Nondetrimental impact of two concomitant entomopathogenic fungi on life history parameters of a generalist predator, Coccinella septempunctata (Coleoptera: Coccinellidae)

The non-persistent impact of biocontrol agents can be revealed for pest control when associated entomopathogenic fungi (EPFs) negatively affect the natural enemies. In this assay, impacts of Beauvaria bassiana (Balsamo) Vuillemin, and Metarhizium anisopliae (Metschnikoff) Sorokin were studied for their compatibility or side effects on life table parameters of an important generalist predator, Coccinella septempunctata L. The results indicated non-significant impacts of both EPFs on life table parameters of C. septempunctata. The development time (egg-adult) was not significantly different in control (69.79 days) and EPFs treated C. septempunctata (69.35–80.07 days). Both fungi did not induce any significant changes in the fecundity, adult pre-oviposition period (APOP), total preoviposition period (TPOP), and mean generation time (T) as compared to control treatment. Similarly, no difference in fecundity rate of C. septempunctata was observed after EPFs treatment (287.7–288.5) compared to control (290.0). The highest net reproductive rate (R0) occurred in control (87.05 offspring individual−1) and M. anisopliae (86.31 offspring individual−1) as compared to B. bassiana treated beetles (76.97 offspring individual−1). The age-specific fecundity curves indicated that the C. septempunctata had a similar fecundity rate in both EPFs treatments and control. This study demonstrates no significant side effects of B. bassiana and M. anispoliae on the performance and biology of C. septempunctata. Considering the compatibility of both EPFs with C. septempunctata, their combinations can be recommended in various integrated pest management programs.

Entomopathogenic fungi. Both entomopathogenic fungi, B. bassiana and M. anisopliae were obtained from AgriLife SOM Phytopharma (India) Limited (http:// www. agril ife. in) in talc form and were tested at 1 × 10 8 CFU/ml recommended by the manufacturer to control various insect pests. Both fungi have been reported effective in controlling insect pests such as Diaphorina citri Kuwayama (Hem., Liviidae) 45 , Cnaphalocrocis medinalis (Guenee) (Lep., Pyralidae) 21 , and Nilaparvata Lugens (Stål) (Hom., Delphacidae) 22 . The quality of conidia was determined by counting the conidial concentration in a Neubauer chamber, hemacytometer. The germination of conidia was determined on Potato dextrose agar (PDA) based on the counts of 200 random conidia per plate, 18 h post-incubation at 25 ± 2 °C 46 , and the suspensions with 90% conidial germination were used in the bioassay.

Life table study.
To study the pre-imaginal development and survival, 60 eggs of beetles were obtained from the reared culture for each treatment (2 EPFs and control) and placed separately in clean Petri plates. The egg incubation period was recorded at 12 h intervals. Newly molted 2nd instar larvae of coccinellid beetle were treated with EPFs and distilled water was used in the control group. Sprayed larvae were shifted into new glass Petri dishes, and were kept without food for 3 h to avoid EPFs ingestion through food material. Data for the duration of each developmental stage from larvae to adults were recorded at 12 h intervals. Adults were selected from the corresponding treatment with the immature stages and were sexed to record the longevity and fecundity rate till the death of adults. The adult pairs were kept in a clean plastic jars and provided aphids daily. The Life table analysis. Using raw data, the stage mean, age-stage-specific survival rate (s xj ), age-stage reproductive value (v xj ), age-stage-specific fecundity (f xj ), age-stage life expectancy (e xj ), age-specific survival rate (l x ), age-specific fecundity (m x ), age-specific net maternity (l x m x ), and life table parameters (R 0 , net reproductive rate; r, intrinsic rate of increase; λ, finite rate of increase; and T, mean generation time) were calculated using TWO-SEX-MSChart program 47 . The quick paired bootstrapping (Paired 1 by 1) technique with 100,000 replications was used to minimize the variation in the results for calculating the mean and standard error of the population 48 by TWOSEX-MSChart program 47 . The statistical significance of the observed differences between three treatments was evaluated by TWOSEX-MSChart software. The age-specific survival rate (l x , m x, and R 0 ) was calculated as: where k denotes the number of stages, x = age in days, j = stage, R 0 (net reproductive rate) is the average number of offspring per female during its whole life cycle. The intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T) is calculated as: The life expectancy (e xj ) is referred to as the expected life of an individual of age x and stage j is calculated by the equation suggested by Chi and Su 44 : where S ′ iy is the probability that individuals of age x and stage j will survive to age i and stage y, and is calculated by assuming S xj = 1.
The reproductive value (v xj ) was calculated by the equation suggested by Tuan et al. 49 : The projection of the population growth of C. septumpunctata was calculated through Timing-MSChart 47 . The population growth was simulated for an initial population of 10 eggs over a period of 60 days.

