Oecologia (2015) 179:487–494 DOI 10.1007/s00442-015-3358-7
PLANT-MICROBE-ANIMAL INTERACTIONS - ORIGINAL RESEARCH
Endophyte‑mediated interactions between cauliflower, the herbivore Spodoptera litura, and the ectoparasitoid Bracon hebetor Tamanreet Kaur1 · Bahaderjeet Singh2 · Amarjeet Kaur2 · Sanehdeep Kaur1
Received: 1 January 2015 / Accepted: 19 May 2015 / Published online: 4 June 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Fungal endosymbionts in plants may influence interactions among plants, herbivores and their parasitoids through the production of secondary metabolites. We used a lepidopteran pest and its generalist parasitoid to test the effect of endophyte-infected plants on a third trophic level. Endophytic fungi, Aspergillus flavus and Aspergillus niger, isolated from Acacia arabica, were used to infect cauliflower plants. We found that the presence of the endophyte in the plants significantly extended the development period of Spodoptera litura (Fab.) larvae. Feeding of the host on endophyte-infected plants further adversely affected the development and performance of its parasitoid, Bracon hebetor (Say). A negative impact was also recorded for longevity and fecundity of endophyte-naive parasitoid females due to the parasitization of host larvae fed on endophyte-infected plants. The presence of endophytes in the diet of the host larvae significantly prolonged the development of the parasitoid. A strong detrimental effect was also recorded for larval survival and emergence of parasitoid adults. The longevity and parasitism rate of female wasps were reduced significantly due to the ingestion of endophyte-infected cauliflower plants by S. litura larvae. Overall, we found that both endophytic fungi had a negative impact on the parasitoid.
Communicated by Corné Pieterse. * Sanehdeep Kaur [email protected] 1
Department of Zoology, Guru Nanak Dev University, Amritsar, India
2
Department of Microbiology, Guru Nanak Dev University, Amritsar, India
Keywords Bottom-up effects · Multitrophic-level interactions · Aspergillus flavus · Aspergillus niger · Secondary metabolites
Introduction Tritrophic interactions consisting of plant secondary chemicals, insect herbivores, and parasitoids play a crucial role in structuring natural systems (Price et al. 1980; Barbosa and Saunders 1985; Turlings and Benrey 1998; Heil 2008). Plant chemistry can have profound bottom-up effects on the interactions between herbivores and their natural enemies. Natural enemies may benefit by exploiting constitutive or induced phytochemicals as kairomones in host finding (Fukushima et al. 2002; Heil 2008), or their fitness may be reduced when hosts are stunted from the metabolic stress of coping with ingested phytochemicals (van der Meijden 1980; Slansky 1986), or when larval parasitoids encounter such toxins in the body of their hosts (Gauld and Gaston 1994; Lampert and Bowers 2010). Most studies investigating the mediating effects of plants on herbivore–natural enemy interactions focus on plant secondary compounds that are either permanently expressed or induced by herbivory (Karban and Baldwin 1997). However, plant quality and plant chemistry can be altered by the presence of microorganisms such as fungal endophytes (Clay 1990) that grow inter- and intracellularly in the tissues of higher plants without causing overt symptoms in the plants in which they live. While the fungus benefits from access to plant nutrients, infected plants may benefit from a number of ecological advantages including resistance to insect herbivory (Richmond 2007). The basis for endophyte-mediated resistance lies in the synthesis of a number of defensive compounds which may deter feeding (antixenosis) or
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reduce insect performance (antibiosis) (Braman et al. 2002; Crawford et al. 2010; Breen 1994). Previous studies indicate that endophyte-mediated resistance may also influence interaction between insect herbivores and their natural enemies (Goldson et al. 2000; Bultman et al. 1997; Grewal et al. 1995), which may have community-wide implications (Omacini et al. 2001). Most of the research work on endophyte-mediated interactions has been focused on the effect of endophytes on insect physiology. However, the insect natural enemies may be adversely affected by direct exposure to fungalderived toxins accumulated in the tissues of their herbivore host or benefit from the resulting prolongation of herbivore development (Faeth and Bultman 2002; Breen 1994) and weakened physiological state (Grewal et al. 1995). Understanding of these multitrophic-level interactions is very important in explaining the success or failure of integration of plant resistance and biological control agents. Most of the studies on trophic interactions involving endophyte-infected plants, insect herbivores and their parasitoids have been conducted on grass endophytes (Bultman et al. 2003; Harri et al. 2008; Bixby-Brosi and Potter 2011). However, the relationship between fungal endophytes of tropical plants and their effect on insect herbivores and their parasitoids has not been previously much described. Endophytic fungi isolated from one plant have been shown to exhibit insecticidal activity when artificially inoculated into another plant (Azevedo et al. 2000). In light of this, in the present study, endophytic fungi isolated from Acacia arabica (Lam.) Willd. were used to inoculate cauliflower plants, and the trophic interactions between endophyte-infected plants, the insect herbivore Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) and its ectoparasitoid Bracon hebetor (Say) (Hymenoptera: Braconidae) were studied. S. litura is a polyphagous pest causing economic losses to a number of crops including cabbage, cauliflower, cotton, potato, etc. B. hebetor, which is a cosmopolitan gregarious ectoparasitoid, plays a significant role in the suppression of insect pests. We measured the larval development time of S. litura feeding on endophyteinfected and uninfected cauliflower plants. A range of life history traits of B. hebetor were compared when the host larvae were fed either on endophyte-infected or uninfected plants. The studies on parasitoids were conducted for two generations: the parental generation never exposed to endophyte and the first offspring generation that developed on S. litura larvae reared on endophyte-infected or uninfected cauliflower plants. We hypothesized that endophyteinfected plants have a negative effect on the development of the herbivore host and the survival and fitness of the parasitoid developing on it.
