ARTICLE IN PRESS Journal of Invertebrate Pathology xxx (2010) xxx–xxx Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus Óscar Dembilio a, Enrique Quesada-Moraga c, Cándido Santiago-Álvarez c, Josep A. Jacas b,* a Institut Valencià d’Investigacions Agràries (IVIA), Unitat Associada d’Entomologia Agrícola, Universitat Jaume I (UJI), Deptartament de Protecció Vegetal i Biotecnologia, Ctra Montcada-Nàquera km 4.5, E-46113 Montcada, Spain Universitat Jaume I (UJI), Unitat Associada d’Entomologia Agrícola, Institut Valencià d’Investigacions Agràries (IVIA), Departament de Ciències Agràries i del Medi Natural, Campus del Riu Sec, E-12071 Castelló de la Plana, Spain c Universidad de Córdoba, Departamento de Ciencias y Recursos Agrícolas y Forestales, ETSIAM, E-14071 Córdoba, Spain b a r t i c l e i n f o Article history: Received 22 February 2010 Accepted 10 April 2010 Available online xxxx Keywords: Beauveria bassiana Rhynchophorus ferrugineus Palmae Curculionidae Hypocreales Pathogenicity Virulence Horizontal transmission Efficacy Entomopathogenic fungi a b s t r a c t The potential of a strain of Beauveria bassiana (Ascomycota: Clavicipitaceae) obtained from a naturally infected Rhynchophorus ferrugineus (Coleoptera: Curculionidae) pupa as a biological control agent against this weevil was evaluated both in the laboratory and in semi-field assays. Laboratory results indicate that this strain of B. bassiana can infect eggs, larvae and adults of R. ferrugineus (LC50 from 6.3  107 to 3.0  109 conidia per ml). However, mortality was not the only indicator of treatment efficacy because adults of either sex inoculated with the fungus efficiently transmitted the disease to untreated adults of the opposite sex, with male-to-female and female-to-male rates of transmission of 55% and 60%, respectively. In addition, treatment with B. bassiana significantly reduced fecundity (up to 62.6%) and egg hatching (32.8%) in pairing combinations with fungus-challenged males, females or both sexes. Likewise, 30–35% increase in larval mortality was observed in larvae obtained from eggs from funguschallenged females or from untreated females coupled with inoculated males, resulting in an overall 78% progeny reduction. Semi-field preventive assays on potted 5-year old Phoenix canariensis palms, with efficacies up to 85.7%, confirmed the potential of this strain as a biological control agent against R. ferrugineus. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The invasive red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), has become the major pest of palms in the Mediterranean Basin, where it spread slowly during the mid 1990s and very quickly during the last five years. This pest is at present widely distributed in Oceania, Asia, Africa and Europe (EPPO, 2008) and was found in Curaçao, Netherlands Antilles, in December 2008 (EPPO, 2009). Females lay eggs at the base of the fronds in separate holes made with their rostrum. Neonate larvae bore into the palm core and upon completion of development move back to the base of the fronds to pupate. A new generation emerges and adults may remain within the same host and reproduce until the palm eventually dies. Subsequently, adults move to new hosts. R. ferrugineus has been reported on 19 palm species belonging to 15 different genera (EPPO, 2008; Dembilio et al., 2009). Several control methods have been applied against this pest * Corresponding author. Fax: +34 964728216. E-mail address: jacas@camn.uji.es (J.A. Jacas). within an Integrated Pest Management strategy. Its main components are phyto-sanitation, which involves cutting down and burning infested palms, use of insecticides and use of pheromone traps for adult monitoring and mass trapping (Murphy and Briscoe, 1999; Faleiro, 2006). Few studies have been conducted on the natural enemies of R. ferrugineus or other Rhynchophorus species (Murphy and Briscoe, 1999; Faleiro, 2006). Steinernema carpocapsae (Weiser) (Nematoda: Steinernematidae) proved effective against R. ferrugineus in semifield and field trials including both preventive and curative assays (Llácer et al., 2009; Dembilio et al., 2010). Different strains of Metarhizium anisopliae (Metschnikoff) Sokorin (Ascomycota: Clavicipitaceae) and Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Clavicipitaceae) were tested against R. ferrugineus (Gindin et al., 2006). The former proved more virulent than B. bassiana and achieved 100% larval mortality within 6–7 days. However, none of the strains tested was originally obtained from diseased R. ferrugineus specimens. In 2005–2006, El-Sufty et al. (2009) obtained a mortality of 12.8–47.1% in adult R. ferrugineus population in field assays using a strain of B. bassiana isolated in the United Arab 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.04.006 Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS 2 Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx Emirates. More recently, Sewify et al. (2009) successfully reduced the incidence of R. ferrugineus under field conditions in Egypt using a native strain of B. bassiana isolated from a R. ferrugineus cadaver. In 2007, R. ferrugineus pupae presumed to be infected with entomopathogenic fungi were collected in a date palm grove in Spain near the town of Catral (lat.: 38°090 2900 N; long.: 00°460 3300 W; alt.: 12 m). One of these pupae proved to be infected with the entomopathogenic fungus B. bassiana. This species is one of the most widely distributed entomopathogenic fungi in the world and it infects insects in tropical, temperate, humid, and desert areas (Zimmermann, 2007), some of them of agricultural importance, such as the coffee berry borer Hypothenemus hampei Ferrari (De la Rosa et al., 1997) and other Curculionidae (Adane et al., 1996). In these studies adults were the most suitable or the only possible stage for treatment. Unlike other insect pathogens, entomopathogenic fungi infect the host by contact, penetrating the insect cuticle. The host can be infected both by direct treatment and by horizontal transmission from infected insects or cadavers to untreated insects or to subsequent developmental stages via the new generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). Passive mechanical transmission of fungi within insect populations has been observed for various entomopathogenic