Effects of single and combined applications of entomopathogenic fungi and nematodes against Rhynchophorus ferrugineus (Olivier)

This study was carried out to investigate the insecticidal properties of Beauveria bassiana, Metarhizium anisopliae and Heterorhabditis bacteriophora for their virulence against different larval instars of Rhynchophorus ferrugineus (Olivier). Both fungi were either applied alone or in combination, with H. bacteriophora simultaneously or 1 and 2 weeks after fungal application; EPN were also applied alone. Moreover, assessment of host development, diet consumption, frass production and weight gain were observed at sub-lethal dose rates. In combined treatments, additive and synergistic interactions were observed. Synergism was observed more frequently in H. bacteriophora + B. bassiana combinations than in H. bacteriophora + M. anisopliae combinations, and was higher in early instars than old instars. In 2nd and 4th instars, synergy was noted in H. bacteriophora + B. bassiana combinations at 0, 7 and 14 d intervals and in 6th instar synergy was observed only in H. bacteriophora + B. bassiana combinations (at 0 and 7 d intervals). A decrease in pupation, adult emergence and egg hatching was enhanced in the combined treatments. Furthermore, reduced weights and variation in duration of insect developmental stages were observed among entomopathogens and enhanced in H. bacteriophora + B. bassiana combinations. Larvae treated with sub-lethal concentrations exhibited reductions in food consumption, growth and frass production and weight gain.


Results
Entomopathogenic fungi and nematodes interaction. In integrated application of H. bacteriophora with B. bassiana or M. anisopliae, additive and synergistic interactions were observed in all the three instars tested when the microbial agents were applied simultaneously or following delayed nematode application (Tables 1, 2 and 3). Antagonism was not observed in any of the combinations tested (Tables 1, 2 and 3). During simultaneous application, 2 nd instar larvae exhibited additive effects for B. bassiana and H. bacteriophora for the first two weeks, while synergistic interaction was observed at the third week of application. The degree of synergism increased with the delayed application of H. bacteriophora one or two weeks after B. bassiana treatments. For M. anisopliae additive effects were recorded for simultaneous application, while interactions were shifted towards synergism when delayed nematode application was made after one and two weeks of fungal spore application (Table 1). A similar trend was recorded for 4 th and 6 th instar larvae but susceptibility was lower among treatments as compared with the 2 nd instar larvae, and the instances of synergy also decreased as instar size increased (Tables 1, 2 and 3). Also, synergy was observed in H. bacteriophora and B. bassiana combinations more than in H. bacteriophora and M. anisopliae combinations (Tables 1, 2 and 3). In a factorial analysis the main effects for pupation adult emergence and egg eclosion were significant while their interaction effects were non-significant except pupation (Table 4). A decrease in pupation, adult emergence and egg hatching was caused by all treatments and the effects were enhanced in the combined treatments (particularly with H. bacteriophora + B. bassiana) ( Table 5). Development of R. ferrugineus: larvae through adult stages. Growth and development of RPW (larvae to adult stage) was adversely affected by the microbial agents. When larvae were exposed to the sub-lethal doses of H. bacteriophora, B. bassiana and M. anisopliae, significant variations were recorded for larval duration, larval weight, pre-pupal duration, pre-pupal weight, pupal duration, pupal weight, adult longevity and adult weight (larval duration: F 5, 53 = 9.92, P <0.01; larval weight F 5, 53 = 27.3, P <0.01; pre-pupal duration: F 5, 53 = 6.59, P <0.01; pre-pupal weight: F 5, 53 = 6.94, P<0.01; pupal duration F 5, 53 = 5.15, P < 0.01; pupal weight F 5, 53 = 11.10, P <0.01; adult longevity (female F 5, 53 = 3.93, P < 0.01 and male F 5, 53 = 5.58, P < 0.01); adult weight (female F 5, 53 = 4.26, P < 0.01 and male F 5, 53 = 12.7, P < 0.01). For the most part, increases in larval, pre-pupal and pupal duration occurred while decreases in weight among stages were observed for all the treatments tested. On the other hand a decrease in adult life span and weight (male and female) was also recorded. The numerically highest detrimental effect on growth tended to be for combined application of B. bassiana and H. bacteriophora (e.g., see larval and adult weights) followed by M. anisopliae and H. bacteriophora, H. bacteriophora alone, B. bassiana and M. anisopliae (Table 6).  (Fig. 1). Similarly frass production was influenced by treatments applied (F 3, 69 = 386.22, P <0.01), with the lowest frass production for the combined treatment of H. bacteriophora and B. bassiana (0.57 ± 0.04 to 0 ± 0.00 g) and highest in the control (the single-applied treatments were intermediate and not different from each other) (Fig. 2). Untreated larvae (control) and larvae treated with sub-lethal concentrations of B. bassiana and H. bacteriophora gained more weight as compared to their combined application (F 3, 24 = 56.77, P <0.01) (Fig. 3); the single applied treatments were not different from the control whereas the combination treatment was statistically separated from all three others.