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Materials and methods Insect cultures Spodoptera litura The culture of S. litura was maintained in the laboratory at 25 ± 2 °C, 65 ± 5 % relative humidity (RH) and 12-h:12-h (day:light) photoperiod. Different larval stages and egg masses of S. litura were collected from the cauliflower fields around Amritsar (Punjab), India, and subsequent generations were maintained in the laboratory. The mass rearing was carried out in glass jars (15 × 10 cm) on Ricinus communis (L.) leaves. The diet was changed daily to maintain hygienic conditions till pupation. Pupae were transferred to pupation jars containing moist sterilized sand, and the freshly emerged adults were shifted to oviposition jars (15 × 15 cm) lined with filter paper on the inner side to facilitate egg laying. Adults were fed with a mixture of water and honey solution (4:1) soaked on a cotton swab which was replenished daily. At black head stage the egg masses were cut from the filter paper and kept in Petri plates on R. communis leaves to facilitate larval feeding after hatching. The larvae developed from this culture were used for various experiments. Bracon hebetor The mass rearing of B. hebetor was carried out on 5th instar larvae of Corcyra cephalonica (Stainton). The culture of C. cephalonica was maintained in the laboratory on partially crushed sorghum grains at 25 ± 2 °C and 65 ± 5 % RH. The freshly emerged adult parasitoids were transferred to glass chimneys and provided with water:honey (4:1) mixture as food. Isolation of endophytic fungi The leaves of visibly healthy plants of A. arabica growing in the botanical garden of Guru Nanak Dev University, Amritsar, were used for the isolation of endophytic fungi. The isolation was carried out as per the protocol of Singh et al. (2012). The leaves were thoroughly washed with distilled water followed by treatment with 70 % ethanol for 3 min and 5 % sodium hypochlorite solution for 2 min to accomplish surface sterilization. The surfacesterilized samples were rinsed in sterile distilled water prior to plating. The water obtained after the last washing was also plated to ensure complete surface sterilization. After cutting them into five or six pieces (4–6 mm), the leaf samples were placed on water agar plates [distilled water, 1.5 % (w/v) agar–agar, pH 5.5] supplemented with
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ampicillin (200 mg/ml; Himedia, Mumbai, India) to inhibit bacterial growth. These plates were incubated at 30 °C for 3–4 days to a few weeks till fungal growth appeared. The fungal hyphae that emerged from the plated samples were isolated, purified and maintained on potato dextrose agar (PDA) plants for further studies. A total of 21 fungal cultures were isolated. Preliminary screening of isolated cultures for insecticidal potential was carried out as per the protocol of Thakur et al. (2013). Larval mortality ranging from 3.33 to 56.66 %was exhibited by ten cultures. Among these, PJ-1 and PJ-2, which were identified as Aspergillus flavus and Aspergillus niger, respectively (data submitted elsewhere), were selected for the present study because of their higher insecticidal potential. Inoculation of cauliflower plants
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one of E− plants. The experiment was conducted at controlled temperature and humidity conditions of 25 ± 2 °C and 65 ± 5 %, respectively. The experiment was replicated five times with six larvae per replicate (n = 30). The larvae were provided with fresh leaves daily and kept individually in rearing containers (4 × 6 cm) to avoid cannibalism. The observations on larval development period were recorded. Effect of endophytic fungi on the development and survival of B. hebetor To test the effect of endophyte-infected cauliflower plants on the third trophic level, i.e., the parasitoid, second-instar larvae of S. litura were fed on leaves of E+ or E− cauliflower plants for 10 days. All the experiments were conducted at 25 ± 2 °C and 65 ± 5 % humidity.
Cauliflower, being a preferred host of S. litura, was selected to study the effect of endophyte-infected plants on B. hebetor. Cauliflower seedlings were transplanted into pots (one plant/pot) filled with a mixture of soil and vermicompost (4:1). Twenty-one days after transplantation, the plants were inoculated with A. flavus or A. niger spore suspension prepared from 3-week-old fungal cultures. In each slant, 10 ml of distilled water and one drop of 0.01 % Tween 80 (HiMedia, Mumbai) were added to get a homogeneous suspension, and spores were counted with a haemocytometer. Each plant was inoculated with 150 ml of spore suspension consisting of 4.45 × 106 spores/ml of A. flavus or 4.68 × 106 spores/ml of A. niger, whereas control plants were treated with 150 ml of water containing the same concentration of 0.01 % Tween 80. For each treatment 25 plants were taken and placed randomly in the glasshouse at 20 ± 2 °C. To confirm the endophyte infection, after 3 weeks of fungal infection, stem and leaf samples were collected from six randomly selected endophyte-inoculated (E+) and uninoculated (E−) plants. The endophytic fungi were re-isolated, as explained previously, in the isolation method. The establishment of endophytes was considered successful when more than 20 % endophytes obtained in the E+ plants were the same as the inoculated ones (Jallow et al. 2008), while they were completely absent in the E− plants.
Parental generation
Effect of endophytic fungi on the larval period of S. litura
To study the effect of the endophyte on the development of B. hebetor, S. litura larvae reared on E+ or E− plants for 10 days were exposed to endophyte-naive parasitoid females. Two-day-old mated females were transferred into glass chimneys (two females/chimney) and allowed to parasitize the 3rd instar larvae of S. litura (two larvae/chimney) reared either on E+ or E− plants. After exposure for 24 h, the larvae were removed from the chimneys and fresh larvae were introduced. The experiment was replicated three
Second-instar larvae from the stock culture were used to study the effect of endophytic fungi on larval development of S. litura. Three weeks after inoculation, the older leaves from the lower parts of E+ and E− plants were used to feed the larvae. There were three treatments i.e., two of E+ plants (A. niger- or A. flavus-infected plants), and
From the stock culture of B. hebetor, the freshly emerged adults having no prior exposure to endophytes were collected and allowed to mate for 24 h. The adults were provided with a water:honey (4:1) mixture as food impregnated on a cotton swab. Two female parasitoid adults were transferred to a glass chimney containing 3rd instar larvae of S. litura (two larvae/chimney). This comprised a nochoice experiment as the female wasps were provided with larvae reared either on E+ or E− plants. There were three replications with two females per replicate. After exposure for 24 h the host larvae were removed and transferred to Petri plates (90 × 14 mm). In this way, S. litura larvae fed on E+ or E− plants were exposed daily till the female died in each treatment. A total of 101, 100 and 116 larvae of S. litura fed on leaves of A. flavus- and A. niger-infected, and endophyte-uninfected plants, respectively, were exposed to adults of B. hebetor depending upon the longevity of the female adults in each treatment. Observations were recorded on adult longevity, parasitism rate (number of host larvae parasitized by the female wasp throughout its life span), fecundity and number of parasitoid eggs per host larvae. First offspring generation
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times with two female wasps per replicate. Throughout the experiment a total of 84 larvae of S. litura were exposed for each treatment. The parasitized larvae were kept individually in Petri plates and checked daily for the development of B. hebetor. Observations were recorded on hatching of eggs, larval survival, larval, pupal and total developmental period of B. hebetor. Percent cocoon formation and adult emergence were also recorded. On emergence the number and sex of the progeny were observed. Data were also recorded for adult longevity, parasitism rate, fecundity and number of eggs per host. Statistical analysis The statistical analyses were performed using SPSS software for WINDOWS version 16.0 (SPSS, Chicago, Ill.). All data sets were checked for normality and homogeneity of variance. All the values were presented as non-transformed mean ± SE. There were three treatments i.e., two of E+ plants and one of E− plants. The experiment of the effect of endophytes on the larval development period of S. litura was replicated five times with six larvae per replication for each treatment. The data on larval development were analyzed using one-way ANOVA and Tukey’s test. All the bioassay studies for B. hebetor were performed in three replicates for each treatment. The data on all biological parameters of B. hebetor were analyzed using one-way ANOVA and Tukey’s test.