Results
Entomopathogenic fungi lasted no significant impact on the duration of different life stages of the coccinellid beetle when compared to the control group (P > 0.05, Supplementary Table S1). Coccinella septumpunctata larvae infected with the two fungi (B. basiana and M. anisopilae) possessed the same larval duration (4.25-4.66 d and 4.21-4.61 d, respectively) and no significant difference (P > 0.05) was noticed with those of the control (4.25-4.68 d). Similarly, the pupal period was recorded as 5.18 and 5.08 d when larvae were infected with B. basiana and M. anisopilae, respectively as compared to the control group (5.17 d). Adults who emerged from EPFs-infected immature stages survived for 40.2 to 41.2 d. while adults' longevity from the control group was 40.6 d. Female adults lived more days than males in both EPF treatment and control (Table 1).
Population parameters (R 0 , T, r, λ) of C. septumpunctata recorded in treated and control groups are displayed in Table 2. Not only APOP, TPOP, and oviposition period remained unaffected by the application of EPFs, fecundity rate of coccinellid beetle also remained unchanged (P > 0.05, Supplementary Table S2) in EFPtreated (287.7-288.5 eggs) and control group (290.0 eggs). Net reproductive rate (R 0 ), was significantly affected (P < 0.05) by B. basiana (76.97 offspring/individual), while no significant difference (P = 0.6791) was recorded in M. anisopilae treated (86.31 offspring/individual) and control group (87.05 offspring/individual). In comparison www.nature.com/scientificreports/ to control treatment, no significant difference (P > 0.05) was recorded in mean generation time (T), intrinsic rate of increase (r), and finite rate of increase (λ) when larvae were infected with EPFs ( Table 2) (Fig. 2). The highest age-stage specific reproductive rate (v xj ) value for females was recorded as 92.07 at 43rd day in control, 88.68 at 42nd day in B. bassiana, and 91.67 at 43rd day in M. anisopliae treatments (Fig. 3). The l x , f xj , and m x curves indicated that the coccinellid beetle had a similar survival rate and fecundity in both EPFs treatments and control. The f xj curve indicates age-stage-specific female fecundity of a female and the following trend of maximum egg-laying was found; 15.67 eggs at the age-stage of 52nd day in control, 15.66 eggs at the age-stage of 55th day in M. anisopliae, and 14.84 egg at the age-stage of 52nd day in B. bassiana (Fig. 4). The population projection reveals the projected growth of an insect over a specific period under the same conditions. In this analysis, we have calculated the population of C. septempunctata for 60 days. The initials size of the population was recorded 10 eggs for each treatment. The highest population size of beetle (573.43 individuals) was in the control group followed by 553.21 individuals in M. anisopliae treatment. The total population size in B. bassiana treatment was 510.46 individuals (Fig. 5).

