001). The LT50 value resulting from
Fig. 2. Cumulative proportional survival of B. germanica adults unexposed (solid line and ·) and exposed to infected dead cockroaches at a ratio of 1:10 infected to healthy adults (dashed line and s). Data are means ± SE.
mixing healthy and dead M. anisopliae-infected adults was 12.2 days [(9.0–16.7 days) (slope ± SE = 2.51 ± 0.34, v2 = 11.78)]. Newly emerged adult cockroach males and females were treated with 1.7 · 105 spores per insect (nearly LD60) of M. anisopliae EAMa 01/121-Su strain to study its effects on cockroach reproduction. Average survival time (AST ± SE) values of treated adult males, 40.10 ± 5.6 days, and females, 36.19 ± 5.7 days, were significantly lower (log-rank statistic, P < 0.05) than those of untreated males, 89.93 ± 4.2 days, and females, 94.59 ± 2.8 days, respectively. We observed no differences in AST neither between treated nor untreated males and females. For all combinations, mortality of treated males and females was significantly higher than the one of untreated adults (F3, 54, P = 0.03 for males; F5, 75 = 11.45, P < 0.001 for females) (Table 1). By 103 days, mortality had reached 66–70 and 29–54% for control males and females and 85–100 and 100% for treated males and females, respectively. However, Fig. 3 indicates that significant mortality in the treated insects began toward 10 days after treatment, whereas mortality in controls was significant toward 80 days after fungal application. The longevity of males and females treated with M. anisopliae was significantly lower (F3, 54 = 19.13, P < 0.001 for males; F5, 75 = 28.85, P < 0.001 for females) than that of the untreated ones (Table 1). Mean mortality and longevity values for treated and non-treated individuals in virgin and paired females did not differ significantly (Table 1). The cumulative number of oothecae laid per surviving female over the study period is shown in Fig. 4. Two-way repeated measures ANOVA for this parameter showed significant effects of treatment (F5, 29 = 4.71, P = 0.0029), time of observation (F103, 515 = 92.61, P < 0.001), and interaction between both factors (F515, 5771=5.34, P = 0.0039) throughout the assessment period. First oviposition was recorded in the range of 6–10 days after treatment for most paired females and
Table 1 Lethal and sublethal reproductive effects of EAMa 01/121-Su strain on B. germanica adults at different pairing conditions after 103 days posttreatment Pairing combinationb
Number of pairs
Mortality %
Longevity (days)
Oothecal production
Oothecal viability
Males
Females
Males
N
Mean number per female
N
% Hatched oothecae per female
#nt · $nt #t · $t #t · $nt #nt · $t $nt $t
13 16 14 15 12 11
61.54 ± 14.04 a 100.00 ± 0.00 b 85.71 ± 9.71 ab 66.67 ± 12.60 a — —
53.85 ± 14.39 a 100.00 ± 0.00 b 28.57 ± 12.53 a 100.00 ± 0.00 b 50.00 ± 15.08 a 100.00 ± 0.00 b
95.92 ± 2.53 30.75 ± 6.46 50.79 ± 9.25 84.73 ± 7.19 — —
43 26 46 23 39 9
3.31 ± 0.21 1.63 ± 0.33 3.29 ± 0.19 1.53 ± 0.35 3.25 ± 0.35 0.82 ± 0.26
21 3 17 6 0 —
51.92 ± 10.64 a 7.69 ± 5.21 b 42.86 ± 13.72 a 27.10 ± 11.47 ab 0.00 ± 0.00 b —
Females a c b a
92.08 ± 4.39 36.81 ± 7.69 96.93 ± 3.76 35.53 ± 8.68 99.25 ± 1.44 26.09 ± 7.24
a b a b a b
a b a b a b
Data are means ± EE.a a Means within columns with the same letter are not significantly different (least significant difference P > 0.05). b nt, untreated; t, treated with 1.7 · 105 conidia per insect of M. anisoplia EAMa 01/121-Su strain.
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Fig. 3. Cumulative proportional survival of B. germanica newly emerged untreated adult males (solid line and ·) and females (dashed line and ·) and of newly emerged adults males (solid line and s) and females (dashed line and s) treated with 1.7 · 105 conidia per insect of M. anisoplia EAMa 01/121-Su strain. Data are means ± SE.
was delayed for 4–5 days in females kept apart from males (Fig. 4). One-way ANOVA showed that the mean number of oothecae laid by female did not vary significantly among the treatments after first oviposition period (F5, 75 = 1.63, P = 0.1629 for day 25 after treatment) while it was progressively and significantly reduced by fungal treatment from second oviposition period onwards, irrespective of whatever females were kept apart from males or paired and of whatever their partner were treated or not (F5, 75 = 6.97, P < 0.001 for day 50 after treatment; F5, 75 = 12.4, P < 0.001 for day 75 after treatment). The mean per capita number of oothecae and their viability for the overall experiment are shown in Table 1. At the crosses in which females were treated, they produced only 46.2 (paired with untreated males) and 49.2% (paired with treated males) of the total oothecae produced by the control (3.31/female); thus when both parents were treated with M. anisopliae EAMa 01/121-Su strain, only females showed an effect on
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oothecal production (Table 1). For both untreated and treated females, the mean per capita number of oothecae of paired and virgin individuals did not differ significantly (Table 1). The sublethal dose of EAMa 01/121-Su strain caused a significant decline in oothecal viability (F5, 65 = 5.24, P < 0.001). The mean hatched oothecae produced by treated females were only 14.8–52.2% of the total hatched oothecae produced by the untreated pairs (Table 1). Oothecal viability of treated females paired with untreated males (27.1%) and with treated ones (7.7%) did not differ significantly at the 5% level (Table 1), but it approached significance at the 10% level. Non-viable oothecae from all pairing combinations were half dropped prematurely and the other half discarded after the normal development period. It can be observed that none of the oothecae laid by unfertilized (virgin) females hatched (Table 1). When mean incubation time (mean carrying time until hatch) was examined, no significant differences were found between treatments (F3, 43 = 0.48, P = 0.69) (Table 2). In contrast, treatment with M. anisopliae EAMa Table 2 Hatch of oothecae laid by B. germanica females at different pairing conditions after 103 days post-treatment Pairing combinationb
#nt · $nt #t · $t #t · $nt #nt · $t
Oothecal hatch Mean carrying time until hatch
Nymphs hatched per oothecae
23.43 ± 1.45 27.67 ± 4.37 23.65 ± 1.60 25.67 ± 2.97
36.62 ± 1.95 28.33 ± 4.25 37.47 ± 2.63 23.83 ± 7.23
a a a a
a ab a b
Data are means ± EE.a a Means within columns with the same letter are not significantly different (least significant difference P > 0.05). b nt, untreated; t, treated with 1.7 · 105 conidia per insect of M. anisoplia EAMa 01/121-Su strain.