Results Effect of endophytes on the host insect The presence of endophytes in cauliflower plants had a significant effect on the development of S. litura larvae. As compared to E− plants, the larvae took 3.45 and 2.60 days more to complete their development on A. flavus- and A. niger-infected plants, respectively (ANOVA F2,12 = 21.52, P ≤ 0.001) (Fig. 1). Effect of endophytes on parasitoid Parental generation Indirect inhibitory effects of endophyte-infected plants were recorded on longevity of female parasitoids. The life span of female wasps parasitizing the host larvae reared on E+ plants decreased significantly as compared to those parasitizing the larvae reared on E− plants (ANOVA F2,6 = 7.30, P = 0.025) (Table 1). No significant differences were observed in parasitism rate of larvae feeding on E+ and E− cauliflower plants. However, a significant
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Fig. 1 Larval development period of Spodoptera litura reared on endophyte-uninfected (E−) and endophyte-infected (E+; Aspergillus flavus or Aspergillus niger) cauliflower plants. Columns Mean, bars ±SE. Different letters above the columns representing each treatment indicate significant differences
reduction in fecundity was observed (ANOVA F2,6 = 5.18, P = 0.049). The females deposited fewer eggs per E+ hosts (4.75 ± 0.19 and 4.74 ± 0.20 eggs in A. flavus and A. niger, respectively) as compared to E− hosts (6.63 ± 0.33 eggs) (ANOVA F2,6 = 17.94, P = 0.0029) (Table 1). First offspring generation Endophyte-infected cauliflower plants did not have a significant effect on the hatching of eggs (Fig. 2). The survival of parasitoid larvae developing on hosts fed on E+ plants decreased significantly, from 54.40 ± 1.99 and 58.81 ± 2.23 % on A. flavus and A. niger, respectively, in comparison to 67.43 ± 1.36 % on E− plants (ANOVA F2,6 = 12.18, P = 0.0077) (Fig. 2). The larval, pupal and overall total developmental period of the parasitoid extended significantly under the influence of E+ plants. The total developmental time was 1.99 and 1.60 days longer on A. flavus- and A. niger-infected plants, respectively, than E− plants (ANOVA F2,6 = 26.27, P = 0.0011) (Fig. 3). Similarly, inhibitory effects were also recorded on adult emergence, which reduced from 71.92 ± 3.38 % in the case of E− plants to 49.73 ± 2.67 and 53.34 ± 0.30 % on A. flavus- and A. niger-infected plants, respectively (ANOVA F2,6 = 22.75, P = 0.0016) (Fig. 2). A. flavusinfected plants significantly influenced the sex ratio, as fewer females emerged when the host larvae were reared on these plants, while A. niger-infected plants did not differ significantly from E− plants (ANOVA F2,6 = 20.21, P = 0.0022) (Table 2). Longevity of first-generation male and female parasitoid adults was decreased significantly by the presence of endophytes. Although the parasitism rate of these female parasitoids was not significantly influenced by the endophytes,
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Table 1 Longevity, parasitism rate, fecundity and eggs per host of Bracon hebetor adults of the parental generation on Spodoptera litura larvae fed on endophyte-uninfected (E−) and endophyte-infected (E+; Aspergillus flavus or Aspergillus niger) cauliflower plants Treatment
Female longevity (days)
Parasitism rate (%)
Fecundity (no. of eggs per female)
No. of parasitoid eggs per host
E− E+ (A. flavus)
20.66 ± 0.33a 15.33 ± 1.45b
53.03 ± 6.47a 46.82 ± 5.72a
73.00 ± 9.77a 44.83 ± 6.69b
6.63 ± 0.33a 4.75 ± 0.19b
E+ (A. niger)
15.66 ± 1.20b
42.04 ± 4.59a
41.83 ± 5.54b
4.74 ± 0.20b
Mean ± SE. Means followed by different letters within a column are significantly different
(ANOVA F2,6 = 8.64, P = 0.017). The number of eggs per host was also significantly low due to the presence of endophytes (ANOVA F2,6 = 19.25, P = 0.0025) (Table 2).
Discussion
Fig. 2 Egg hatching, larval survival and adult emergence of first offspring generation of B. hebetor developed on S. litura larvae fed on E− and E+ cauliflower plants. Columns Mean, bars ± SE. Different letters above the columns representing each treatment indicate significant differences. For abbreviations, see Fig. 1
Fig. 3 Developmental period of first offspring generation of B. hebetor developed on S. litura larvae fed on E− and E+ cauliflower plants. Columns Mean, bars ± SE. Different letters above the columns representing each treatment indicate significant differences. For abbreviations, see Fig. 1
the fecundity rate of female wasps decreased significantly in the E+ plants. As compared to 66.00 ± 9.56 eggs/female on host larvae reared on E− plants, only 32.33 ± 5.13 and 36.16 ± 6.49 eggs/female were recorded, respectively, on larvae fed on A. flavus- and A. niger-infected plants
Fungal endophytes are known to enhance plant resistance to insect herbivores. The capacity of endophytic fungi to repel insects, induce weight loss, growth and development reduction and increased mortality in insects has been correlated with toxin production by the fungi or by the plant due to interaction with the fungi (Clay 1988a, b; Azevedo et al. 2000). The effect of such mycotoxins also moves up the food chain and adversely affects the development and reproductive potential of parasitoids (de Sassi et al. 2006; Harri et al. 2008). In accordance with our prediction, we found that the negative effects of host plant endophytes on S. litura host larvae permeated up to the parasitoid. Although endophytes have been known to protect their host plant from herbivores, natural enemies of insect herbivores may also be adversely affected by the endophytic fungi, as indicated by our results. The larval development of S. litura was significantly prolonged on endophyte-infected plants. The prolonged development of insects under the influence of endophytes has also been reported earlier (Jallow et al. 2004; Jaber and Vidal 2010). Further deleterious effects of endophytes at the third trophic level were manifested throughout the life of B. hebetor via longer developmental time, reduced survival rate and longevity and an overall reduced reproductive success. The parental females, when allowed to parasitize host larvae reared on E+ and E− plants, did not show significant difference in parasitism rate. However, fecundity and longevity of these parental female parasitoids decreased significantly due to parasitization of E+ hosts as compared to E− hosts. Although these females did not have any prior exposure to endophytes, their decreased life span may be due to host feeding, which has been reported in a large number of hymenopteran parasitoid adults (Campbell and Duffey 1981; Kidd and Jervis 1989; Roth et al. 1997). Host feeding is very important in synovigenic parasitoid females
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Table 2 Inhibitory effects of endophytes on adults of B. hebetor descended from S. litura larvae fed on E− and E+ cauliflower plants Treatment