fungi, e.g. B. bassiana, M. anisopliae and Isaria fumosorosea (Lacey et al., 1999; Quesada-Moraga et al., 2004, 2008). These unique characters make entomopathogenic fungi especially important for the control of concealed insects. In the case of R. ferrugineus, most of its life cycle occurs within the palm, making the pest inaccessible to direct-contact treatments. Adults are the only exposed stage and can be infected upon emergence. Evidence for successful application of fungi via adults was obtained from experiments on the pinhole borer, Platypus spp. (Coleoptera: Curculionidae). Adults that were contaminated with B. bassiana or M. anisopliae spores transferred the fungal infection to larvae, which resulted in 50–100% larval mortality (Glare et al., 2002). The aim of this work was to evaluate in the laboratory both lethal and sublethal effects of an indigenous strain of B. bassiana against different stages of R. ferrugineus and to determine the efficacy of a formulation of this strain in a semi-field trial as a first step to reveal the biocontrol potential of this strain. of conidia was over 95%. Suspensions were kept in 4 °C dark storage before use (Goettel and Inglis, 1997). 2.2. Stock colonies Adult weevils collected in the province of Valencia in traps baited with ferrugineol (the male R. ferrugineus aggregation pheromone) and plant kairomones (ethyl acetate and pieces of palm fronds) were used to start the stock colonies. These colonies were established in 2007 and have been periodically supplemented with the introduction of additional wild specimens. Adult weevils were reared in a controlled environment cabinet at 25 ± 1 °C, 75 ± 5% R.H. and a 16-h light photoperiod in perspex cages (30  30  45 cm depth) with a density of 120–150 weevils per cage. These cages had a round hole (8 cm in diameter) on the upper side covered by a mesh used for manipulation of the specimens and its bottom side consisted of a 2 mm metal mesh used by females for oviposition. Cages were set on top of a tray containing a folded piece of moistened filter paper containing thin apple slices used by female weevils as oviposition substrate and by both males and females as food. Apple slices were replaced three times per week (Dembilio et al., 2009). 2.3. Experimental insects Eggs, less than 24-h old, were obtained from the stock colonies and used in our assays. When necessary, eggs were further kept on apple slices until hatching or reared up until the fourth instar or to adulthood (assays with neonate larvae, fourth instars and laboratory-reared adults, respectively). Upon hatching, neonate larvae were individually transferred to 125 ml vials containing 45–50 ml of the weevil’s artificial diet (Martín and Cabello, 2006). Larvae were moved to a new vial fortnightly until ready for pupation (45-day old larvae). At that moment, larvae were moved to another vial containing strands of dry esparto grass (Stipa tenacissima L.) used by the larvae to build a cocoon. About one month later, laboratory-reared adults were obtained. Adults collected in field-traps as described were also used in our laboratory assays. 2.4. Plant material 2. Materials and methods 2.1. Entomopathogenic fungus The B. bassiana strain used in the experiment was isolated from an infected pupa originally collected in a date palm grove near the town of Catral, Spain, and belongs to the fungal collection of the Departamento de Ciencias y Recursos Agrícolas y Forestales of the University of Córdoba (Spain) with the reference code EABb 07/06-Rf. This strain was deposited with accession No. CECT20752 on May 13, 2009, following the Budapest Treaty, in the Spanish Collection of Culture Types (CECT) at the University of Valencia (Burjassot, Spain). Fungal cultures were grown on malt agar (12.75 g/l malt extract, 2.75 g/l dextrine, 2.35 g/l glycerol, 0.78 g/l gelatine peptone and 15.0 g/l agar) at 25 °C in the dark. Conidial suspensions for experiments were prepared by scraping conidia from 15-day old, well sporulated cultures into an aqueous solution of 0.2% Tween 80. Suspensions were then filtered through several layers of cheesecloth to remove mycelium. To homogenize the inoculum, they were sonicated for 10 min (P-selecta ultrasounds, Barcelona, Spain). Suspensions were prepared independently for each assay and therefore conidial concentrations used were not exactly the same. Conidial concentrations were determined using a hemocytometer. Viable germinating conidia were counted after 36 h of incubation at 25 °C in malt agar. Germination Semi-field assays were performed on 5-year old potted Phoenix canariensis palms obtained from an officially inspected nursery (EU, 2007) and therefore were presumed to be free of R. ferrugineus. The stipe of these palms was 0.35–0.55 m high and 0.30–0.40 m wide. Plants were watered twice a week and kept inside a double mesh security enclosure containing 24 independent cages (4  3  3 m). Palms were kept under natural light and temperature conditions during summer-fall 2009. Mean temperature during the assays was 24.5 °C (max: 42.2 °C; min: 6.3 °C). A plastic roof protected the enclosure from the rain. 2.5. Bioassays R. ferrugineus is quite a difficult and expensive insect to rear in the laboratory. Therefore, the number of insects used in our experiments had to be considered very carefully. Preliminary pathogenicity tests were conducted on both adult and immature stages of R. ferrugienus firstly to perform the Koch postulates with the indigenous strain against all instars and secondly to define the range of dosages to be used in the biological activity assays. Therefore, each stage was tested at least twice before undertaking the laboratory assays described below. Laboratory assays took place in a controlled environment cabinet at 25 ± 1 °C, 75 ± 5% R.H. and a 16-h light photoperiod, whereas semi-field trials took place at Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx the Institut Valencià d’Investigacions Agràries (IVIA) in the double security mesh enclosure described above. 