Fig. 4. Mean cumulative number of oothecae laid per surviving female over the study period.
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01/121-Su strain caused the mean number of nymphs hatched per oothecae to be reduced significantly (between 22.6 and 34.9%) in the two crosses in which females were treated (F3, 43 = 2.89, P = 0.047) (Table 2). By 20 days after hatching, most of the nymphs were alive; we only observed two dead nymphs, none of which presented fungal growth.
4. Discussion The present work indicates that M. anisopliae EAMa01/121-Su strain can provide a good control of B. germanica. As expected for any fungal strain, the virulence of strain EAMa01/121-Su to cause mortality in B. germanica was directly related to spore concentration (Pachamuthu et al., 1999). We obtained a LD50 of 5 · 104 spores per cockroach, which it is tenfold lower than the one obtained by Pachamuthu et al. (1999) for the M. anisopliae ESC-1 isolate on the German cockroach (4.18 · 105 spores per cockroach). These authors found that ESC-1 strain caused appreciable mortality at 21 days following treatment while we have obtained LT50 values of 14.8 and 5.3 days for 1.7 · 106 and 1.7 · 107 spores per cockroach, respectively. Therefore, our strain seems to be more pathogenic and to kill faster than the commercialized ESC-1 strain. From our study, it is clear that horizontal transmission of M. anisopliae may occur between B. germanica infected and healthy adults. Kaakeh et al. (1996) obtained a LT50 of 20 days and a cumulative mortality of 67% after mixing healthy nymphs with dead-infected cockroaches. In our experiment, the mortality rate (87.5%) was higher and the LT50 value, 12.2 days, was clearly lower. In our experiment cockroaches were exposed to infected cadavers during the whole experiment while those authors used exposure times of 6 and 48 h (Kaakeh et al., 1996). However, they also found that the rate of mortality increased significantly by increasing the ratio of dead-infected cockroaches to unexposed ones (i.e., 1:1 ratio > 1:10 ratio) (Kaakeh et al., 1996); thus our results have been obtained in the worst case scenario (1:10 ratio). Interestingly, these authors observed that mycosed cadavers were not cannibalized, and they suggested an avoidance behavior of unexposed nymphs. On the opposite, we have observed mycosed cockroaches to be cannibalized by unexposed ones. As cannibalism and necrophagy are within the main ways which facilitate secondary kill of German cockroaches (Durier and Rivault, 2000), we would expect the humid conditions prevailing in the ecological niches of cockroaches to improve the development of fungal infection, the further mycosis of the cadavers and the rapid spread of the infection in the insect population. Oothecal production, oothecal hatchability, and nymphal production of female cockroaches in this study de-
clined upon exposure to a sublethal dose of M. anisopliae EAMa 01/121-Su strain. The reduction of the per capita mean number of oothecae in fungally challenged females began significant by the second oviposition onwards. Arthurs and Thomas (2000) observed that females of Locustana pardalina (Walker) treated with M. anisopliae var. acridum exhibited a reduced pre-oviposition period resulting in them laying more eggs than untreated females within the first weeks following fledging. As a consequence of that, no significant reductions in fecundity were recorded by the end of the assessment period (Arthurs and Thomas, 2000). In our study, no effect of fungal exposure on pre-oviposition period has been observed and by the end of the experiment the treated females showed a 46–49% reduction in the mean oothecal production. Interestingly, females kept apart from males showed a delay of 4–5 days in the first oviposition, which is commonly detected in females from many insect species when are avoided or delayed from mating (Huang and Subramanyam, 2003). None of the oothecae laid by unfertilized females hatched in agreement with Roth and Stay (1962), who stated that German cockroaches can not reproduce parthenogenetically being oothecae either dropped prematurely or discarded after the normal developmental period. Our results indicate that only treated females showed an effect on oothecal production, oothecal viability, and nymphal production. Lee et al. (1998) observed that only treated females of B. germanica surviving insecticide exposure showed a reduction of oothecal production while oothecal hatch and nymphal production were governed by both maternal and paternal factors. Our data suggest such male effect in oothecal viability, but the reduction from 27.1% when treated females were paired with untreated males to 7.7% when they were presented to treated males, approached significance only at the 10% level. Information on sublethal reproductive effects of entomopathogenic fungi on insects is very scarce and particularly on orthopteroid insects. Blanford and Thomas (2001) observed a significant reduction of the total number of egg pods laid by females of desert locust treated with M. anisopliae var acridum 3 days post-fledging, but they observed no effect of treatment neither on per capita number of pods nor on number of eggs per pod. From this work, it is not possible to conclude by which sex is governed this reproductive effect. Reproduction of non-orthopteroids insects has been also reported to be impaired by fungal treatments. Interestingly, the main sublethal reproductive effect is the reduction of fecundity while egg fertility is not affected or moderately affected. Fargues et al. (1991) observed a 20–50% reduction in fecundity of adult Colorado potato beetles females surviving Beauveria bassiana infection at 22 C, whereas at 25 C the fecundity of survivors was not affected by the fungal infection. At both temperatures, egg fertility of surviving females