E−
Sex ratio female (%)
Longevity (days) Male
Female
47.34 ± 1.82a
19.33 ± 1.20a 17.33 ± 1.20ab
20.83 ± 1.16a 14.33 ± 0.88b
52.27 ± 6.81a 42.10 ± 5.47a
66.00 ± 9.56a 32.33 ± 5.13b
5.83 ± 0.04a 4.31 ± 0.12b
14.00 ± 0.57b
14.00 ± 0.86b
43.13 ± 8.02a
36.16 ± 6.49b
4.81 ± 0.27b
b
E+ (A. flavus)
32.16 ± 1.14
E+ (A. niger)
49.26 ± 2.89a
Parasitization (%) Fecundity (no. of eggs per No. of parasitoid female) eggs per host
Mean ± SE. Means followed by different letters within a column are significantly different. For abbreviations, see Table 1
like B. hebetor, which emerge with a limited number of eggs and egg production and maturation continue throughout the life of the female (Jervis and Kidd 1986; Godfray 1994). Nutritional demands associated with egg production are obtained through host feeding by adult females. The adverse effects of E+ plants on the longevity of female wasps subsequently resulted in reduced fecundity and egg hatchability. Our results are in agreement with previous studies on aphids where negative effects were found on endophyte-naive Aphidius ervi (Haliday) females (Harri et al. 2008). Although E+ plants did not influence the parasitism rate of female wasps, they negatively affected their longevity and fecundity. The E+ host did not influence the hatching of parasitoid eggs; however, the survival rate of larvae reduced significantly on both the endophytes. Because the insect parasitoids complete their development on a single host, the development of parasitoids is significantly influenced by the diet of their host. This may affect the developing parasitoid indirectly by altering host suitability or directly by exposing the parasitoid to unmetabolized defensive chemicals (Lampert et al. 2008), which the herbivorous insects may accumulate within their hemolymph for their own defense (Barbosa et al. 1991, Francis et al. 2001). Feeding on E+ plants thus may have rendered the S. litura larvae nutritionally less suitable for the development of the parasitoid. The larval, pupal, as well as the total development period of B. hebetor extended significantly on E+ hosts as compared to E− hosts. The toxicity of the E+ hosts also manifested as reduced larval survival and adult emergence. Barker and Addison (1996) documented that Acremonium lolii-infected plants reduced the nutritional quality of the weevil Listronotus bonariensis (Kuschel), resulting in retarded development of Microctonus hyperodae (Loan) larvae. Contrary to this, Bultman et al. (1997) reported that Euplectrus comstockii (Howard) developed faster when its host fall armyworm, Spodoptera frugiperda (J. E. Smith), was reared on E+ plants as compared to E− plants. The impact of the endophyte on the sex ratio of the parasitoid was observed only in the case of A. flavus, where significantly fewer females emerged as compared to on E− hosts. The reproductive potential of these first-generation parasitoid adults was adversely affected by the endophytes.
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Although the parasitism rate of the female parasitoid did not differ between E+ and E− hosts, they laid significantly fewer eggs per host. A significant reduction was also observed in overall fecundity rate due to endophytes. The decreased number of eggs on the E+ host may be due to the ability of the parasitoid to assess the suitability of the host by using chemical cues (Fisher 1971). There are some reports indicating that parasitoids developing on hosts fed on a diet having a higher concentration of plant allelochemicals produced smaller primary clutch sizes than those developed on hosts fed on a diet with a lower concentration of allelochemicals (Lampert et al. 2008; Kaur and Kaur 2013). Toxic effects of fungal endophytes were also observed on the hatching of eggs, which significantly decreased in number. Coudron et al. (2009) documented a significant interaction between the host and the parasitoid egg prior to hatching, as they recorded a reduction in the hatching of eggs of E. comstockii due to a depletion of ascorbic acid from the diet of its larval host Heliothis virescens (Fabricius). It is a kind of host immunity to ectoparasitoids, a phenomenon well documented in endoparasitoids (Schmidt et al. 2001), that may have resulted in this reduced hatching of eggs. B. hebetor suffered greatly from endophyte infection in almost all the life history parameters we measured. Our results are in agreement with previous studies on parasitoids where mainly negative effects were recorded (Barker and Addison 1996; Bultman et al. 1997, 2003; de Sassi et al. 2006). Similar detrimental effects of endophytes on the reproductive ability of the aphid parasitoid A. ervi and coccinellid predator have been demonstrated (Harri et al. 2008; de Sassi et al. 2006). This reduction in reproductive success could have been due to parasitoid exposure during larval development to the fungal toxin present in the host insect that weakened the parasitoid females, which laid fewer eggs with reduced viability. Contrary to this, Krauss et al. (2007) did not find a negative effect of endophyteinfected plants on aphids. Although we have not investigated the mechanism by which the endophytes A. flavus and A. niger influenced the parasitoid B. hebetor, our results suggest that secondary metabolites produced by the endophytic fungi or due to the interaction of the plants with fungi, may play a role in mediating the effects we found on
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parasitoid performance. Moreover, the negative effects of endophytes on generalist parasitoids like B. hebetor, may have greater impacts on the entire community because they are more strongly affected by the plant defensive chemistry than specialist parasitoids (Harvey et al. 2003; Harvey 2005; Barbosa et al. 1991; Sznajder and Harvey 2003). The net effect of endophyte-produced toxins on plants will depend upon their relative impact on herbivores and their natural enemies. However, when natural enemies of insect herbivores are negatively affected by endophytic fungi, the benefits of protection by fungal endophytes may be diminished, as indicated by our results. Further studies based on field experiments that simultaneously quantify the effects of endophytes on an insect and its parasitoid, and relate those effects to plant performance, are needed to assess the overall impact of endophytes on plants. This information will help us to understand the ways in which plant symbionts can modify multitrophic interactions. Author contribution statement S. K. and A. K. conceived and designed the experiments. T. K. and B. S. performed the experiments, analyzed the data and prepared the manuscript with the help of S. K. and A. K. Acknowledgments Financial assistance was received from the University Grants Commission, New Delhi, India, under the University with Potential for Excellence scheme, which is duly acknowledged.