Discussion
This is the first study to investigate the combined effects of fungal isolates and H. bacteriophora against larvae of RPW. The results revealed that the microbial agents applied alone displayed pathogenicity. However, when fungi and nematodes were combined (either applied simultaneously or delayed application of H. bacteriophora), the level of virulence was enhanced in an additive or synergistic fashion. Synergistic levels of virulence were observed more frequently in H. bacteriophora + B. bassiana combinations than in H. bacteriophora and M. anisopliae combinations. Additionally, synergy appeared to be more prevalent in the younger instars tested than the later instars, and our findings are similar to the findings of Ansari et al. 48 In prior research, synergistic interactions were also observed between entomopathogenic nematodes and fungi. For example 51 laboratory trials were conducted against Otiorhynchus sulcatus (the black vine weevil) using a combination of M. anisopliae and EPN. Synergy occurred with either H. bacteriophora or S. kraussei and mortality with S. feltiae was recorded as additive. They also performed greenhouse trials with the combination of these agents applied at various concentrations. The combination of M. anisopliae and H. bacteriophora resulted in synergistic interactions in all treatments. The combinations of M. anisopliae and S. feltiae only resulted in synergy when a high concentration of fungus was used with a low concentration of nematodes; all other interactions were additive 51 . When overwintering black vine weevils in greenhouses were exposed to M. anisopliae and S. kraussei the interaction recorded was synergistic in the first trial, but when the trial was repeated results were additive 52 .  This shows that the combination of these pathogens may not be consistent. It was hypothesised that the inconsistent results may be due to the sensitivity of the fungus to temperature 52 .
We speculate that the longer grubs are exposed to the fungus, the more debilitated they become and subsequently are more susceptible to the EPN. It is also conceivable that the debilitated insects respire more, attracting the EPNs, which follow a CO 2 gradient to their hosts 51 . It is also suggested that the stressed insects are more vulnerable to pathogen infection, which thus enhances insect mortality or facilitates a higher speed of kill leading to synergistic effects in combined treatments 53 . For example, Paenibacillus popilliae (Dutky) applied against scarab larvae acted as a stressor to nematode infection which caused elevated larval mortality 34,35 . Similarly entomopathogenic fungi have been shown to alter the behavior of the host insect, reducing locomotion, feeding and increasing irritability 54 . Grubs suffering from a fungal infection may not be able to feed or utilize food normally. This could adversely affect morphological, behavioral and physiological mechanism that grubs have evolved in order to defend against natural enemies in the soil 55 , thereby making the grub more susceptible to nematode penetration. Synergistic interactions between fungi and nematodes have been reported by many researchers [34][35][36][37][56][57][58] . Contrarily 59 found antagonism between EPNs and Isaria fumosorosea (Wize) when virulence to pecan weevil, Curculio caryae (Horn). Also, in contrast to this study 59 , also observed antagonism between certain combinations of B. bassiana and EPN, Steinernema carpocapsae (Weiser) or Heterorhabditis indica Poinar, Karunakar & David, and antagonism in combinations of S. carpocapsae and M. anisopliae. These discrepancies emphasize that synergistic interactions depend heavily on the host target as well as the pathogens that are being combined, and timing of applications (simultaneous versus sequential).
Growth and development are required for successful completion of the insect life-cycle and reproduction. Any delay may render the insect susceptible to biotic and abiotic factors (such as natural enemies or environmental regimes) that ultimately limit their growth and development. In this regard larval stages are vulnerable towards such phenomena 60 . In our study, treatments caused reduced food consumption, reduced longevity of the adult stage, and reduced weights in life-stages, which thus affected the insect's fecundity and survival into the next generation.
The present study showed that B. bassiana and M. anisopliae isolates in integration with H. bacteriophora under laboratory conditions caused high mortality against larvae of the red palm weevil. The pathogens exerted detrimental effects on survival, growth and development of different developmental stages of R. ferrugineus. Hence, integrated application of H. bacteriophora in sequential manners with B. bassiana and M. anisopliae might be effectively used for the successful control of red palm weevil. This approach (of combined applications) promises to provide superior efficacy than previous attempts to control the insect pest with singly applied treatments but further research is needed (e.g. under field conditions) for confirmation of the method's success in date palm orchards.