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was similar to that of control insect (Fargues et al., 1991). The fecundity of Western Corn Rootworm, Diabrotica virginifera virginifera, has also been reported to be reduced by 70% by B. bassiana infection (Mulock and Chandler, 2001). Fecundity of Mediterranean fruit fly Ceratitis capitata Wiedemann adult females exposed to M. anisopliae and Paecilomyces fumosoroseus (Wize) Brown & Smith was reduced 65 and 40–50%, respectively, but egg fertility was only moderately affected by fungal treatments (Castillo et al., 2000). In other insect hosts/entomopathogens systems, i.e., lepidopterans and baculoviruses, the male reproductive capacity is also impaired (Matthews et al., 2002; Santiago-Alvarez and Vargas-Osuna, 1988). Sublethal reproductive effects of Bacillus thuringiensis (Berliner) have also been reported on some insect pests. Adult females of Colorado potato beetle surviving exposure to delta-endotoxin of B. thuringiensis tended to produce around 45% fewer eggs (Costa et al., 2000). Similarly, adult females of spruce budworm Choristoneura fumiferana Clemens coming from larvae surviving delta-endotoxin treatments laid a lower number of eggs even if egg fertility was not affected (Pedersen et al., 1997). In contrast, B. thuringiensis b-exotoxin seems to cause no negative effects on fecundity and fertility of survivors (Toledo et al., 1999). In untreated German cockroach females, we have obtained values of mean nymphs hatched per oothecae and of mean carrying time that can be considered normal for German cockroach laboratory populations (Barson and Renn, 1983; Lee et al., 1998). However, in a normal culture of B. germanica an oothecal viability over 75% can be expected, which is higher than the one obtained in our work for untreated females and than that over 65% observed by Lee et al. (1998). This is probably a consequence of the experimental design of our work and this latter work in which females from a high density laboratory culture were individualized in boxes either paired or kept apart from a male. Moreover, relative humidity within the experimental boxes raises by 20% over that of the laboratory culture, which could also reduce the oothecal viability as reported by Barson and Renn (1983), who found a 25% reduction in this parameter after increasing the relative humidity from 45 to 70%. The reduction in number of oothecae laid by survivors of M. anisopliae infection observed in our study could be due to the reduction in adult longevity upon fungal treatment that might cooperate on the subsequent fecundity reduction. In fact, in Orthopteroid insects, the number of egg pods or oothecae laid per female is directly related to its longevity (Albrecht, 1967). In the case of American cockroach P. americana, the 50% reduction in number of oothecae per female coming from nymphs treated with azadirachtin was associated to their decreased ingestion (Richter et al., 1997). In. our work, we have treated adults and we have
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not observed a reduction of food ingestion. The reasons for the 36–85% reduction in oothecal viability and for the 22.6–34.9% reduction in nymphal production in fungally challenged females are more difficult to find. The insect host exhibits various types of cellular and humoral immune reactions against the fungus (Vey and Go¨tz, 1986), but the pathogen may overcome them through a combination of events including mechanical damage by hyphal growth and production of toxins (Inglis et al., 2001). At a sublethal level, any of these processes together with the nutrient depletion caused by the fungal growth could directly or indirectly impair the host reproduction. A further consequence of these changes may be a prolongation of the mean oothecal incubation time; we did not found significant differences in this parameter between treated and untreated females but interestingly, it approached significance (P < 0.10). Our results on pathogenicity, virulence and horizontal transmission of the fungal conidia of M. anisopliae EAMa 01/121-Su strain aim its use in biologically based mycoinsecticides for German cockroach control even without its further integration with sublethal doses of conventional insecticides in an integrated pest management approach (Kaakeh et al., 1996; Pachamuthu et al., 1999; Zurek et al., 2002). This is also supported by the reproductive effects of this strain on B. germanica, decrease in oothecal production, oothecal viability and hatchability, which can be seen as highly effective to decrease the pest status of adult B. germanica in the long term and even its suppression. The research discussed here has been conducted under controlled laboratory conditions; thus there is a need for additional research at field conditions that will hopefully aid to assess the real contribution of this fungal strain to German cockroach management programs.
Acknowledgments The authors thank Professor B. Federici from the University of Riverside (California) for critically reviewing the manuscript and the English phraseology and Mrs. Ma Victoria Paredes for her technical support.
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Arthurs, S., Thomas, M.B., 2000. Effects of a mycoinsecticide on feeding and fecundity of the Brown locust Locustana pardalina. Biocontrol Sci. Technol. 10, 321–329. Barson, G., Renn, N., 1983. Hatching from oothecae of the German cockroach (Blatella germanica) under laboratory culture conditions and after premature removal. Entomol. Exp. Appl. 34, 179– 185. Blanford, S., Thomas, M., 2001. Adult survival, maturation, and reproduction of the desert locust Schistocerca gregaria infected with the fungus Metarhizium anisopliae var acridum. J. Invertebr. Pathol. 78, 1–8. Castillo, M.A., Moya, P., Herna´ndez, E., Primo-Yu´fera, E., 2000. Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to entomopathogenic fungi and their extracts. Biol. Control 19, 274–282. Cochran, D.G., 2003. Blattodea. In: Resh, V.H., Carde´, R.T. (Eds.), Encyclopedia of Insects. Academic Press, New York, pp. 123–126. Costa, S.D., Barbercheck, M.E., Kennedy, G.G., 2000. Sublethal acute and chronic exposure of Colorado potato beetle (Coleoptera: Chrysomelidae) to the delta-endotoxin of Bacillus thuringiensis. J. Econ. Entomol. 93, 680–689. Durier, V., Rivault, C., 2000. Secondary transmission of toxic baits in German cockroach (Dictyoptera: Blatellidae). J. Econ. Entomol. 93, 434–440. Fargues, J., Delmas, J.C., Auge, J., Lebrun, R.A., 1991. Fecundity and egg fertility in the adult Colorado beetle Leptinotarsa decemlineata surviving larval infection by the fungus Beauveria bassiana. Entomol. Exp. Appl. 61, 45–51. Go´mez, K.A., Go´mez, A.A., 1984. Statistical Procedures for Agricultural Research. Wiley, New York. Harmon, J.D., Ross, M., 1987. Effects of propoxur on females of the German cockroach, Blatella germanica and their oothecae. Entomol. Exp. Appl. 44, 269–275. Huang, F., Subramanyam, B., 2003. Effects of delayed mating on reproductive performance of Plodia interpunctella (Hu¨bner) (Lepidoptera: Pyralidae). J. Stored Prod. Res. 39, 53–63. Inglis, G.D., Goettel, M.S., Butt, T.M., Strasser, H., 2001. Use of Hyphomycetous fungi for managing insects pest. In: Butt, T.M., Jackson, C.W., Magan, N. (Eds.), Fungi as Biocontrol Agents Progress, Problems and Potential. CABI Publishing, pp. 23–70. Kaakeh, W., Reid, B.L., Bohnert, T.J., Bennett, G., 1996. Horizontal transmission of the entomopathogenic fungus Metarhizium anisopliae (Imperfect Fungi: Hyphomycetes) and Hydramethylnon among German cockroaches (Dictyoptera: Blatellidae). J. Entomol. Sci. 31, 378–390. Kaakeh, W., Reid, B.L., Bohnert, T.J., Bennett, G., 1997. Toxicity of imidacloprid in the German cockroach (Dyctioptera: Blatellidae), and the synergism between imidacloprid and Metarhizium anisopliae (Imperfect Fungi: Hyphomycetes). J. Econ. Entomol. 90, 473– 482. Lee, Y.C., Yap, H.H., Chong, N.L., 1998. Sublethal effects of deltamethrin and propoxur on longevity and reproduction of