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Oecologia (2015) 179:487–494 Price PW, Bouton CE, Gross P, McPheron BA, Thompson JN, Weis AE (1980) Interactions among three trophic levels: influence of plants on interactions between insect herbivores and natural enemies. Annu Rev Ecol Evol Syst 11:41–65 Richmond DS (2007) Mediation of herbivore-natural enemy interactions by Neotyphodium endophytes: the role of insect behavioural response. In: Popay AJ, Thom ER (eds) Proceedings of the 6th International Symposium on Fungal Endophytes of Grasses. New Zealand Grassland Association, Dunedin, pp 313–319 Roth S, Knorr C, Lindroth RL (1997) Dietary phenolics affect performance of the gypsy moth (Lepidoptera: Lymantriidae) and its parasitoid Cotesia melanoscela (Hymenoptera: Braconidae). Environ Entomol 26:668–671 Schmidt O, Theopold U, Strand M (2001) Innate immunity and its evasion and suppression by hymenopteran endoparasitoids. BioEssays 23:344–351 Singh B, Thakur A, Kaur S, Chadha BS, Kaur A (2012) Acetylcholinesterase inhibitory potential and insecticidal activity of an endophytic Alternaria sp. from Ricinus communis. Appl Biochem Biotechnol 168:991–1002 Slansky F (1986) Nutritional ecology of endoparasitic insects and their hosts: an overview. J Insect Physiol 32:255–261 Sznajder B, Harvey JA (2003) Second and third trophic level effects of differences in plant species reflect dietary specialisation of herbivores and their endoparasitoids. Entomol Exp Appl 109:73–82 Thakur A, Singh V, Kaur A, Kaur S (2013) Insecticidal potential of an endophytic fungus, Cladosporium uredinicola, against Spodoptera litura. Phytoparasitica 41:373–382 Turlings TCJ, Benrey B (1998) Effects of plant metabolites on the behavior and development of parasitic wasps. Ecoscience 5:321–333 van der Meijden E (1980) Can hosts escape from their parasitoids? The effects of food shortage on the braconid parasitoid Apanteles popularis and its host Tyria jacobaeae. Neth J Zool 30:382–391
PLANT-MICROBE-ANIMAL INTERACTIONS - ORIGINAL RESEARCH
Endophyte‑mediated interactions between cauliflower, the herbivore Spodoptera litura, and the ectoparasitoid Bracon hebetor Tamanreet Kaur1 · Bahaderjeet Singh2 · Amarjeet Kaur2 · Sanehdeep Kaur1
Received: 1 January 2015 / Accepted: 19 May 2015 / Published online: 4 June 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Fungal endosymbionts in plants may influence interactions among plants, herbivores and their parasitoids through the production of secondary metabolites. We used a lepidopteran pest and its generalist parasitoid to test the effect of endophyte-infected plants on a third trophic level. Endophytic fungi, Aspergillus flavus and Aspergillus niger, isolated from Acacia arabica, were used to infect cauliflower plants. We found that the presence of the endophyte in the plants significantly extended the development period of Spodoptera litura (Fab.) larvae. Feeding of the host on endophyte-infected plants further adversely affected the development and performance of its parasitoid, Bracon hebetor (Say). A negative impact was also recorded for longevity and fecundity of endophyte-naive parasitoid females due to the parasitization of host larvae fed on endophyte-infected plants. The presence of endophytes in the diet of the host larvae significantly prolonged the development of the parasitoid. A strong detrimental effect was also recorded for larval survival and emergence of parasitoid adults. The longevity and parasitism rate of female wasps were reduced significantly due to the ingestion of endophyte-infected cauliflower plants by S. litura larvae. Overall, we found that both endophytic fungi had a negative impact on the parasitoid.
Communicated by Corné Pieterse. * Sanehdeep Kaur [email protected] 1
Department of Zoology, Guru Nanak Dev University, Amritsar, India
2
Department of Microbiology, Guru Nanak Dev University, Amritsar, India
Keywords Bottom-up effects · Multitrophic-level interactions · Aspergillus flavus · Aspergillus niger · Secondary metabolites
Introduction Tritrophic interactions consisting of plant secondary chemicals, insect herbivores, and parasitoids play a crucial role in structuring natural systems (Price et al. 1980; Barbosa and Saunders 1985; Turlings and Benrey 1998; Heil 2008). Plant chemistry can have profound bottom-up effects on the interactions between herbivores and their natural enemies. Natural enemies may benefit by exploiting constitutive or induced phytochemicals as kairomones in host finding (Fukushima et al. 2002; Heil 2008), or their fitness may be reduced when hosts are stunted from the metabolic stress of coping with ingested phytochemicals (van der Meijden 1980; Slansky 1986), or when larval parasitoids encounter such toxins in the body of their hosts (Gauld and Gaston 1994; Lampert and Bowers 2010). Most studies investigating the mediating effects of plants on herbivore–natural enemy interactions focus on plant secondary compounds that are either permanently expressed or induced by herbivory (Karban and Baldwin 1997). However, plant quality and plant chemistry can be altered by the presence of microorganisms such as fungal endophytes (Clay 1990) that grow inter- and intracellularly in the tissues of higher plants without causing overt symptoms in the plants in which they live. While the fungus benefits from access to plant nutrients, infected plants may benefit from a number of ecological advantages including resistance to insect herbivory (Richmond 2007). The basis for endophyte-mediated resistance lies in the synthesis of a number of defensive compounds which may deter feeding (antixenosis) or
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reduce insect performance (antibiosis) (Braman et al. 2002; Crawford et al. 2010; Breen 1994). Previous studies indicate that endophyte-mediated resistance may also influence interaction between insect herbivores and their natural enemies (Goldson et al. 2000; Bultman et al. 1997; Grewal et al. 1995), which may have community-wide implications (Omacini et al. 2001). Most of the research work on endophyte-mediated interactions has been focused on the effect of endophytes on insect physiology. However, the insect natural enemies may be adversely affected by direct exposure to fungalderived toxins accumulated in the tissues of their herbivore host or benefit from the resulting prolongation of herbivore development (Faeth and Bultman 2002; Breen 1994) and weakened physiological state (Grewal et al. 1995). Understanding of these multitrophic-level interactions is very important in explaining the success or failure of integration of plant resistance and biological control agents. Most of the studies on trophic interactions involving endophyte-infected plants, insect herbivores and their parasitoids have been conducted on grass endophytes (Bultman et al. 2003; Harri et al. 2008; Bixby-Brosi and Potter 2011). However, the relationship between fungal endophytes of tropical plants and their effect on insect herbivores and their parasitoids has not been previously much described. Endophytic fungi isolated from one plant have been shown to exhibit insecticidal activity when artificially inoculated into another plant (Azevedo et al. 2000). In light of this, in the present study, endophytic fungi isolated from Acacia arabica (Lam.) Willd. were used to inoculate cauliflower plants, and the trophic interactions between endophyte-infected plants, the insect herbivore Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) and its ectoparasitoid Bracon hebetor (Say) (Hymenoptera: Braconidae) were studied. S. litura is a polyphagous pest causing economic losses to a number of crops including cabbage, cauliflower, cotton, potato, etc. B. hebetor, which is a cosmopolitan gregarious ectoparasitoid, plays a significant role in the suppression of insect pests. We measured the larval development time of S. litura feeding on endophyteinfected and uninfected cauliflower plants. A range of life history traits of B. hebetor were compared when the host larvae were fed either on endophyte-infected or uninfected plants. The studies on parasitoids were conducted for two generations: the parental generation never exposed to endophyte and the first offspring generation that developed on S. litura larvae reared on endophyte-infected or uninfected cauliflower plants. We hypothesized that endophyteinfected plants have a negative effect on the development of the herbivore host and the survival and fitness of the parasitoid developing on it.