Materials and Methods
RPW collection and rearing. A survey was conducted for collection of R. ferrugineus in date palm growing areas of west Punjab, Pakistan. Different developmental stages (larvae, pupae and adults) were collected from fallen and infested date palm trees with the permission of farmers (owners). All the stages collected were kept separately in plastic jars until brought to Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad (UAF), Pakistan. Larvae were fed with sets of sugarcane (Saccharum officinarum L.; Poales: Poaceae) and pupation occurred within the stalks, while shredded sugarcane pieces were offered to adults for feeding and as a substrate for oviposition. After pupation, pupal cocoons were kept in separate plastic jars for adult emergence. After adult emergence beetles were shifted to the adult's jar for feeding, mating and oviposition. The colony was developed in plastic boxes (30 × 60 × 60 cm) having a lid covered with mesh wire gauze (60 mesh size, 10 cm diameter) in the middle for aeration. The adult's diet was changed every three days, and used sugarcane pieces were kept in separate jars (8 × 8 × 12 cm) for egg hatching. After egg hatching neonate larvae were allowed to feed for some time in the same set, after 3 days larvae were transferred to new sugarcane sets for feeding and pupation. Larvae were shifted to the new sugarcane sets after every week until pupation. The rearing conditions were maintained at 25 ± 2 °C, 65 ± 5% RH and a 12:12 (D: L) hour photoperiod. Entomopathogenic fungi. Two isolates of entomopathogenic fungi B. bassiana (WG-11) and M. anisopliae (WG-02) used in the study were obtained from the culture collection of the Microbial Control Laboratory; the strains were originally isolated from soils of vegetables and crop fields, respectively. Culturing of the fungi was accomplished by inoculating Petri plates containing Potato Dextrose Agar (PDA) media (BD, France) 62 . Spore concentration of 1 × 10 6 spore ml −1 was determined with a Neubauer haemocytometer.
Treatment with entomopathogenic fungi. The larvae (2 nd , 4 th and 6 th instars) were directly immersed in 100 ml conidial suspension for 60 s individually and both treatments and the control were applied in an aqueous solution of 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) 63 ; control larvae were dipped into a suspension of 0.01% Tween-80 (without fungal spores). The treated and control larvae were then individually moved to 150 ml cylindrical plastic cups, each measuring 6 cm in height and 6 cm diameter. The top of the cups were covered with a fine mesh in order to prevent the insects from escaping. A piece of 2 × 2 cm 2 artificial diet (Agar, brewer's yeast, wheat germ, corn flour, ascorbic acid, benzoic acid, amino acid-vitamin mix, chloramphenicol and nipagin) 64 was kept in the center of each cup. The cups were kept at 27 ± 2 °C, 65 ± 5% RH and a 12:12 (D:L) hour photoperiod in an incubator (Sanyo, Japan). Three replicates of 10 larvae were treated to the fungal suspensions or control. Each cup was opened daily and checked for mortality, and the old diet was replaced with fresh artificial diet until insect mortality or pupation was observed. After the insect reached the last instar stage, dry coir (coconut) was provided to the surviving larvae for pupation. The entire bioassay was repeated thrice.
Treatment with H. bacteriophora. Nematode suspensions were prepared with concentrations of 100 IJs ml −1 in glass jars and 1 ml of suspension was poured into the cylindrical plastic cups lined with Whatman filter paper (described above). After pouring 30 minutes allowing the nematodes to distribute evenly on the filter paper, a small piece of artificial diet 2 × 2 cm 2 was placed in the middle of the cups as a food source. Ten larvae for each treatment were used individually in each cup and each treatment was replicated three times, while control treatment received 1 ml of distilled water (without nematodes). The cups were maintained at above mentioned environmental conditions. The cups were opened daily to note mortality until insect mortality or pupation was observed. After the insect reached the last instar stage, dry coir was provided to the surviving larvae for pupation. The entire bioassay was repeated thrice.