German cockroaches, Blatella germanica. Entomol. Exp. Appl. 89, 137–145. Matthews, J.H., Smith, I., Edwards, P., 2002. Lethal and sublethal effects of a granulovirus on the tomato moth Lacanobia oleracea. J. Invertebr. Pathol. 80, 73–80. Mulock, B.S., Chandler, L.D., 2001. Effect of Beauveria bassiana on the fecundity of western corn rootworm, Diabrotica virginifera virginifera (Coleoptera: Chrysomelidae). Biol. Control 22, 16–21. Murali Mohan, C.H., Aruna Lakshmi, K., Uma Devi, K., 1999. Laboratory evaluation of the pathogenicity of three isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin on the American cockroach (Periplaneta americana). Biocontrol Sci. Technol. 9, 29–33. Pachamuthu, P., Kamble, S.T., 2000. In vivo study of combined toxicity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) strain ESC-1 with sublethal doses of chlorpyrifos, propetamphos, and cyfluthrin against German cockroach (Dictyoptera: Blatellidae). J. Econ. Entomol. 93, 60–70. Pachamuthu, P., Kamble, S.T., Yuen, G.Y., 1999. Virulence of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) strain ESC-1 to the German cockroach (Dictyoptera: Blattellidae) and its compatibility with insecticides. J. Econ. Entomol. 92, 340–346. Pedersen, A., Dedes, J., Gauthier, D., vanFrankenhuyzen, K., 1997. Sublethal effects of Bacillus thuringiensis on the spruce budworm, Choristoneura fumiferana. Entomol. Exp. Appl. 83, 253–262. Richter, K., Bohm, G.A., Kleeberg, H., 1997. Effect of NeemAzal, a natural azadirachtin-containing preparation, on Periplaneta americana (L.) (Orthopt., Blattidae). J. Appl. Entomol. 121, 59–64. Roth, L.M., Stay, B., 1962. Oocyte development in Blatella germanica and Blatella vaga (Blattaria). Ann. Entomol. Soc. Am. 55, 633– 642. Santiago-Alvarez, C., Vargas-Osuna, E., 1988. Reduction of reproductive capacity of Spodoptera littoralis males by a Nuclear Polyhedrosis Virus (NPV). J. Invertebr. Pathol. 52, 142–146. SPSS Inc., 1997. SPSS 8.0 for Windows. SPSS Inc. Headquarters, 233 S. Wacker Drive, 11th floor. Chicago, Illinois 60606. Throne, J.E., Weaver, D.K., Chew, V., Baker, J.E., 1995. Probit analysis of correlated data: multiple observations over time at one concentration. J. Econ. Entomol. 88, 1510–1512. Toledo, J., Liedo, P., Williams, T., Ibarra, J., 1999. Toxicity of Bacillus thuringiensis beta-exotoxin to three species of fruit flies (Diptera: Tephritidae). J. Econ. Entomol. 92, 1052–1056. Vey, A., Go¨tz, P., 1986. Antifungal cellular defense mechanisms in insects. In: Gupta, A.P. (Ed.), Hemocytic and Humoral Immunity in Arthropods. Wiley, New York, pp. 89–115. Zimmermann, G., 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential as a Biocontrol Agent. Pestic. Sci. 37, 375–379. Zurek, L., Wes Watson, D., Schal, C., 2002. Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and boric acid against the German Cockroach (Dictyoptera: Blatellidae). Biol. Control 23, 296–302.
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 87 (2004) 51–58 www.elsevier.com/locate/yjipa
Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae) ´ lvarez* E. Quesada-Moraga, R. Santos-Quiro´s, P. Valverde-Garcı´a, C. Santiago-A Departamento de Ciencias y Recursos Agrı´colas y Forestales, ETSIAM, Universidad de Co´rdoba, Apartado 3048, 14080 Co´rdoba, Spain Received 25 March 2004; accepted 22 July 2004 Available online 12 September 2004
Abstract Virulence of Metarhizium anisopliae (Metschnikoff) Sorokin strain EAMa 01/121-Su against the German Cockroach, Blatella germanica (L.), was determined using four concentrations ranging from 4.2 · 106 to 4.2 · 109 spores per milliliter. The LD50 value was 1.4 · 107 spores per milliliter (56,000 spores per cockroach) and LT50 values were 14.8 days and 5.3 days for 4.2 · 108 and 4.2 · 109 spores per milliliter, respectively. An experiment was conducted to evaluate whether a fungal transmission could exist among infected and healthy cockroaches. Percentage mortality at a ratio of 1:10 of infected to unexposed cockroaches was 87.5% and LT50 was 12.2 days, which indicated the potential of this strain to be horizontally transmitted and to rapidly spread the infection in the insect population. The effect of a sublethal dose (ca. LD60) of M. anisopliae EAMa 01/121-Su strain, applied topically on German cockroaches, was studied by reciprocal crossing. Othecal production, oothecal hatchability, and nymphal production declined upon exposure to M. anisopliae EAMa 01/121-Su strain. The mean number of oothecae laid by female was progressively and significantly reduced by fungal treatment from second oviposition period onwards. Oothecal hatch of fungally challenged females was reduced by 46–49%, oothecal viability by 48–85%, and nymphal production by 22–35%. Only treated females showed an effect on oothecal production, oothecal hatch, and nymphal production, although oothecal hatch was also governed by treated males at a higher significance level. Our results on virulence and horizontal transmission of fungal conidia of M. anisopliae EAMa 01/121-Su strain and its sublethal reproductive effects on German cockroach females are discussed in terms of its potential to decrease the pest status of B. germanica in the short and long terms. 2004 Elsevier Inc. All rights reserved. Keywords: Blatella germanica; German cockroach; Blattodea; Metarhizium anisopliae; Entomopathogenic fungi; Virulence; Horizontal transmission; Reproductive effects; Sublethal effects; Biological control
1. Introduction The German cockroach, Blatella germanica, is a worldwide distributed species surviving well in association with any human habitation that provides warmth, moisture, and food in places such as apartments, homes and food-handling facilities (Cochran, 2003). German *
Corresponding author. Fax: +34 957218440. ´ lvarez). E-mail address: [email protected] (C. Santiago-A
0022-2011/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.07.002
cockroaches are primarily controlled through the use of synthetic organic insecticides (organophosphates, pyrethroids, and carbamates), but many factors including insecticide resistance, concerns about human and environmental safety, and increased developmental cost of new insecticides have intensified the search for new control methods. One potential alternative is the use of biologically based insecticides, such as those containing entomopathogenic fungi (Kaakeh et al., 1996, 1997; Murali Mohan et al., 1999).