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Materials and methods Insect cultures Spodoptera litura The culture of S. litura was maintained in the laboratory at 25 ± 2 °C, 65 ± 5 % relative humidity (RH) and 12-h:12-h (day:light) photoperiod. Different larval stages and egg masses of S. litura were collected from the cauliflower fields around Amritsar (Punjab), India, and subsequent generations were maintained in the laboratory. The mass rearing was carried out in glass jars (15 × 10 cm) on Ricinus communis (L.) leaves. The diet was changed daily to maintain hygienic conditions till pupation. Pupae were transferred to pupation jars containing moist sterilized sand, and the freshly emerged adults were shifted to oviposition jars (15 × 15 cm) lined with filter paper on the inner side to facilitate egg laying. Adults were fed with a mixture of water and honey solution (4:1) soaked on a cotton swab which was replenished daily. At black head stage the egg masses were cut from the filter paper and kept in Petri plates on R. communis leaves to facilitate larval feeding after hatching. The larvae developed from this culture were used for various experiments. Bracon hebetor The mass rearing of B. hebetor was carried out on 5th instar larvae of Corcyra cephalonica (Stainton). The culture of C. cephalonica was maintained in the laboratory on partially crushed sorghum grains at 25 ± 2 °C and 65 ± 5 % RH. The freshly emerged adult parasitoids were transferred to glass chimneys and provided with water:honey (4:1) mixture as food. Isolation of endophytic fungi The leaves of visibly healthy plants of A. arabica growing in the botanical garden of Guru Nanak Dev University, Amritsar, were used for the isolation of endophytic fungi. The isolation was carried out as per the protocol of Singh et al. (2012). The leaves were thoroughly washed with distilled water followed by treatment with 70 % ethanol for 3 min and 5 % sodium hypochlorite solution for 2 min to accomplish surface sterilization. The surfacesterilized samples were rinsed in sterile distilled water prior to plating. The water obtained after the last washing was also plated to ensure complete surface sterilization. After cutting them into five or six pieces (4–6 mm), the leaf samples were placed on water agar plates [distilled water, 1.5 % (w/v) agar–agar, pH 5.5] supplemented with
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ampicillin (200 mg/ml; Himedia, Mumbai, India) to inhibit bacterial growth. These plates were incubated at 30 °C for 3–4 days to a few weeks till fungal growth appeared. The fungal hyphae that emerged from the plated samples were isolated, purified and maintained on potato dextrose agar (PDA) plants for further studies. A total of 21 fungal cultures were isolated. Preliminary screening of isolated cultures for insecticidal potential was carried out as per the protocol of Thakur et al. (2013). Larval mortality ranging from 3.33 to 56.66 %was exhibited by ten cultures. Among these, PJ-1 and PJ-2, which were identified as Aspergillus flavus and Aspergillus niger, respectively (data submitted elsewhere), were selected for the present study because of their higher insecticidal potential. Inoculation of cauliflower plants
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one of E− plants. The experiment was conducted at controlled temperature and humidity conditions of 25 ± 2 °C and 65 ± 5 %, respectively. The experiment was replicated five times with six larvae per replicate (n = 30). The larvae were provided with fresh leaves daily and kept individually in rearing containers (4 × 6 cm) to avoid cannibalism. The observations on larval development period were recorded. Effect of endophytic fungi on the development and survival of B. hebetor To test the effect of endophyte-infected cauliflower plants on the third trophic level, i.e., the parasitoid, second-instar larvae of S. litura were fed on leaves of E+ or E− cauliflower plants for 10 days. All the experiments were conducted at 25 ± 2 °C and 65 ± 5 % humidity.
Cauliflower, being a preferred host of S. litura, was selected to study the effect of endophyte-infected plants on B. hebetor. Cauliflower seedlings were transplanted into pots (one plant/pot) filled with a mixture of soil and vermicompost (4:1). Twenty-one days after transplantation, the plants were inoculated with A. flavus or A. niger spore suspension prepared from 3-week-old fungal cultures. In each slant, 10 ml of distilled water and one drop of 0.01 % Tween 80 (HiMedia, Mumbai) were added to get a homogeneous suspension, and spores were counted with a haemocytometer. Each plant was inoculated with 150 ml of spore suspension consisting of 4.45 × 106 spores/ml of A. flavus or 4.68 × 106 spores/ml of A. niger, whereas control plants were treated with 150 ml of water containing the same concentration of 0.01 % Tween 80. For each treatment 25 plants were taken and placed randomly in the glasshouse at 20 ± 2 °C. To confirm the endophyte infection, after 3 weeks of fungal infection, stem and leaf samples were collected from six randomly selected endophyte-inoculated (E+) and uninoculated (E−) plants. The endophytic fungi were re-isolated, as explained previously, in the isolation method. The establishment of endophytes was considered successful when more than 20 % endophytes obtained in the E+ plants were the same as the inoculated ones (Jallow et al. 2008), while they were completely absent in the E− plants.
Parental generation
Effect of endophytic fungi on the larval period of S. litura
To study the effect of the endophyte on the development of B. hebetor, S. litura larvae reared on E+ or E− plants for 10 days were exposed to endophyte-naive parasitoid females. Two-day-old mated females were transferred into glass chimneys (two females/chimney) and allowed to parasitize the 3rd instar larvae of S. litura (two larvae/chimney) reared either on E+ or E− plants. After exposure for 24 h, the larvae were removed from the chimneys and fresh larvae were introduced. The experiment was replicated three
Second-instar larvae from the stock culture were used to study the effect of endophytic fungi on larval development of S. litura. Three weeks after inoculation, the older leaves from the lower parts of E+ and E− plants were used to feed the larvae. There were three treatments i.e., two of E+ plants (A. niger- or A. flavus-infected plants), and
From the stock culture of B. hebetor, the freshly emerged adults having no prior exposure to endophytes were collected and allowed to mate for 24 h. The adults were provided with a water:honey (4:1) mixture as food impregnated on a cotton swab. Two female parasitoid adults were transferred to a glass chimney containing 3rd instar larvae of S. litura (two larvae/chimney). This comprised a nochoice experiment as the female wasps were provided with larvae reared either on E+ or E− plants. There were three replications with two females per replicate. After exposure for 24 h the host larvae were removed and transferred to Petri plates (90 × 14 mm). In this way, S. litura larvae fed on E+ or E− plants were exposed daily till the female died in each treatment. A total of 101, 100 and 116 larvae of S. litura fed on leaves of A. flavus- and A. niger-infected, and endophyte-uninfected plants, respectively, were exposed to adults of B. hebetor depending upon the longevity of the female adults in each treatment. Observations were recorded on adult longevity, parasitism rate (number of host larvae parasitized by the female wasp throughout its life span), fecundity and number of parasitoid eggs per host larvae. First offspring generation
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times with two female wasps per replicate. Throughout the experiment a total of 84 larvae of S. litura were exposed for each treatment. The parasitized larvae were kept individually in Petri plates and checked daily for the development of B. hebetor. Observations were recorded on hatching of eggs, larval survival, larval, pupal and total developmental period of B. hebetor. Percent cocoon formation and adult emergence were also recorded. On emergence the number and sex of the progeny were observed. Data were also recorded for adult longevity, parasitism rate, fecundity and number of eggs per host. Statistical analysis The statistical analyses were performed using SPSS software for WINDOWS version 16.0 (SPSS, Chicago, Ill.). All data sets were checked for normality and homogeneity of variance. All the values were presented as non-transformed mean ± SE. There were three treatments i.e., two of E+ plants and one of E− plants. The experiment of the effect of endophytes on the larval development period of S. litura was replicated five times with six larvae per replication for each treatment. The data on larval development were analyzed using one-way ANOVA and Tukey’s test. All the bioassay studies for B. hebetor were performed in three replicates for each treatment. The data on all biological parameters of B. hebetor were analyzed using one-way ANOVA and Tukey’s test.