2.5.1. Virulence against eggs Eggs, less than 24-h old, were removed from the apple slices with a fine paintbrush. Replicates consisted of groups of 12 eggs exposed together to conidial suspensions of B. bassiana in a cleaning stainless steel mesh basket (16 mm diam.; 17 mm h.). The basket was immersed for 60 s in an aqueous conidial suspension of either 6.73  106, 6.73  107, 6.73  108 or 6.73  109 conidia/ml or in a control aqueous solution. Both treated and control solutions contained 0.01% Tween 80 (Marannino et al., 2006). Three replicates per concentration were used. Subsequently, eggs were transferred onto a moistened sterile filter paper inside an unsealed Petri dish (2.5 cm diam.). The moistened filter paper prevented egg desiccation. Egg hatching was recorded daily for up to 6 days and, when present, neonate larvae were provided apple slices on alternate days. Dead larvae were individually transferred onto a moistened filter paper in a Petri dish. When external signs of fungal infection on larvae were not observed during the subsequent 24 h, cadavers were surface sterilized with 1% sodium hypochlorite followed by three rinses with sterile distilled water, placed on a sterile moistened filter paper in an individual sterile Petri dish sealed with parafilm and kept at room temperature to ascertain the involvement of B. bassiana in their death. In total, 144 eggs were used in this experiment. Additional B. bassiana-infected eggs were fixed overnight at 4° C with 0.25% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, and then dehydrated in an ethanol series to 100% ethanol. Specimens were critical point dried in a drying unit with CO2 (Balzers CPD-030), coated with gold–palladium (20:80) in a Polaron E5100 sputter coating unit and viewed on a JEOL JSM 6300 SEM at 39 kV. 2.5.2. Virulence against neonate larvae Neonate larvae less than 48-h were processed in groups of 12 as above and exposed to a conidial aqueous suspension of either 3.14  106, 3.14  107, 3.14  108 or 3.14  109 conidia/ml or a control aqueous solution. Both treated and control suspensions contained 0.01% Tween 80. Three replicates of 12 larvae per concentration tested were considered. Mortality was recorded daily for up to 8 days and dead specimens were processed as above. In total, 144 neonate larvae were used in this experiment. 2.5.3. Virulence against fourth instar larvae Fourth instar larvae were directly immersed in groups of 5 for 60 s in a conidial aqueous suspension of either 4.6  106, 4.6  107, 4.6  108 or 4.6  109 conidia/ml or in a control aqueous solution. Both treated and control solutions contained 0.01% Tween 80. Three replicates of 10 larvae per concentration were treated. After treatment, larvae were individually transferred onto a moistened filter paper in a Petri dish (2.5 cm diam.) for 24 h. Then they were further individually transferred to 100 ml rearing vials. Mortality was recorded daily and dead specimens were processed as above. In total, 120 larvae were used in this experiment. 2.5.4. Virulence against laboratory-reared and field-collected adults Laboratory-reared weevils, less than 24–48-h old, were immersed in groups of four for 90 s in a conidial aqueous suspension of either 5.16  106, 5.16  107, 5.16  108 or 5.16  109 conidia/ ml or in a control aqueous solutions. Both control and treated solutions contained 0.02% Tween 80. Adults were transferred to a plastic box (15  10  10 cm) on moistened filter paper and apple slices. Mortality was checked daily. Three replicates of 12 adults per concentration were considered. The same procedure was applied to field-collected adults, which were exposed to either 3 6.73  106, 6.73  107, 6.73  108 or 6.73  109 conidia/ml or to a control solution. In total, 288 adults were used in these experiments. 2.5.5. Horizontal transmission among adults and sublethal reproductive effects Laboratory-reared adults less than 24-h old were immersed individually for 90 s in a conidial aqueous suspension adjusted to a final concentration of 1.5  109 conidia/ml. Both control and treated solutions contained 0.02% Tween 80. Four mating combinations were established: (1) untreated females  untreated males, (2) treated females  untreated males, (3) untreated females  treated males and (4) treated females  treated males. Each pair was subsequently transferred to a plastic box (15  10  10 cm) on moistened filter paper and apple slices. Mortality and oviposition were checked daily for 21 days. Eggs were further transferred to an unsealed Petri dish (2.5 cm diam.) on a moistened sterile filter paper. Egg hatching was recorded daily for up to 6 additional days. Four replicates of 10 couples per mating combination were carried out. Dead specimens were processed as above to confirm the involvement of B. bassiana in their death. In total, 40 males and 40 females were used in this experiment. 2.5.6. Semi-field assays Two different preventive assays using two application methods were performed. The product (a B. bassiana conidial aqueous suspension adjusted to a final concentration of 6.4  108 conidia/ml by diluting with 0.02% Tween 80) was applied either by painting with a brush ensuring that both the outer stipe and the base of fronds were covered (2000 ml per palm), or by directly spraying onto the stipe with a Manual Knapsack Sprayer (MauricioÒ 18 l Pulverizadores Mauricio S.A., Valencia, Spain) (2000 ml per palm). The first assay consisted of applying up to four treatments at 15-day intervals on 45 palms infested at day 1. Twenty palms were painted and twenty palms were sprayed, whereas the remaining five palms received no treatment and constituted the control group. Two hours after product application all palms were individually exposed to three presumably-mated females and two males of R. ferrugineus per plant in three separate cages (one cage per application method and control). One week later, when found, adults were removed. Treated palms in groups of five received from 1 to 3 additional treatments at 15-day intervals. One month after last treatment, palms were carefully dissected and checked for the presence of R. ferrugineus. All specimens found were counted. The second trial, where product persistence was evaluated, consisted of applying just one treatment per palm and subsequent infestations starting 15 days after the treatment and then every 15 days up to day 45. Fifteen uninfested palms were painted and 15 more were sprayed as previously described. Fifteen additional palms constituted the control group. Fifteen days after product application, five palms per application method and the control were infested as above. The remaining 10 palms per application method and control were infested at subsequent 15-day intervals in groups of five. Palms were processed as before. Control palms and those infested 2 h after the treatment in the first assay were included in the statistical analyses of this trial. 