Discussion
Interaction between natural enemy species of an organism present in the field is one of the most important factors to determine the biological control programs 50

. Various studies have been performed on interactions between
EPFs and other biological control agents (parasitoids and predators) 34,51,52 ; however, little information is available about the effects of EPFs on the demography of coccinellid species. Even though the EPFs and coccinellids may occupy the same habitat, it is important to understand their interspecific interactions during coexistence.
In the present study, we investigated the biological parameters of C. septempunctata beetles infected with two EPFs (B. basiana and M. anisopilae). The effects were shown through surface contact of EPFs to newly emerged larvae of the generalist predator. Such information could be useful to assess the compatibility of this coccinellid beetle with EPFs in IPM programs of aphid species. Using EPFs in pest control programs, their selection is very important to obtain effective control of agricultural pests 53 . The compatibility of EPFs with other biocontrol agents could be useful to achieve higher pest control by decreasing the application of synthetic insecticides and minimizing the resistance to insecticide 54 . Our findings showed that the life span of the coccinellid beetle remained unaffected when infected with both EPFs, as no significant difference was noticed in the developmental period of each larval stage, pupa, and adult beetles when compared to control treatment. Both fungi were found to have a negligible threat on this coccinellid beetle and hence their recommended concentrations are suggested as compatible with coccinellid predators. www.nature.com/scientificreports/ In general, the safety of EPFs to non-target organisms has been reported earlier 55,56 . Zaki 57 and Ormond et al. 52 documented that B. bassiana did not affect the developmental period of Coccinella spp. The larval mortality was noticed in both the treatments as well as control in the reported experiment, however, it may be attributed to natural death because all grubs of beetle did not reach adulthood. However, EPFs might have a negative effect on newly emerged larvae of predators. As reported by Sayed et al. 58 , the first larval instar of C. undecimpunctata was affected negatively by B. bassiana. Our findings showed a maximum age stage-specific survival rate (s xj ) in control and B. bassiana than M. anisopliae treatment. The higher mortality in M. anisopliae may be attributed to its higher pathogenicity as compared to B. bassiana. The lower net reproductive rate in M. anisopliae treated ladybird beetle may also be attributed to the pathogenicity factor as fewer numbers of larvae were able to reach adulthood as compared to B. bassiana and control. However, no significant difference was found in the values of e xj and v xj in the EPFs-infected population and control group. Similarly, l x , f xj , and m x curves also directed similar survival rates and fecundity of beetle in both EPFs treatments and control. Our findings suggest that EPFs do not affect the biological parameters of the ladybird beetle, C. septempunctata. It could be the ability of coccinellid to detect and avoid the EPFs conidia in terms of adaptation that enhances the survival rate and ultimately their fitness. The non-target effect  However, laboratory-reared insects are more susceptible to infection by pathogens 64 . To explain the low incidence of EPFs infecting coccinellids under natural field conditions, the behavioral responses of the predators should be taken into consideration. According to Ormond et al. 52 both male and female adult C. septempunctata avoid B. bassiana through contact with leaf surfaces and soil inoculations. Combined use of endophytic EPFs and entomophagous insects described low risks for predators and parasitoids in aphid IPM programs 65 . Use of M. anisopliae with Nabis pseudoferus (Hem., Nabidae) was reported a useful combination in controlling tomato   40 . Our findings also demonstrated that the projection populations of coccinellid beetle were almost consistent in control and EPFs treatment. The projection of insect population growth using life table data is a vital tool in pest management and decision-making. Based on the findings of this study, it is suggested that EPFs are eco-friendly and don't have any negative effect on coccinellid predators, the environment, and human health. Thus, these EPFs can be recommended to farmers to control aphids as an alternative to synthetic insecticides.

Conclusion
The application of B. bassiana and M. anisopliae did not affect the generalist predator, C. septempunctata. Biological control agents and microbial pesticides are two such tools that can be used simultaneously due to their effectiveness, less risk to the environment and human health, and especially their potential compatibility. Our results indicate that commercial formulations of both EPFs are safer for coccinellid predators, suggesting these EPFs as compatible with other biological control agents (predator) and can be used in integrated pest management programs.

Data availability
The datasets generated during and/or analysed during the current study are available from thefirst author on reasonable request. www.nature.com/scientificreports/