Results Effect of endophytes on the host insect The presence of endophytes in cauliflower plants had a significant effect on the development of S. litura larvae. As compared to E− plants, the larvae took 3.45 and 2.60 days more to complete their development on A. flavus- and A. niger-infected plants, respectively (ANOVA F2,12 = 21.52, P ≤ 0.001) (Fig. 1). Effect of endophytes on parasitoid Parental generation Indirect inhibitory effects of endophyte-infected plants were recorded on longevity of female parasitoids. The life span of female wasps parasitizing the host larvae reared on E+ plants decreased significantly as compared to those parasitizing the larvae reared on E− plants (ANOVA F2,6 = 7.30, P = 0.025) (Table 1). No significant differences were observed in parasitism rate of larvae feeding on E+ and E− cauliflower plants. However, a significant
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Fig. 1 Larval development period of Spodoptera litura reared on endophyte-uninfected (E−) and endophyte-infected (E+; Aspergillus flavus or Aspergillus niger) cauliflower plants. Columns Mean, bars ±SE. Different letters above the columns representing each treatment indicate significant differences
reduction in fecundity was observed (ANOVA F2,6 = 5.18, P = 0.049). The females deposited fewer eggs per E+ hosts (4.75 ± 0.19 and 4.74 ± 0.20 eggs in A. flavus and A. niger, respectively) as compared to E− hosts (6.63 ± 0.33 eggs) (ANOVA F2,6 = 17.94, P = 0.0029) (Table 1). First offspring generation Endophyte-infected cauliflower plants did not have a significant effect on the hatching of eggs (Fig. 2). The survival of parasitoid larvae developing on hosts fed on E+ plants decreased significantly, from 54.40 ± 1.99 and 58.81 ± 2.23 % on A. flavus and A. niger, respectively, in comparison to 67.43 ± 1.36 % on E− plants (ANOVA F2,6 = 12.18, P = 0.0077) (Fig. 2). The larval, pupal and overall total developmental period of the parasitoid extended significantly under the influence of E+ plants. The total developmental time was 1.99 and 1.60 days longer on A. flavus- and A. niger-infected plants, respectively, than E− plants (ANOVA F2,6 = 26.27, P = 0.0011) (Fig. 3). Similarly, inhibitory effects were also recorded on adult emergence, which reduced from 71.92 ± 3.38 % in the case of E− plants to 49.73 ± 2.67 and 53.34 ± 0.30 % on A. flavus- and A. niger-infected plants, respectively (ANOVA F2,6 = 22.75, P = 0.0016) (Fig. 2). A. flavusinfected plants significantly influenced the sex ratio, as fewer females emerged when the host larvae were reared on these plants, while A. niger-infected plants did not differ significantly from E− plants (ANOVA F2,6 = 20.21, P = 0.0022) (Table 2). Longevity of first-generation male and female parasitoid adults was decreased significantly by the presence of endophytes. Although the parasitism rate of these female parasitoids was not significantly influenced by the endophytes,
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Table 1 Longevity, parasitism rate, fecundity and eggs per host of Bracon hebetor adults of the parental generation on Spodoptera litura larvae fed on endophyte-uninfected (E−) and endophyte-infected (E+; Aspergillus flavus or Aspergillus niger) cauliflower plants Treatment
Female longevity (days)
Parasitism rate (%)
Fecundity (no. of eggs per female)
No. of parasitoid eggs per host
E− E+ (A. flavus)
20.66 ± 0.33a 15.33 ± 1.45b
53.03 ± 6.47a 46.82 ± 5.72a
73.00 ± 9.77a 44.83 ± 6.69b
6.63 ± 0.33a 4.75 ± 0.19b
E+ (A. niger)
15.66 ± 1.20b
42.04 ± 4.59a
41.83 ± 5.54b
4.74 ± 0.20b
Mean ± SE. Means followed by different letters within a column are significantly different
(ANOVA F2,6 = 8.64, P = 0.017). The number of eggs per host was also significantly low due to the presence of endophytes (ANOVA F2,6 = 19.25, P = 0.0025) (Table 2).