2.6. Statistical analyses In the laboratory tests, for each stage/sex and concentration tested, the Average Survival Times (AST) in days were calculated using the Kaplan–Meier survival analysis (Kaplan and Meier, 1958). Fecundity, egg hatching and AST values were further subjected to analysis of variance (ANOVA; P < 0.05) using Statgraphics Plus 4.1 (Manugistics Group Inc., Rockville, MD, USA). Egg hatching Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS 4 Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx results were subjected to the angular transformation prior to the analysis to meet the requirements of ANOVA. Where appropriate, means were separated by Duncan’s test (P = 0.05). For each tested stage, percent mortality was corrected (Abbott, 1925), transformed into probits and the corresponding probit lines fitted (LeOra Software Inc., 1987). A chi-square test was used to prove the goodness-of-fit. In addition, plots of standardized residuals were checked for their location within a horizontal band of ±2 units about zero (Robertson et al., 1994). In semi-field trials, mean number of insects per palm were subjected to ANOVA. Where appropriate, means were separated by Duncan’s test (P = 0.05). When significant differences relative to control palms were found, efficacies were calculated according to Abbott (1925). adults. In general, the higher the dose the lower the egg hatching rates and the shorter the AST values recorded for any given stage (Table 1). Larvae responded faster to infection than adults and for fourth instar larvae there were no significant differences between the AST values recorded for the two higher doses tested. The same applied to field-collected adults. When the corresponding probit lines were adjusted (Table 2), fourth instar larvae yielded the lowest LC50 and field-collected adults yielded the highest. Laboratory-reared adults resulted more homogeneous in their response than field-collected specimens, as indicated by the steeper probit line adjusted, and almost four times more sensitive to B. bassiana than field-collected specimens, as indicated by the relative potency (3.805). 3.2. Horizontal transmission among adults and sublethal reproductive effects 3. Results Mortality of control insects 21 days after treatment was below 5.0% (Table 3), and was significantly different from that of B. bassiana-treated specimens, which ranged from 55.0% to 90.0%. In all these specimens, B. bassiana outgrowth was observed and therefore, the direct involvement of this fungus in their death was confirmed. Both sex and treatment significantly affected these results and the interaction between these two factors was also significant. For treated pairs, female mortality ranged from 55.0% to 75.0% and differences among treatments were not significant. Therefore, female mortality was the same irrespective of the sex originally infected (treated male or female). However, the origin of the infection was significant for males in treated pairs and male mortality was highest when the infection originated from a treated male (85.0–90.0% versus 60.0%). AST values for insects in treated pairs (AST for control insects could not be calculated because mortality at day 21 was below 50%) did not depend on sex, but were significantly affected by the treatment and the interaction between 3.1. Virulence against R. ferrugineus selected stages B. bassiana infected all stages of R. ferrugineus. Under the microscope, germinated conidia penetrating the chorion of R. ferrugineus egg could be observed at all doses tested as early as 24 h after treatment. The higher the dose and the longer the time elapsed since exposure, the higher the percentage of successfully established conidia. Germinated conidia usually penetrated the chorion directly. However, at higher doses some hyphal growth previous to host penetration could be observed. Most B. bassiana-treated eggs showed internal pink discoloration, loss of turgor, dullness and darkening which appeared 2–3 days after treatment. Subsequently most of the eggs were destroyed and disappeared in the substrate. The few neonate larvae obtained from treated eggs died in less than 3 days at all doses tested. These larvae proved infected by B. bassiana. In most cases, postmortem hyphal growth and sporulation of B. bassiana was observed covering the eggs, larvae and Table 1 Egg hatching at 4 days and Average Survival Time (AST, days after treatment) for R. ferrugineus different instars/stages treated with B. bassiana in the laboratory. The Kaplan–Meier survival analysis method was used to calculate AST values. Dosea (conidia/ml) Egg treatment Egg hatchingb AST for neonate larvae Neonate Fourth instar Laboratory-reared adults Field-collected adults Control X  106 X  107 X  108 X  109 72.2 ± 9.0a 58.3 ± 5.9a 38.9 ± 6.9b 30.6 ± 3.4b 5.6 ± 3.4c – 2.5 ± 0.2a 1.8 ± 0.1b 1.7 ± 0.1b 0.5 ± 0.2c – 5.8 ± 0.2a 4.5 ± 0.1b 3.4 ± 0.3c 2.0 ± 0.0d – 6.6 ± 0.1a 4.6 ± 0.4b 1.5 ± 0.3c 1.4 ± 0.0c – –c –c 21.2 ± 0.5a 16.8 ± 0.2b – –c –c 22.3 ± 2.0a 16.0 ± 3.1a ANOVA (F; df; P) 26.78; 4, 14; <0.0001 28.19; 3, 11; 0.0001 120.75; 3, 11; 0.0001 171.85; 3, 11; <0.0001 84.50; 1, 5; 0.0008 4.40; 1, 5; 0.1039 Average Survival Time, AST AST values limited to the duration of the assay. Within a column, means followed by the same letter are not significantly different from each other (Duncan’s test at P < 0.05). a X = 6.73, 3.14, 4.6, 5.16 and 6.73 for eggs, neonate and fourth instar larvae, laboratory-reared and field-collected adults, respectively. b Actual data were subjected to the angular transformation prior to analysis. c AST values could not be calculated because the observed mortality was below 50%. Table 2 Probit lines adjusted to mortality data of R. ferrugineus different stages when exposed to B. bassiana in the laboratory. Stage Time (days) n Slope ± SE v2 P-valuea LC50 (conidia/ml) 95% FL Eggs Neonate Fourth instar Lab adultsb Field adultb 4 4 4 21 21 144 144 120 144 144 0.706 ± 0.194 1.384 ± 0.260 1.561 ± 0.246 1.471 ± 0.278 0.861 ± 0.236 0.1823 