Treatment with entomopathogenic fungi and nematodes. The fungi (B. bassiana or M. anisopliae)
were tested in combination with H. bacteriophora; combinations of the two fungi together (B. bassiana plus M. anisopliae) were not tested. In combined treatments fungi and nematodes were applied simultaneously or at different time intervals as follows: • B. bassiana or M. anisopliae plus H. bacteriophora were applied simultaneously: larvae were immersed in fungal suspensions and transferred to the cylindrical plastic cups lined with moistened filter paper treated with H. bacteriophora IJs, and maintained at the conditions described above. • Insects were first inoculated with B. bassiana or M. anisopliae, maintained at 27 ± 2 °C and 65 ± 5% RH for one week, then transferred to cylindrical plastic cups lined with moistened filter paper treated with H. bacteriophora IJs, and maintained at above mentioned conditions. • Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27 ± 2 °C and 65 ± 5% RH for two weeks, transferred to cylindrical plastic cups lined with moistened filter paper treated with H. bacteriophora IJs, and maintained at above mentioned conditions. • Control insects were immersed in aqueous solution with 0.01% Tween-80 and maintained in cylindrical plastic cups lined with moistened filter paper using conditions stated above.
Larval mortality was recorded after one, two and three weeks post application. For all treatments, artificial diet was offered to the larvae as food source. Larvae that failed to respond on slight prodding by a blunt needle were considered dead. After the insect reached the last instar stage, dry coir was provided to the surviving larvae for pupation. Percent pupation, adult emergence and egg eclosion were also recorded.
Effects of entomopathogens on R. ferrugineus development. The effects of sub-lethal entomopathogen concentrations on development of RPW was assessed. Fourth instars were exposed to the sub-lethal dose of fungal entomopathogens (1 × 10 4 conidia ml −1 ) and H. bacteriophora (50 IJs ml −1 ). The sub-lethal dosages were determined through preliminary experimentation (unpublished data). The larvae were fed on artificial diet and transferred to the treatment cups. Dry coir was provided to the each larva before pupation for cocoon formation. Adult insects, upon emergence, were offered shredded sugarcane pieces. Developmental parameters of each stage were recorded including larval duration, larval weight, pre-pupal duration, pre-pupal weight, pupal duration, pupal weight, adult longevity (male and female) and adult weight (male and female).
Effects of entomopathogens on larval development. An additional assessment was made on the impact of sub-lethal concentrations on the development of last instar RPW; this experiment only involved B. bassiana and H. bacteriophora (because these pathogens were found to be the most compatible in terms of synergistic interactions, see Results section). Last instar RPW were exposed to sub-lethal doses of B. bassiana (1 × 10 4 conidia ml −1 ) and H. bacteriophora (50 IJs ml −1 ). Before exposure in all the treatments larvae were weighed first and transferred to the rearing cups with artificial diet. Larvae continued to feed until they pupated; the insects were maintained under experimental conditions at 25 ± 2 °C, 65 ± 5% RH and a (12: 12) L: D hour photoperiod. Every day until the larvae pupated in the control (14 d), larvae were changed to a new clean cup and a new piece of artificial diet was offered. Frass produced during this period was separated from vials using a fine camel-hair brush, and weighed. Diet left unused in each vial was recovered, oven dried at 80 °C, and weighed. Prior to the assay, diet in fifteen vials was dried to obtain an estimate of the dry weight. Diet consumption of each larva was thus determined by subtracting the mass after feeding from before feeding estimate. Cumulative weight gain of larvae was also determined. Three replicates of ten insects were used for each treatment and same count of larvae fed on normal diet served as untreated check. The entire experiment was repeated thrice.
Statistical analysis. The fungus-nematode interactions (synergistic, additive or antagonistic) were based on a comparison of observed versus expected values of insect mortality 59 . Expected mortality was calculated using formula P E = P 0 + (1 − P 0 ) (P 1 ) + (1 − P 0 ) (1 − P 1 ) (P 2 ), where P E is the expected mortality of the combination, P 0 is the control mortality, P 1 is the mortality from one pathogen treatment applied alone, and P 2 is the mortality from the other pathogen applied alone. A X 2 test was applied to the observed and expected results: X 2 = (L 0 − L E ) 2 /L E + (D 0 − D E ) 2 /D E , where L 0 is the number of living larvae observed, L E the number of living larvae expected, D 0 the number of dead larvae observed, and D E the number of dead larvae expected. Interactions were additive if X 2 < 3.84, antagonistic if X 2 > 3.84 and P C < P E , and synergistic if X 2 > 3.84 and P C > P E , where P C is the observed mortality from the combination and P E is the expected mortality from the combination. Data for pupation, adult emergence, egg eclosion and developmental parameters were subjected to one way analysis of variance (ANOVA) in Minitab 65 ; means were separated using Tukey's Kramer test (HSD) 66 at a 5% significance level. To inspect the impact of microbial agents on the diet consumption, cumulative weight gain and frass production, data were analyzed by repeated measures (Proc Glimmix); based on residual plots data that were log transformed (back-transformed means are presented in the associated figures).
Ethical approval. This article does not contain any studies with human participants performed by any of the authors.