Discussion
Fig. 2 Egg hatching, larval survival and adult emergence of first offspring generation of B. hebetor developed on S. litura larvae fed on E− and E+ cauliflower plants. Columns Mean, bars ± SE. Different letters above the columns representing each treatment indicate significant differences. For abbreviations, see Fig. 1
Fig. 3 Developmental period of first offspring generation of B. hebetor developed on S. litura larvae fed on E− and E+ cauliflower plants. Columns Mean, bars ± SE. Different letters above the columns representing each treatment indicate significant differences. For abbreviations, see Fig. 1
the fecundity rate of female wasps decreased significantly in the E+ plants. As compared to 66.00 ± 9.56 eggs/female on host larvae reared on E− plants, only 32.33 ± 5.13 and 36.16 ± 6.49 eggs/female were recorded, respectively, on larvae fed on A. flavus- and A. niger-infected plants
Fungal endophytes are known to enhance plant resistance to insect herbivores. The capacity of endophytic fungi to repel insects, induce weight loss, growth and development reduction and increased mortality in insects has been correlated with toxin production by the fungi or by the plant due to interaction with the fungi (Clay 1988a, b; Azevedo et al. 2000). The effect of such mycotoxins also moves up the food chain and adversely affects the development and reproductive potential of parasitoids (de Sassi et al. 2006; Harri et al. 2008). In accordance with our prediction, we found that the negative effects of host plant endophytes on S. litura host larvae permeated up to the parasitoid. Although endophytes have been known to protect their host plant from herbivores, natural enemies of insect herbivores may also be adversely affected by the endophytic fungi, as indicated by our results. The larval development of S. litura was significantly prolonged on endophyte-infected plants. The prolonged development of insects under the influence of endophytes has also been reported earlier (Jallow et al. 2004; Jaber and Vidal 2010). Further deleterious effects of endophytes at the third trophic level were manifested throughout the life of B. hebetor via longer developmental time, reduced survival rate and longevity and an overall reduced reproductive success. The parental females, when allowed to parasitize host larvae reared on E+ and E− plants, did not show significant difference in parasitism rate. However, fecundity and longevity of these parental female parasitoids decreased significantly due to parasitization of E+ hosts as compared to E− hosts. Although these females did not have any prior exposure to endophytes, their decreased life span may be due to host feeding, which has been reported in a large number of hymenopteran parasitoid adults (Campbell and Duffey 1981; Kidd and Jervis 1989; Roth et al. 1997). Host feeding is very important in synovigenic parasitoid females
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Table 2 Inhibitory effects of endophytes on adults of B. hebetor descended from S. litura larvae fed on E− and E+ cauliflower plants Treatment
E−
Sex ratio female (%)
Longevity (days) Male
Female
47.34 ± 1.82a
19.33 ± 1.20a 17.33 ± 1.20ab
20.83 ± 1.16a 14.33 ± 0.88b
52.27 ± 6.81a 42.10 ± 5.47a
66.00 ± 9.56a 32.33 ± 5.13b
5.83 ± 0.04a 4.31 ± 0.12b
14.00 ± 0.57b
14.00 ± 0.86b
43.13 ± 8.02a
36.16 ± 6.49b
4.81 ± 0.27b
b
E+ (A. flavus)
32.16 ± 1.14
E+ (A. niger)
49.26 ± 2.89a
Parasitization (%) Fecundity (no. of eggs per No. of parasitoid female) eggs per host
Mean ± SE. Means followed by different letters within a column are significantly different. For abbreviations, see Table 1
like B. hebetor, which emerge with a limited number of eggs and egg production and maturation continue throughout the life of the female (Jervis and Kidd 1986; Godfray 1994). Nutritional demands associated with egg production are obtained through host feeding by adult females. The adverse effects of E+ plants on the longevity of female wasps subsequently resulted in reduced fecundity and egg hatchability. Our results are in agreement with previous studies on aphids where negative effects were found on endophyte-naive Aphidius ervi (Haliday) females (Harri et al. 2008). Although E+ plants did not influence the parasitism rate of female wasps, they negatively affected their longevity and fecundity. The E+ host did not influence the hatching of parasitoid eggs; however, the survival rate of larvae reduced significantly on both the endophytes. Because the insect parasitoids complete their development on a single host, the development of parasitoids is significantly influenced by the diet of their host. This may affect the developing parasitoid indirectly by altering host suitability or directly by exposing the parasitoid to unmetabolized defensive chemicals (Lampert et al. 2008), which the herbivorous insects may accumulate within their hemolymph for their own defense (Barbosa et al. 1991, Francis et al. 2001). Feeding on E+ plants thus may have rendered the S. litura larvae nutritionally less suitable for the development of the parasitoid. The larval, pupal, as well as the total development period of B. hebetor extended significantly on E+ hosts as compared to E− hosts. The toxicity of the E+ hosts also manifested as reduced larval survival and adult emergence. Barker and Addison (1996) documented that Acremonium lolii-infected plants reduced the nutritional quality of the weevil Listronotus bonariensis (Kuschel), resulting in retarded development of Microctonus hyperodae (Loan) larvae. Contrary to this, Bultman et al. (1997) reported that Euplectrus comstockii (Howard) developed faster when its host fall armyworm, Spodoptera frugiperda (J. E. Smith), was reared on E+ plants as compared to E− plants. The impact of the endophyte on the sex ratio of the parasitoid was observed only in the case of A. flavus, where significantly fewer females emerged as compared to on E− hosts. The reproductive potential of these first-generation parasitoid adults was adversely affected by the endophytes.
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Although the parasitism rate of the female parasitoid did not differ between E+ and E− hosts, they laid significantly fewer eggs per host. A significant reduction was also observed in overall fecundity rate due to endophytes. The decreased number of eggs on the E+ host may be due to the ability of the parasitoid to assess the suitability of the host by using chemical cues (Fisher 1971). There are some reports indicating that parasitoids developing on hosts fed on a diet having a higher concentration of plant allelochemicals produced smaller primary clutch sizes than those developed on hosts fed on a diet with a lower concentration of allelochemicals (Lampert et al. 2008; Kaur and Kaur 2013). Toxic effects of fungal endophytes were also observed on the hatching of eggs, which significantly decreased in number. Coudron et al. (2009) documented a significant interaction between the host and the parasitoid egg prior to hatching, as they recorded a reduction in the hatching of eggs of E. comstockii due to a depletion of ascorbic acid from the diet of its larval host Heliothis virescens (Fabricius). It is a kind of host immunity to ectoparasitoids, a phenomenon well documented in endoparasitoids (Schmidt et al. 2001), that may have resulted in this reduced hatching of eggs. B. hebetor suffered greatly from endophyte infection in almost all the life history parameters we measured. Our results are in agreement with previous studies on parasitoids where mainly negative effects were recorded (Barker and Addison 1996; Bultman et al. 1997, 2003; de Sassi et al. 2006). Similar detrimental effects of endophytes on the reproductive ability of the aphid parasitoid A. ervi and coccinellid predator have been demonstrated (Harri et al. 2008; de Sassi et al. 2006). This reduction in reproductive success could have been due to parasitoid exposure during larval development to the fungal toxin present in the host insect that weakened the parasitoid females, which laid fewer eggs with reduced viability. Contrary to this, Krauss et al. (2007) did not find a negative effect of endophyteinfected plants on aphids. Although we have not investigated the mechanism by which the endophytes A. flavus and A. niger influenced the parasitoid B. hebetor, our results suggest that secondary metabolites produced by the endophytic fungi or due to the interaction of the plants with fungi, may play a role in mediating the effects we found on
Oecologia (2015) 179:487–494
parasitoid performance. Moreover, the negative effects of endophytes on generalist parasitoids like B. hebetor, may have greater impacts on the entire community because they are more strongly affected by the plant defensive chemistry than specialist parasitoids (Harvey et al. 2003; Harvey 2005; Barbosa et al. 1991; Sznajder and Harvey 2003). The net effect of endophyte-produced toxins on plants will depend upon their relative impact on herbivores and their natural enemies. However, when natural enemies of insect herbivores are negatively affected by endophytic fungi, the benefits of protection by fungal endophytes may be diminished, as indicated by our results. Further studies based on field experiments that simultaneously quantify the effects of endophytes on an insect and its parasitoid, and relate those effects to plant performance, are needed to assess the overall impact of endophytes on plants. This information will help us to understand the ways in which plant symbionts can modify multitrophic interactions. Author contribution statement S. K. and A. K. conceived and designed the experiments. T. K. and B. S. performed the experiments, analyzed the data and prepared the manuscript with the help of S. K. and A. K. Acknowledgments Financial assistance was received from the University Grants Commission, New Delhi, India, under the University with Potential for Excellence scheme, which is duly acknowledged.
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