0.5184 0.1575 0.8021 0.1217 0.9129 0.7717 0.9243 0.6696 0.9410 1.5  108 3.7  108 6.3  107 7.2  108 3.0  109 1.1  107–5.6  108 1.8  108–6.5  108 3.7  107–1.0  108 3.7  108–1.2  109 1.1  109–1.1  1010 a Degrees of freedom were two in all cases. Lines obtained for field-collected and laboratory-reared adults were statistically different ({2 = 11.3846; df = 2; P < 0.0030). However, they could be satisfactorily forced to parallelism ({2 = 3.0838; df = 1; P = 0.079) and the relative potency of field-collected adults compared to laboratory-reared adults resulted 3.8055 (fiducial limits 95%: 1.5086–9.7317). b Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS 5 Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx Table 3 Mortality (%) 21 days after treatment and Average Survival Times (AST, days after treatment), for different types of R. ferrugineus pairs either treated or not with B. bassiana in the laboratory. The Kaplan–Meier survival analysis method was used to calculate AST values (Kaplan and Meier, 1958). Each value is the mean of 4 replicates including 10 couples each. Treatment Mortality AST $ # $ # $ # Untreated Untreated Treated Treated Untreated Treated Untreated Treated 5.0 ± 2.8 55.0 ± 2.9 70.0 ± 4.0 75.0 ± 2.9 2.5 ± 2.5 85.0 ± 2.9 60.0 ± 4.0 90.0 ± 4.1 – 19.6 ± 0.4 17.2 ± 0.7 17.0 ± 0.4 – 16.2 ± 0.2 19.2 ± 0.2 16.1 ± 0.6 2-Factor ANOVA (sex and treatment): F; df; P Sex: 11.8; 1, 31; 0.0022 Treatment: 220.7; 3, 31; <0.0001 Interaction: 14.4; 3, 31; <0.0001 Sex: 3.3; 1, 23; 0.0837 Treatment: 6.4; 2, 23; 0.0079 Interaction: 14.5; 2, 23; 0.0002 AST values limited to the duration of the assay. For treated pairs, fungal outgrowth proving the direct involvement of B. bassiana in their death was confirmed. Table 4 Oviposition rate (eggs per female and day), fecundity (eggs per female), egg hatching (%) and larval survival 6 days after hatching (%) for different types of R. ferrugineus pairs either treated or not with B. bassiana in the laboratory. Each value is the mean of 4 replicates including 10 couples each. Only ovipositing females were taken into account to calculate the oviposition rate. Pair $ # Untreated Untreated Treated Treated Untreated Treated Untreated Treated ANOVA (F; df; P) Oviposition rate Fecundity Egg hatching Larval survival 2.09 ± 0.07a 1.49 ± 0.01b 1.43 ± 0.05b 1.50 ± 0.03b 37.25 ± 0.8a 20.63 ± 0.78b 18.60 ± 0.57c 13.95 ± 0.75d 69.2 ± 1.8a 45.1 ± 1.5b 48.8 ± 1.4b 45.4 ± 3.3b 84.9 ± 1.3a 54.6 ± 1.8b 59.2 ± 2.2b 58.1 ± 3.2b 58.6; 3, 15; <0.0001 258.0; 3, 15; <0.0001 272.3; 3, 15; <0.0001 422.4; 3, 15; <0.0001 Within a column, data followed by the same letter are not different (P < 0.005). Fig. 1. Oviposition rates (mean number of eggs per female per day) of R. ferrugineus females fed on apple slices under four mating combinations: (1) untreated female  untreated male, (2) treated female  untreated male, (3) untreated female  treated male and (4) treated female  treated male. both factors was also significant. Survival was significantly higher when the originally infected insect was from the opposite sex (19.2–19.6 d versus 16.1–17.2 d, Table 3). Reproductive parameters of untreated control pairs were always significantly different from those of treated insects (Table 4). Fecundity and egg hatching were significantly lower in treated pairs (52.4% and 32.8%, mean reductions respectively) and maximum reductions were observed when both males and females had been originally treated with B. bassiana (62.6% fecundity reduction). Significant differences were observed in the oviposition rate of control and treated pairs starting 14 days after treatment (Fig. 1). From that day onwards fecundity of control females increased up to more than 3.5 eggs per female per day whereas that of B. bassiana-exposed ones decreased to around 1.5 eggs per female per day irrespective of the sex of the parental originally treated. Differences in egg hatching were observed much earlier (Fig. 2). From day 4 onwards, eggs obtained from treated pairs hatched significantly less than control ones. Likewise, survival of larvae obtained from eggs laid by treated pairs was significantly lower than that of control insects (Table 4) and the reduction (32.5%) was the same irrespective of the sex of the parental originally treated. Overall, these reductions resulted in a mean 78.4% progeny reduction in treated pairs. 3.3. Semi-field assays Results obtained showed that in both assays, treated palms contained significantly less living specimens than control ones Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS 6 Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx Fig. 2. Percentage egg hatching of eggs obtained from R. ferrugineus females fed on apple slices under four mating combinations: (1) untreated female  untreated male, (2) treated female  untreated male, (3) untreated female  treated male and (4) treated female  treated male. Table 5 Palm infestation (%) and mean number of immature stages of R. ferrugineus found in 5-year old P. canariensis after infestation with 3 females and 2 males per palm and subsequent treatment with B. bassiana (6.4  108 conidia/ml). Palms received one to four treatments of 2000 ml per palm as either a paint or a spray at 15-day intervals. Efficacies (%) were calculated based on the total number of individuals found. Five palms (=replicates) per application method and number of treatments and the control were considered. Treatment # Applications Infested palms # Larvae # Pupae a # Total Efficacy a 100 37.8 ± 4.7 – 37.8 ± 4.7 – Paint 1 2 3 4 100 100 100 100 15.2 ± 2.9bcd 11.2 ± 3.3cde 23.0 ± 1.5b 16.8 ± 3.1bc – – – 1.3 ± 0.3 15.2 ± 2.9bcd 11.2 ± 3.3cd 23.0 ± 1.5b 17.6 ± 2.7bc 59.8 ± 7.7abc 70.4 ± 8.8ab 39.2 ± 4.1c 53.4 ± 7.0bc Spray 1 2 3 4 80 60 80 80 9.6 ± 4.2cde 8.6 ± 5.2cde 6.2 ± 2.0de 3.8 ± 2.0e – – – 2.0 ± 0.4 9.6 ± 4.2cd 8.6 ± 5.2cd 6.2 ± 2.0d 5.4 ± 2.1d 74.6 ± 11.1ab 77.2 ± 13.8a 83.6 ± 5.4a 85.7 ± 5.5a 11.50; 8, 44; <0.0001 – 11.35; 8, 44; <0.0001 3.43; 7, 39; 0.0075 Control ANOVA results (F, df, P) Within a column, data followed by the same letter are not different (P < 0.005). Table 6 Palm infestation (%) and mean number of immature stages of R. ferrugineus found in 5-year old P. canariensis palms. Palms were initially treated with 2000 ml of an aqueous solution of B. bassiana (6.4  108 conidia/ml) as either a