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The entomopathogenic fungus, Metarhizium anisopliae, is a common fungal pathogen showing a wide insect host range including B. germanica (Zimmermann, 1993). Despite considerable research on the use of Metarhizium sp. for crop insect pest control, there are few reports on the application of this fungus to control B. germanica. From them, it can be concluded that although M. anisopliae has the potential to be an effective control agent, its slow action has limited effective use of this fungus under field conditions, as reported for the Bio-Path Cockroach Control Chamber (Pachamuthu et al., 1999). Therefore, efforts have been made to enhance the biological activity of M. anisopliae by integrating it with sublethal doses of chemical insecticides (Pachamuthu and Kamble, 2000; Zurek et al., 2002). To date, a majority of the work has focused on mortality of the target pest caused directly by the fungus. This was due to the urgent need for more active fungal strains. In addition, mortality due to initial fungus application may be followed by horizontal transmission within the target population (Kaakeh et al., 1996), a phenomenon that may be attributable to the gregarious behavior of cockroaches. Horizontal transmission may contribute greatly to the spread of infection and thus should be studied as part of a commercial development effort. However, to make an accurate estimation of the influence that a fungal pathogen has on a cockroach population is necessary to consider all effects, other than mortality on target individuals (Arthurs and Thomas, 2000; Blanford and Thomas, 2001; Fargues et al., 1991; Mulock and Chandler, 2001). It has been demonstrated that sublethal effects of fungal infection can have important implications for the population dynamics of the host, which ultimately contribute to the status of the target insect as pest (Arthurs and Thomas, 2000; Blanford and Thomas, 2001). There is some documentation on the effects of different chemical insecticides on oothecae carried by German cockroach at application time (Abd-Elghafar et al., 1991; Appel and Abd-Elghafar, 1990; Harmon and Ross, 1987), and on the sublethal effects of azadirachtin on reproduction of Periplaneta americana (L.) (Richter et al., 1997), and of deltamethrin and propoxur on longevity and reproduction of B. germanica (Lee et al., 1998), but to our knowledge, no information is available on the sublethal effects of entomopathogenic fungi on cockroach reproduction. From our collection of strains of M. anisopliae, we have selected strain EAMa 01/121-Su for its pathogenicity to B. germanica. This research was undertaken to evaluate the virulence of M. anisopliae strain EAMa01/ 121-Su against B. germanica, to study whether it may be transmitted from infected to healthy cockroaches and to assess the sublethal effects of fungal infection on German cockroach reproduction.
2. Materials and methods 2.1. Insects The insects used in this study came from a stock colony established in our laboratory. The colony was reared at 27 ± 2 C, 60 ± 10% RH, and photoperiod of 12:12 h light:dark cycle and fed with Purina dog chow and water provided in glass tubes with cotton stoppers. Each plastic cage (30 · 20 · 20 cm) was provided with a paper egg carton shelter. 2.2. Conidial production The M. anisopliae strain used was originally isolated from a cotton field soil at Marchena, Sevilla (Spain) in 2001. This strain is deposited in the fungal collection of CRAF Department of the Universidad de Co´rdoba with the access number EAMa 01/121-Su. Conidia were produced on Malt Agar (Panreac No. 413781) following the instructions of the fabricant. After autoclaving at 121 C for 20 min, approximately 15 ml of media was poured into sterile petri dishes (10 · 15 cm). Ten microliter of the spore suspension prepared in 0.05% Triton X-100 was placed in the center of each Petri dish containing the media and sealed with parafilm. Spore cultures in sealed Petri dishes were incubated at constant temperature (27 ± 2 C) in darkness for 21 days. Conidia were harvested in sterile water containing 0.05% Triton X-100. Conidia from each plate were scraped with a sterile spatula, and the spore suspension was filtered through an eight-layered cheese cloth, centrifuged (4000g for 15 min at 4 C), and resuspended in sterile water containing 0.05% Triton X-100. The spore concentration was determined using a hemocytometer. Germination of conidia was over 95%. 2.3. Bioassay to determine the biological activity of M. anisopliae strain EAMa 01/121-Su Four spore concentrations (4.2 · 106, 4.2 · 107, 4.2 · 108, and 4.2 · 109 spores per milliliter) were prepared from the stock suspension, based on the results of a preliminary experiment. Then, by means of a micropipette, 4 ll from each spore dilution was applied topically on the first ventral abdominal segment of each cool immobilized adult German cockroach. Controls were treated with 4 ll of sterile distiller water plus 0.05% Triton X-100. The treated cockroaches were placed in plastic containers (12 · 12 · 6 cm) conditioned as described above. Cockroaches were fed with Purina dog chow and water provided in glass tubes with cotton stoppers. The bioassay was conducted at 27 ± 2 C and 75 ± 10% RH. Mortality was monitored daily for 31 days, and dead cockroaches were removed daily. Each treatment was replicated four times with 10 adult cock-
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roaches per treatment, and the entire experiment was conducted twice. Cadavers were immediately removed and placed into sterile Petri dishes. Afterwards, they were surface sterilized with 70% ethanol and placed on sterile wet filter paper in sterile Petri dishes that were then sealed with parafilm and kept at room temperature. The emergence of hyphae was monitored for 10 days. The mortality data were pooled and LD50 calculated by probit analysis using SPSS (Statistical Package for Social Sciences in personal computers) 8.0 for Windows (SPSS, 1997). The LT50 values were determined using the probit analysis method for correlation data (Throne et al., 1995). 2.4. Transmission of M. anisopliae from sporulating adult cadavers to healthy adults Single EAMa 01/121-Su-killed adult cockroach with profuse mycosis was placed at the center of plastic containers (12 · 12 · 6 cm) with 10 newly emerged healthy adult cockroaches (one container per replication) and incubated at 27 ± 2 C and 75 ± 10% RH. Mortality was monitored daily for 31 days, and dead cockroaches were removed daily. The treatment was replicated four times and the entire experiment was conducted twice. Kaplan–Meier survival analysis was performed using SPSS (Statistical Package for Social Sciences in personal computers) 8.0 for Windows (SPSS, 1997). The LT50 values were determined using the probit analysis method for correlation data (Throne et al., 1995). 2.5. Bioassay to determine the sublethal effects of M. anisopliae strain EAMa 01/121-Su on B. germanica Based in the biological activity bioassay, we selected a spore concentration of 4.2 · 107 spores per milliliter (ca. LD60) to study the sublethal effects of M. anisopliae EAMa 01/121-Su strain on German cockroach reproduction. Newly emerged adult females and males were treated topically as above. As controls, newly emerged adult females and males were treated with 4 ll of sterile distilled water plus 0.05% Triton X-100. We established six mating combinations: Untreated females · untreated males, treated females · untreated males, untreated females · treated males, treated females · treated males, untreated virgin females, and treated virgin females. Couples were placed in plastic containers (12 · 12 · 6 cm), one couple per replication except for virgin females, conditioned as described above and incubated at 27 ± 2 C and 75 ± 10% RH. Each reproductive combination was replicated 14 times with one couple per treatment. Couples were checked daily for death and offspring for 100 days, and dead cockroaches were immediately removed and processed as above to evaluate fungal growth. Insects in the orthopteroid orders Blattodea, Mantodea, and Orthoptera secrete an egg case or pod
53
surrounding the eggs, to give additional protection from desiccation and predation which it is commonly named as oothecae. The percentage of dropped oothecae from both live and dead females was determined. Comparisons of cumulative oothecae per surviving female over time were analyzed by a two-way repeated measures ANOVA for multi-observation data (Go´mez and Go´mez, 1984). Multiple comparisons between treatments for each sample date were performed by one-way ANOVA. Both analyses were made using SPSS (Statistical Package for Social Sciences in personal computers) 8.0 for Windows (SPSS, 1997). Surviving females with oothecae, dead females with oothecae, and dropped oothecae from live or dead females were held individually in 5 cm diameter plastic Petri dishes with a water wick and a piece of Purina dog chow. Cockroaches and oothecae were held at 27 ± 2 C and 75 ± 10% and photoperiod of 12:12 h light:dark cycle. We recorded the mean percentage of oothecal hatch and the mean carrying time until hatch. Nymph mortality was recorded for 20 days after hatching. Cadavers were immediately removed and processed as described before to evaluate fungal growth.
3. Results Based on initial mortality data, four tenfold concentrations from 4.2 · 106 to 4.2 · 109 spores per milliliter were selected for the biological activity bioassay. The average cockroach mortality ranged from 42.3 to 93.3% in the treated insects, whereas 13.6% mortality was observed in the untreated cockroaches. The dose– mortality response regression equation from assay of the four concentrations of EAMa 01/121-SV-strain against B. germanica newly emerged adults is shown in Fig. 1. The LD50 value was 1.4 · 107 spores per milliliter (56,000 spores per cockroach) [(95% CI: 6.0 · 106 spores
Fig. 1. Dose–mortality response of B. germanica newly emerged adults to four tenfold doses of M. anisopliae EAMa 01/121-Su strain (4.2 · 106–4.2 · 109 spores/ml). For each point N = 80 insects. Each insect was treated topically with 4 ll from each spore dilution.
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per milliliter (24,000 spores per cockroach)–2.7 · 107 spores per milliliter (108,000 spores per cockroach)]. The LT50 values were 14.8 days for 4.2 · 108 spores per milliliter [(95% CI: 10.8–20.6 days) (slope ± SE = 2.50 ± 0.38, v2 = 18.41)], and 5.3 days for 4.2 · 109 spores per milliliter [(95% CI: 3.3–7.3 days) (slope ± SE = 2.28 ± 0.40, v2 = 12.05)]. M. anisopliae grew from all cadavers in the 4.2 · 108 and 4.2 · 109 fungal treatments and from 20 to 60% of them in the 4.2 · 106 and 4.2 · 107 treatments. The fungus was horizontally transmitted from infected to healthy adult cockroaches. Mortality rates observed for unexposed and exposed cadavers were 10 and 87.5%, respectively (Fig. 2). Average survival time of healthy adults mixed with dead-infected cockroaches at a 1:10 ratio was 16.1 ± 0.96 days (means ± SE), which it is significantly lower than the one of 29.03 ± 0.96 days showed by unexposed cockroaches (log-rank statistic = 46.9, P < 0.001). The LT50 value resulting from
Fig. 2. Cumulative proportional survival of B. germanica adults unexposed (solid line and ·) and exposed to infected dead cockroaches at a ratio of 1:10 infected to healthy adults (dashed line and s). Data are means ± SE.
mixing healthy and dead M. anisopliae-infected adults was 12.2 days [(9.0–16.7 days) (slope ± SE = 2.51 ± 0.34, v2 = 11.78)]. Newly emerged adult cockroach males and females were treated with 1.7 · 105 spores per insect (nearly LD60) of M. anisopliae EAMa 01/121-Su strain to study its effects on cockroach reproduction. Average survival time (AST ± SE) values of treated adult males, 40.10 ± 5.6 days, and females, 36.19 ± 5.7 days, were significantly lower (log-rank statistic, P < 0.05) than those of untreated males, 89.93 ± 4.2 days, and females, 94.59 ± 2.8 days, respectively. We observed no differences in AST neither between treated nor untreated males and females. For all combinations, mortality of treated males and females was significantly higher than the one of untreated adults (F3, 54, P = 0.03 for males; F5, 75 = 11.45, P < 0.001 for females) (Table 1). By 103 days, mortality had reached 66–70 and 29–54% for control males and females and 85–100 and 100% for treated males and females, respectively. However, Fig. 3 indicates that significant mortality in the treated insects began toward 10 days after treatment, whereas mortality in controls was significant toward 80 days after fungal application. The longevity of males and females treated with M. anisopliae was significantly lower (F3, 54 = 19.13, P < 0.001 for males; F5, 75 = 28.85, P < 0.001 for females) than that of the untreated ones (Table 1). Mean mortality and longevity values for treated and non-treated individuals in virgin and paired females did not differ significantly (Table 1). The cumulative number of oothecae laid per surviving female over the study period is shown in Fig. 4. Two-way repeated measures ANOVA for this parameter showed significant effects of treatment (F5, 29 = 4.71, P = 0.0029), time of observation (F103, 515 = 92.61, P < 0.001), and interaction between both factors (F515, 5771=5.34, P = 0.0039) throughout the assessment period. First oviposition was recorded in the range of 6–10 days after treatment for most paired females and
Table 1 Lethal and sublethal reproductive effects of EAMa 01/121-Su strain on B. germanica adults at different pairing conditions after 103 days posttreatment Pairing combinationb
Number of pairs
Mortality %
Longevity (days)
Oothecal production
Oothecal viability
Males
Females
Males
N
Mean number per female
N
% Hatched oothecae per female
#nt · $nt #t · $t #t · $nt #nt · $t $nt $t
13 16 14 15 12 11
61.54 ± 14.04 a 100.00 ± 0.00 b 85.71 ± 9.71 ab 66.67 ± 12.60 a — —
53.85 ± 14.39 a 100.00 ± 0.00 b 28.57 ± 12.53 a 100.00 ± 0.00 b 50.00 ± 15.08 a 100.00 ± 0.00 b
95.92 ± 2.53 30.75 ± 6.46 50.79 ± 9.25 84.73 ± 7.19 — —
43 26 46 23 39 9
3.31 ± 0.21 1.63 ± 0.33 3.29 ± 0.19 1.53 ± 0.35 3.25 ± 0.35 0.82 ± 0.26
21 3 17 6 0 —
51.92 ± 10.64 a 7.69 ± 5.21 b 42.86 ± 13.72 a 27.10 ± 11.47 ab 0.00 ± 0.00 b —
Females a c b a
92.08 ± 4.39 36.81 ± 7.69 96.93 ± 3.76 35.53 ± 8.68 99.25 ± 1.44 26.09 ± 7.24
a b a b a b
a b a b a b
Data are means ± EE.a a Means within columns with the same letter are not significantly different (least significant difference P > 0.05). b nt, untreated; t, treated with 1.7 · 105 conidia per insect of M. anisoplia EAMa 01/121-Su strain.