paint or a spray. Subsequently, they were infested with 3 females and 2 males per palm at different dates. Efficacies (%) were calculated based on the total number of individuals found. Five palms (=replicates) per application method and date and control were considered. Infest. day Treatment Infested palms # Larvae # Pupae # Total Efficacy Day 1 Control Paint Spray 100 100 80 37.8 ± 4.7 15.2 ± 2.9 9.6 ± 4.2 – – – 37.8 ± 4.7 15.2 ± 2.9 9.6 ± 4.2 – 59.8 ± 7.7Bab 74.6 ± 11.1Aab Day 15 Control Paint Spray 100 100 60 22.0 ± 1.3 12.4 ± 3.0 3.8 ± 2.3 – – – 22.0 ± 1.3 12.4 ± 3.0 3.8 ± 2.3 – 43.6 ± 13.7Bb 82.7 ± 10.3Ab Day 30 Control Paint Spray 100 100 80 20.6 ± 1.0 14.4 ± 3.1 4.4 ± 2.5 – – – 20.6 ± 1.0 14.4 ± 3.1 4.4 ± 2.5 – 30.1 ± 12.2Bb 78.6 ± 12.1Ab Day 45 Control Paint Spray 100 60 40 10.4 ± 1.2 2.0 ± 1.1 1.4 ± 1.1 2.0 ± 0.4 1.0 – 12.4 ± 1.2 2.2 ± 1.2 1.4 ± 1.1 – 82.3 ± 10.0Ba 88.7 ± 8.8Aa Treatment Date Interaction 55.9; 2, 58, <0.0001 22.4; 3, 58; <0.0001 3.7; 6, 58; 0.0041 – 590; 2, 58; <0.0001 20.4; 3, 58; <0.0001 3.3; 6, 58; 0.0083 14.65; 1, 39; 0.0006 3.3; 3, 39; 0.0322 1.8; 3, 39; 0.1603 ANOVA results (F, df, P) Data followed by the same upper-case letter are not different in relation to treatment, as are those followed by the same lower-case letter in relation to date. (Tables 5 and 6). Incomplete small galleries and other signs of early R. ferrugienus development where larvae had prematurely died were frequently observed in treated palms. However, it was impossible to recover any immature cadaver from these palms because they had already decayed and therefore the involvement of B. bassiana on these deaths could not be directly proved. In both assays, the efficacy of the treatment depended on the application method used, and the spray was consistently more effective than the paint. Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx In the first assay (Table 5), there was no clear relationship between efficacy and the number of treatments applied (up to four). Efficacies in painted palms ranged between 39.2% and 70.4%, for 3 and 2 applications, respectively. In the case of the spray, there were no significant differences among treatments and efficacies ranged from 74.6% to 85.7%. In the second assay (Table 6), palms infested at the end of August, in coincidence with the B. bassiana treatment, had significantly more individuals than those infested either 15 or 30 days later (37.8 versus 20.6–22.0 in control palms, respectively). Those infested 45 days later, in mid October, had the lowest records (12.4 individuals per control palm) and it was precisely in these palms where efficacies were the highest (82.3 and 88.7% for the paint and the spray treatments, respectively). Lowest efficacy for the paint treatment was 30.1% in palms infested 30 days after the treatment, whereas that of the spray treatment was 74.6% for those infested 2 h after the treatment. 4. Discussion Laboratory results prove that the EABb 07/06-Rf strain of B. bassiana can infect eggs, larvae and adults of R. ferrugineus and in most cases fully complete its life cycle by sporulating on R. ferrugineus cadavers. Insect eggs are commonly the most resistant stage to B. bassiana and this is related to the presence of the chorion (Roberts and Humber, 1981). Accordingly, R. ferrugineus eggs were less susceptible than fourth instar larvae, but contrarily were much more sensitive to B. bassiana infection than adults (Table 2). The B. bassiana strain used in this study killed most of the eggs of R. ferrugineus when directly treated in the laboratory and infected the neonate larvae hatching from the surviving eggs, resulting in a combined effect on both stages of 100% mortality in less than 72 h at the concentrations tested (Table 1). This result could be explained by a direct infection of neonate larvae upon hatching (which in our laboratory assays took less than 3 days), but also by a pre-hatching infection of the embryo within the egg, as observed in some cases and confirmed by the reproduction assays where egg hatching and survival of larvae obtained from eggs from treated couples were significantly reduced (Table 4). In terms of mortality, laboratory-reared adults were almost four times more sensitive to B. bassiana than field-collected specimens. This result may be explained by differences in size (0.48 ± 0.10 versus 1.01 ± 0.11 g for laboratory and field specimens, respectively) (same authors, unpublished results), but differences in cuticle hardiness can not be excluded. Laboratory specimens were 24–48-h old when treated and it is known from other Coleoptera that several days can elapse until elytra reach their maximum hardiness and therefore become less susceptible to penetration (Grosscurt, 1978). Under laboratory conditions, Martín and Cabello (2006) established the longevity of R. ferrugineus as 199.4 days for males and 144.3 days for females. These values are much higher than those reported for this species under natural conditions: 76 and 107 days for males and females, respectively (Menon and Pandalai, 1960), 50–90 days for the adult population in general (Butani, 1975) and 60 days for females (Rahalkar et al., 1985). The AST values obtained in our laboratory assays for treated adults ranged from 16 to 22 d (Tables 1 and 3). Three weeks after treatment mortality of treated insects ranged from 55% to 90%, whereas that of both laboratory-reared and field-collected specimens was below 5% (Table 3). These results indicate that B. bassiana infection in R. ferrugineus adults can reduce their lifespan from half to almost one tenth depending on the reference used for comparison. However, mortality was not the only indicator of the efficacy of the treatment. As in similar situations (Quesada-Moraga et al., 2004, 2006a, 2008), adults of either sex inoculated with the fungus effi- 7 ciently transmitted the disease to untreated adults of the opposite sex (rates of transmission ranged between 55.0% and 60.0%, Table 3). Contrarily to what Gindin et al. (2006) found in their assays with M. anisopliae, infection with strain EABb 07/06-Rf of B. bassiana resulted in a reduction of fecundity around 52% (from 44.6% to 62.6%, depending on the sex of the parental originally treated, Table 3) and the combination of this figure with the aforementioned reductions in egg hatching and larval