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Fig. 3. Cumulative proportional survival of B. germanica newly emerged untreated adult males (solid line and ·) and females (dashed line and ·) and of newly emerged adults males (solid line and s) and females (dashed line and s) treated with 1.7 · 105 conidia per insect of M. anisoplia EAMa 01/121-Su strain. Data are means ± SE.
was delayed for 4–5 days in females kept apart from males (Fig. 4). One-way ANOVA showed that the mean number of oothecae laid by female did not vary significantly among the treatments after first oviposition period (F5, 75 = 1.63, P = 0.1629 for day 25 after treatment) while it was progressively and significantly reduced by fungal treatment from second oviposition period onwards, irrespective of whatever females were kept apart from males or paired and of whatever their partner were treated or not (F5, 75 = 6.97, P < 0.001 for day 50 after treatment; F5, 75 = 12.4, P < 0.001 for day 75 after treatment). The mean per capita number of oothecae and their viability for the overall experiment are shown in Table 1. At the crosses in which females were treated, they produced only 46.2 (paired with untreated males) and 49.2% (paired with treated males) of the total oothecae produced by the control (3.31/female); thus when both parents were treated with M. anisopliae EAMa 01/121-Su strain, only females showed an effect on
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oothecal production (Table 1). For both untreated and treated females, the mean per capita number of oothecae of paired and virgin individuals did not differ significantly (Table 1). The sublethal dose of EAMa 01/121-Su strain caused a significant decline in oothecal viability (F5, 65 = 5.24, P < 0.001). The mean hatched oothecae produced by treated females were only 14.8–52.2% of the total hatched oothecae produced by the untreated pairs (Table 1). Oothecal viability of treated females paired with untreated males (27.1%) and with treated ones (7.7%) did not differ significantly at the 5% level (Table 1), but it approached significance at the 10% level. Non-viable oothecae from all pairing combinations were half dropped prematurely and the other half discarded after the normal development period. It can be observed that none of the oothecae laid by unfertilized (virgin) females hatched (Table 1). When mean incubation time (mean carrying time until hatch) was examined, no significant differences were found between treatments (F3, 43 = 0.48, P = 0.69) (Table 2). In contrast, treatment with M. anisopliae EAMa Table 2 Hatch of oothecae laid by B. germanica females at different pairing conditions after 103 days post-treatment Pairing combinationb
#nt · $nt #t · $t #t · $nt #nt · $t
Oothecal hatch Mean carrying time until hatch
Nymphs hatched per oothecae
23.43 ± 1.45 27.67 ± 4.37 23.65 ± 1.60 25.67 ± 2.97
36.62 ± 1.95 28.33 ± 4.25 37.47 ± 2.63 23.83 ± 7.23
a a a a
a ab a b
Data are means ± EE.a a Means within columns with the same letter are not significantly different (least significant difference P > 0.05). b nt, untreated; t, treated with 1.7 · 105 conidia per insect of M. anisoplia EAMa 01/121-Su strain.
Fig. 4. Mean cumulative number of oothecae laid per surviving female over the study period.
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01/121-Su strain caused the mean number of nymphs hatched per oothecae to be reduced significantly (between 22.6 and 34.9%) in the two crosses in which females were treated (F3, 43 = 2.89, P = 0.047) (Table 2). By 20 days after hatching, most of the nymphs were alive; we only observed two dead nymphs, none of which presented fungal growth.
4. Discussion The present work indicates that M. anisopliae EAMa01/121-Su strain can provide a good control of B. germanica. As expected for any fungal strain, the virulence of strain EAMa01/121-Su to cause mortality in B. germanica was directly related to spore concentration (Pachamuthu et al., 1999). We obtained a LD50 of 5 · 104 spores per cockroach, which it is tenfold lower than the one obtained by Pachamuthu et al. (1999) for the M. anisopliae ESC-1 isolate on the German cockroach (4.18 · 105 spores per cockroach). These authors found that ESC-1 strain caused appreciable mortality at 21 days following treatment while we have obtained LT50 values of 14.8 and 5.3 days for 1.7 · 106 and 1.7 · 107 spores per cockroach, respectively. Therefore, our strain seems to be more pathogenic and to kill faster than the commercialized ESC-1 strain. From our study, it is clear that horizontal transmission of M. anisopliae may occur between B. germanica infected and healthy adults. Kaakeh et al. (1996) obtained a LT50 of 20 days and a cumulative mortality of 67% after mixing healthy nymphs with dead-infected cockroaches. In our experiment, the mortality rate (87.5%) was higher and the LT50 value, 12.2 days, was clearly lower. In our experiment cockroaches were exposed to infected cadavers during the whole experiment while those authors used exposure times of 6 and 48 h (Kaakeh et al., 1996). However, they also found that the rate of mortality increased significantly by increasing the ratio of dead-infected cockroaches to unexposed ones (i.e., 1:1 ratio > 1:10 ratio) (Kaakeh et al., 1996); thus our results have been obtained in the worst case scenario (1:10 ratio). Interestingly, these authors observed that mycosed cadavers were not cannibalized, and they suggested an avoidance behavior of unexposed nymphs. On the opposite, we have observed mycosed cockroaches to be cannibalized by unexposed ones. As cannibalism and necrophagy are within the main ways which facilitate secondary kill of German cockroaches (Durier and Rivault, 2000), we would expect the humid conditions prevailing in the ecological niches of cockroaches to improve the development of fungal infection, the further mycosis of the cadavers and the rapid spread of the infection in the insect population. Oothecal production, oothecal hatchability, and nymphal production of female cockroaches in this study de-
clined upon exposure to a sublethal dose of M. anisopliae EAMa 01/121-Su strain. The reduction of the per capita mean number of oothecae in fungally challenged females began significant by the second oviposition onwards. Arthurs and Thomas (2000) observed that females of Locustana pardalina (Walker) treated with M. anisopliae var. acridum exhibited a reduced pre-oviposition period resulting in them laying more eggs than untreated females within the first weeks following fledging. As a consequence of that, no significant reductions in fecundity were recorded by the end of the assessment period (Arthurs and Thomas, 2000). In our study, no effect of fungal exposure on pre-oviposition period has been observed and by the end of the experiment the treated females showed a 46–49% reduction in the mean oothecal production. Interestingly, females kept apart from males showed a delay of 4–5 days in the first oviposition, which is commonly detected in females from many insect species when are avoided or delayed from mating (Huang and Subramanyam, 2003). None of the oothecae laid by unfertilized females hatched in agreement with Roth and Stay (1962), who stated that German cockroaches can not reproduce parthenogenetically being oothecae either dropped prematurely or discarded after the normal developmental period. Our results indicate that only treated females showed an effect on oothecal production, oothecal viability, and nymphal production. Lee et al. (1998) observed that only treated females of B. germanica surviving insecticide exposure showed a reduction of oothecal production while oothecal hatch and nymphal production were governed by both maternal and paternal factors. Our data suggest such male effect in oothecal viability, but the reduction from 27.1% when treated females were paired with untreated males to 7.7% when they were presented to treated males, approached significance only at the 10% level. Information on sublethal reproductive effects of entomopathogenic fungi on insects is very scarce and particularly on orthopteroid insects. Blanford and Thomas (2001) observed a significant reduction of the total number of egg pods laid by females of desert locust treated with M. anisopliae var acridum 3 days post-fledging, but they observed no effect of treatment neither on per capita number of pods nor on number of eggs per pod. From this work, it is not possible to conclude by which sex is governed this reproductive effect. Reproduction of non-orthopteroids insects has been also reported to be impaired by fungal treatments. Interestingly, the main sublethal reproductive effect is the reduction of fecundity while egg fertility is not affected or moderately affected. Fargues et al. (1991) observed a 20–50% reduction in fecundity of adult Colorado potato beetles females surviving Beauveria bassiana infection at 22 C, whereas at 25 C the fecundity of survivors was not affected by the fungal infection. At both temperatures, egg fertility of surviving females
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was similar to that of control insect (Fargues et al., 1991). The fecundity of Western Corn Rootworm, Diabrotica virginifera virginifera, has also been reported to be reduced by 70% by B. bassiana infection (Mulock and Chandler, 2001). Fecundity of Mediterranean fruit fly Ceratitis capitata Wiedemann adult females exposed to M. anisopliae and Paecilomyces fumosoroseus (Wize) Brown & Smith was reduced 65 and 40–50%, respectively, but egg fertility was only moderately affected by fungal treatments (Castillo et al., 2000). In other insect hosts/entomopathogens systems, i.e., lepidopterans and baculoviruses, the male reproductive capacity is also impaired (Matthews et al., 2002; Santiago-Alvarez and Vargas-Osuna, 1988). Sublethal reproductive effects of Bacillus thuringiensis (Berliner) have also been reported on some insect pests. Adult females of Colorado potato beetle surviving exposure to delta-endotoxin of B. thuringiensis tended to produce around 45% fewer eggs (Costa et al., 2000). Similarly, adult females of spruce budworm Choristoneura fumiferana Clemens coming from larvae surviving delta-endotoxin treatments laid a lower number of eggs even if egg fertility was not affected (Pedersen et al., 1997). In contrast, B. thuringiensis b-exotoxin seems to cause no negative effects on fecundity and fertility of survivors (Toledo et al., 1999). In untreated German cockroach females, we have obtained values of mean nymphs hatched per oothecae and of mean carrying time that can be considered normal for German cockroach laboratory populations (Barson and Renn, 1983; Lee et al., 1998). However, in a normal culture of B. germanica an oothecal viability over 75% can be expected, which is higher than the one obtained in our work for untreated females and than that over 65% observed by Lee et al. (1998). This is probably a consequence of the experimental design of our work and this latter work in which females from a high density laboratory culture were individualized in boxes either paired or kept apart from a male. Moreover, relative humidity within the experimental boxes raises by 20% over that of the laboratory culture, which could also reduce the oothecal viability as reported by Barson and Renn (1983), who found a 25% reduction in this parameter after increasing the relative humidity from 45 to 70%. The reduction in number of oothecae laid by survivors of M. anisopliae infection observed in our study could be due to the reduction in adult longevity upon fungal treatment that might cooperate on the subsequent fecundity reduction. In fact, in Orthopteroid insects, the number of egg pods or oothecae laid per female is directly related to its longevity (Albrecht, 1967). In the case of American cockroach P. americana, the 50% reduction in number of oothecae per female coming from nymphs treated with azadirachtin was associated to their decreased ingestion (Richter et al., 1997). In. our work, we have treated adults and we have
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not observed a reduction of food ingestion. The reasons for the 36–85% reduction in oothecal viability and for the 22.6–34.9% reduction in nymphal production in fungally challenged females are more difficult to find. The insect host exhibits various types of cellular and humoral immune reactions against the fungus (Vey and Go¨tz, 1986), but the pathogen may overcome them through a combination of events including mechanical damage by hyphal growth and production of toxins (Inglis et al., 2001). At a sublethal level, any of these processes together with the nutrient depletion caused by the fungal growth could directly or indirectly impair the host reproduction. A further consequence of these changes may be a prolongation of the mean oothecal incubation time; we did not found significant differences in this parameter between treated and untreated females but interestingly, it approached significance (P < 0.10). Our results on pathogenicity, virulence and horizontal transmission of the fungal conidia of M. anisopliae EAMa 01/121-Su strain aim its use in biologically based mycoinsecticides for German cockroach control even without its further integration with sublethal doses of conventional insecticides in an integrated pest management approach (Kaakeh et al., 1996; Pachamuthu et al., 1999; Zurek et al., 2002). This is also supported by the reproductive effects of this strain on B. germanica, decrease in oothecal production, oothecal viability and hatchability, which can be seen as highly effective to decrease the pest status of adult B. germanica in the long term and even its suppression. The research discussed here has been conducted under controlled laboratory conditions; thus there is a need for additional research at field conditions that will hopefully aid to assess the real contribution of this fungal strain to German cockroach management programs.
Acknowledgments The authors thank Professor B. Federici from the University of Riverside (California) for critically reviewing the manuscript and the English phraseology and Mrs. Ma Victoria Paredes for her technical support.
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