survival of eggs obtained from treated couples, resulted in an overall progeny reduction of around 78%. Therefore, B. bassiana treatments against R. ferrugineus should reduce its populations by both mortality of the primarily infected weevils and their sublethal effects on the reproduction and the offspring of these adults and those in contact with them. The immersion method used in our laboratory assays to infect R. ferrugineus is completely different from the way insects would pick up B. bassiana spores in the field. However, the efficacies obtained in our semi-field assays are indicative that contact infection of adults actually occurred. In the field, adults could be infected either (a) upon emergence or (b) when approaching treated palms for new infestations. The latter is the way we presumed B. bassiana infected R. ferrugineus in our semi-field assays. Because B. bassiana is horizontally transmitted in R. ferrugineus (Table 3) and its effects can be observed even in the following generation (Table 4, Figs. 1 and 2), spreading of the infection to other adults and to the following generation could explain the high efficacies found in our semifield assays. Moreover, dead specimens sporulating within the palm could contribute to further internally spread the disease. Actually, natural internal spreading of some B. bassiana strains which can endophytically establish in plants has been reported (Quesada-Moraga et al., 2006b, 2009; Vega et al., 2008; Vega, 2008). Should this be the case of the EABb 07/06-Rf strain, R. ferruguineus stages within the palm could be directly exposed to the fungus. This is a question though that should be properly addressed as it could provide a new tool for managing R. ferrugineus. Treatment characteristics (formulation, pressure, volume, etc.) and host features (behavior, specific cuticle characteristics, etc.) may influence the pick up of spores and the results from the semi-field assays show that the application method significantly affected the efficacy of our treatments (Tables 5 and 6). Spraying was significantly more effective and yielded more homogeneous results than painting and this may be related to a more thorough coverage of the palm when spraying. Interestingly, the semi-field trials showed that up to four treatments per palm did not result in higher efficacies than just one (Table 5). They also showed that treatment persistence was at least 45 days (Table 6). However, whether the higher efficacies observed in the second assay for palms infested 45 days after treatment is the result of a lower infestation level (under our climatic conditions, October is a poor time of the year for colonization for R. ferrugineus compared to August) deserves further research. Although our results prove that all life stages of R. ferrugineus may be infected by B. bassiana, adults are actually the only free-living stage. Therefore, adults should be considered as the target of any treatment involving this entomopathogenic fungus. Because B. bassiana can fulfil its life cycle in R. ferrugineus and B. bassianainfected adults can transmit the pathogen to other individuals of the same population and even to the following generation, strategies aimed at attracting and infecting adult weevils could prove the most successful. Adult weevils are highly attracted by kairomone leaking during vegetative production practices that include the removal of offshoots and pruning (Giblin-Davis, 2001). These zones are often the most attractive places for oviposition and, therefore, may be the best candidates for localized treatments with fungi. This attraction is also the base for mass trapping strategies currently used against the weevil in some countries (Hallett et al., 1999; Faleiro, 2006). The design of efficient traps aimed Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006 ARTICLE IN PRESS 8 Ó. Dembilio et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx not at killing but at infecting the visiting weevils (Rechcigl and Rechcigl, 2000) could ensure the spreading of the disease within an already established population. Finally, the use of irradiated sterile males (Al-Ayedh and Rasool, 2009) infected with B. bassiana could be used for the same purpose (Sewify et al., 2009). All these strategies, which could be used simultaneously and in combination with mass trapping, should be considered and deserve further research. In summary, our results prove that the EABb 07/06-Rf strain of B. bassiana should be seriously considered as a biological control agent against R. ferrugienus. Acknowledgments The authors thank J.R. Faleiro (ICIAR, Goa, India) and J.E. Peña (TREC, University of Florida) for critically reviewing an earlier draft of this paper, J.J. López-Calatayud (Tragsa S.A., Valencia) for providing the field-collected adults of R. ferrugineus used in our assays, L. Bellver, M. Piquer and E. Llácer (IVIA) and S. Castuera Santacruz (Universidad de Córdoba) for their help during the assays and the palm nurserymen association ASFPLANT for providing the palms used in our assays. This research was partially funded by the Spanish Ministerio de Ciencia e Innovación (project TRT2006-00016C07-05) and the Valencian Conselleria d’Agricultura, Pesca i Alimentació (project IVIA-5611). O. Dembilio was recipient of a predoctoral grant from IVIA. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jip.2010.04.006. References Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Adane, K., Moore, D., Archer, S.A., 1996. Preliminary studies on the use of Beauveria bassiana to control Sitophilus zeamais (Coleoptera: Curculionidae) in the laboratory. J. Stor. Prod. Res. 32, 105–113. Al-Ayedh, H.Y., Rasool, K.G., 2009. Sex ratio and the role of mild relative humidity in mating behaviour of red date palm weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Curculionidae) gamma-irradiated adults. J. Appl. Entomol.. doi:10.1111/j.1439-0418.2009.01460.x. Butani, D.K., 1975. Insect pests of fruit crops and their control, sapota-11: date palm. Pest. Res. J. 9, 40–42. De la Rosa, W., Alatorre, R., Trujillo, J., Barrera, J.F., 1997. Virulence of Beauveria bassiana (Deuteromycetes) strains against the coffee borer (Coleoptera: Scolytidae). J. Econ. Entomol. 90, 1534–1538. Dembilio, Ó., Jacas, J.A., Llácer, E., 2009. Are the palms Washingtonia filifera and Chamaerops humilis suitable hosts for the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)? J. Appl. Entomol. 133, 565–567. Dembilio, Ó., Llácer, E., Martínez de Altube, M.M., Jacas, J.A., 2010. Field efficacy of imidacloprid and Steinernema carpocapsae in a chitosan formulation against the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Phoenix canariensis. Pest Manage. Sci.. doi:10.1002/ps.1882. El-Sufty, R., Al-Awash, S.A., Al Bgham, S., Shahdad, A.S., Al Bathra, A.H., 2009. Pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill to the Red Palm Weevil, Rhynchophorus ferrugineus (Oliv.) (Col.: Curculionidae) under Laboratory and Field Conditions. Egyptian J. Biol. Pest Control 19, 81. EPPO (European and Mediterranean Plant Protection Organization), 2008. Data sheets on quarantine pests. Rhynchophorus ferrugineus. EPPO Bullet. 38, 55–59. EPPO (European and Mediterranean Plant Protection Organization), 2009. First record of Rhynchophorus ferrugineus in Curaçao, Netherlands Antilles, January 26, 2009. EU (European Union), 2007. Commission decision of 25 May 2007 on emergency measures to prevent the introduction into and the spread within the Community of Rhynchophorus ferrugineus (Olivier). 2007/365/EC. Off. J. Eur. Union L 139, 24–27. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Int. J. Trop. Insect Sci. 26, 135–154. Giblin-Davis, R.M., 2001. Borers of palms. In: Howard, F.W., Moore, D., Giblin-Davis, R.M., Abad, R.G. (Eds.), Insects on Palms. CABI Publishing, Wallingford, UK, pp. 267–305. Gindin, G., Levski, S., Glazer, I., Soroker, V., 2006. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against the Red Palm Weevil Rhynchophorus ferrugineus. Phytoparasitica 34, 370–379. Goettel, M.S., Inglis, D.G., 1997. Fungi: Hyphomycetes. In: Lacey, L.A. (Ed.), Manual of techniques in insect pathology. Academic Press, London, UK, pp. 213–249. Grosscurt, A.C., 1978. Effects of diflubenzuron on mechanical penetrability, chitin formation and structure of the elytra of Leptinotarsa decemlineata. J. Insect Physiol. 24, 827–831. Glare, T.R., Placer, C., Nelson, T.L., Reay, S.D., 2002. Potential of Beauveria and Metarhizium as control agents of pinhole borers (Platypus spp.). N. Z. Plant Prot. 55, 73–79. Hallett, R.H., Oehlschlager, A.C., Borden, J.H., 1999. Pheromone-trapping protocols for Rhynchophorus ferrugineus. Int. J. Pest Manage. 45, 231–237. Kaplan, E.L., Meier, P., 1958. Nonparametric estimation from incomplete data. J. Am. Stat. Assoc. 53, 457–481. Lacey, L.A., Kirk, A.A., Millar, L., Mereadier, G., Vidal, C., 1999. Ovicidal and larvicidal activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci. Technol. 9, 9–18. Software, Le.Ora., 1987. Polo-Pc: A User’s Guide to Probit OrLOgit Analysis. LeOra Software, Berkeley. Llácer, E., Martínez, J., Jacas, J.A., 2009. Evaluation of the efficacy of Steinernema carpocapsae in a chitosan formulation against the red palm weevil, Rhynchophorus ferrugineus, in Phoenix canariensis. BioControl 54, 559–565. Marannino, P., Santiago-Álvarez, C., de Lillo, E., Quesada-Moraga, E., 2006. A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera: Buprestidae). J. Invertebr. Pathol. 93, 210–213. Martín, M.M., Cabello, T., 2006. Manejo de la cría del picudo rojo de la palmera, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta artificial y efectos en su biometría y biología. Bol. San. Veg. Plagas 32, 631– 641. Menon, K.P.V., Pandalai, K.M., 1960. A Monograph of the Coconut Palm. Ernakulam, India. p. 384. Murphy, S.T., Briscoe, B.R., 1999. The red palm weevil as an alien invasive: biology and prospects for biological control as a component of IPM. Biocontrol N. Inf. 20, 35–45. Quesada-Moraga, E., Santos-Quiros, R., Valverde-Garcia, P., Santiago-Álvarez, C., 2004. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J. Invertebr. Pathol. 87, 51–58. Quesada-Moraga, E., Ruiz-García, A., Santiago-Álvarez, C., 2006a. Laboratory evaluation of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against puparia and adults of Ceratitis capitata (Diptera: Tephritidae). J. Econ. Entomol. 99, 1955–1966. Quesada-Moraga, E., Landa, B.B., Muñoz-Ledesma, F.J., Jiménez-Díaz, R., SantiagoÁlvarez, C., 2006b. Endophytic colonization of opium poppy Papaver somniferum by an entomopathogenic Beauveria bassiana strain. Mycopathologia 161, 323– 329. Quesada-Moraga, E., Martin-Carballo, I., Garrido-Jurado, I., Santiago-Álvarez, C., 2008. Horizontal transmission of Metarhizium anisopliae among laboratory populations of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Biol. Control 47, 115–124. Quesada-Moraga, E., Muñoz-Ledesma, F.J., Santiago-Álvarez, C., 2009. Systemic protection of the opium poppy, Papaver somniferum L., against Iraella luteipes (Hymenoptera: Cynipidae) by an endophytic strain of Beauveria bassiana (Ascomycota: Hypocreales). Environ. Entomol. 38, 723–730. Rahalkar, G.W., Harwalkar, M.R., Rananavare, H.D., Tamhankar, A.J., Shanthram, K., 1985. Rhynchophorus ferrugineus. In: Singh, P., Moore, R.F. (Eds.), Handbook of Insect Rearing. Elsevier, Amsterdam, NL, pp. 279–286. Rechcigl, J.E., Rechcigl, N.A., 2000. Biological and Biotechnological Control of Insect Pests. Lewis Publishers, Boca Raton, US. 374 pp. Roberts, D.W., Humber, R.A., 1981. Entomogenous fungi. In: Kendrick, G.T., Cole, B. (Eds.), Biology of Conidial Fungi. Academic Press, New York, pp. 201–236. Robertson, J.L., Preisler, H.K., Frampton, E.R., Armstrong, J.W., 1994. Statistical analyses to estimate efficacy of disinfestation treatments. In: Shrarp, J.L., Hallman, G.J. (Eds.), Quarantine Treatments for Pests of Fruit Plants. Westview Press, Boulder, pp. 47–65. Sewify, G.H., Belal, M.H., Al-Awash, S.A., 2009. Use of the Entomopathogenic Fungus, Beauveria bassiana for the Biological Control of the Red Palm Weevil, Rhynchophorus ferrugineus Olivier. Egyptian J. Biol. Pest Control 19, 157–163. Vega, F.E., Posada, F., Aime, M.C., Pava-Ripoll, M., Infante, F., Rehner, S.A., 2008. Entomopathogenic fungal endophytes. Biol. Control 46, 72–82. Vega, F., 2008. Insect pathology and fungal endophytes. J. Invertebr. Pathol. 98, 277– 279. Zimmermann, G., 2007. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Sci. Technol. 17, 553–596. Please cite this article in press as: Dembilio, Ó., et al. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.04.006