BIOLOGICAL AND MICROBIAL CONTROL Acoustic Assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) Effects on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) Larval Activity and Mortality JOHARI JALINAS,1,2 BERENICE GU¨ ERRI-AGULLO´ ,1,3 R. W. MANKIN,4,5,* R. LO´ PEZ-FOLLANA,1,3 1,3 AND L. V. LOPEZ-LLORCA J. Econ. Entomol. 108(2): 444–453 (2015); DOI: 10.1093/jee/tov023 ABSTRACT Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthoridae) is an economically important pest of palm trees in the subtropics. Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypo- creales: Clavicipitaceae), has been shown to be pathogenic against R. ferrugineus in laboratory and field studies. However, because they remain inside the trunks until adulthood, the slowing of feeding and in- creases in mortality of internally feeding R. ferrugineus larvae over time after B. bassiana treatment has not been established. To explore the potential of acoustic methods to assess treatment effects, sound im- pulses produced by untreated, 104-, and 106-conidia mlÀ1 B. bassiana-treated larvae in palms were re- corded for 23 d, after which the palms were dissected and the larvae examined. Analyses were performed to identify trains of impulses with characteristic patterns (bursts) produced frequently by moving and feeding larvae but only rarely (3–8% of the larval rate) by interfering background noise or tree vibrations. The rates of bursts, the counts of larval impulses per burst, and the rates of impulses in bursts decreased significantly over time in both B. bassiana treatments but not in the control. This supports a hypothesis that larvae had briefer movement and feeding bouts as they became weaker after infection, which re- duced the counts of larval impulses per burst, the rates of bursts, and the rates of impulses in bursts. There is considerable potential for use of acoustic methods as tools for nondestructive assessment of effects of biological control treatments against internally feeding insect pests. KEY WORDS detection, entomopathogenic fungi, biological control Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dry- management strategies based on chemical treatments, ophthoridae), the red palm weevil, causes significant pheromone traps (Faleiro and Chellapan 1999), and damage to a wide range of palm species worldwide. In biological control such as entomopathogenic fungi Spain, R. ferrugineus is an important pest of date (Shah and Pell 2003, Gindin et al. 2006, Dembilio [Phoenix dactylifera L. (Arecales: Arecaceae) and can- et al. 2010, Gu¨erri-Agullo´etal.2011). The most com- ary palms (P. canariensis Chabaud; Ferry et al. 2002, monly used control treatments are insecticides such as EPPO 2008). Adults can be monitored and trapped Diazinon, Imidacloprid, and Phosmet (Abbas et al. with pheromone–food attractant baits, but the larvae 2010). However, heavy use of chemical treatments feed hidden inside the trunks, making it difficult to causes environmental damage and harms nontarget detect and control (Fiaboe et al. 2011). organisms, and also leads to the development of insecti- In Spain and other Mediterranean countries where cide resistance. Pheromone traps are excellent monitor- infestation is prevalent in urban areas, there is a strong ing devices but capture only adults, leaving the more emphasis on the development of integrated pest harmful larvae to destroy the trunk and emerge later. Moreover, the potential for adults to be attracted to palm trees located near pheromone traps (Roda et al. 2011) and the capability of adults to escape from dry or 1 Department of Marine Sciences and Applied Biology, Laboratory of Plant Pathology, Multidisciplinary Institute for Environmental nearly dry traps (Fiaboe et al. 2011) suggests that pher- Studies (MIES) Ramon Margalef, University of Alicante, Ap. 99, omone traps may be more useful monitoring or control 03080 Alicante, Spain. tools in commercial palm areas than in zones with 2 School of Environmental and Natural Resource Sciences, Faculty highly valuable historic palms like Elche-Alicante, of Sciences, Universiti Kebangsaan Malaysia (UKM), Selangor, Malay- sia 43600. Spain. Consequently, there remains interest in develop- 3 Glen Biotech, Colegio Mayor Universitario, Ctra. San Vicente del ment of additional alternatives such as entomopatho- Raspeig, 03080 Alicante, Spain. genic fungi for R. ferrugineus management. 4 US Department of Agriculture, Agriculture Research Service, Cen- Beauveria bassiana (Balsamo) Vuillemin (Hypo- ter for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL 32608. creales: Clavicipitaceae) is an entomopathogenic fungus 5 Corresponding author, e-mail: [email protected]. that has been shown to cause R. ferrugineus mortality Published by Oxford University Press on behalf of Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US. April 2015 JALINAS ET AL.: FUNGAL TREATMENT EFFECTS ON RED PALM WEEVIL 445 in the laboratory (Gindin et al. 2006, Gu¨erri-Agullo´ were obtained by shaking 2 g of 15-d-old solid et al. 2010, Rican˜o et al. 2013). A solid formulation of formulation in 20 ml of 0.2% Tween 80. Conidial con- B. bassiana isolate Bb 203 dusted around the crowns, centration was determined using a Neubauer hemocy- as well as the upper stems and petioles of palm trees, tometer and subsequently adjusted to 108 conidia was demonstrated to reduce R. ferrugineus populations mlÀ1. A series dilution was made to 106 conidia mlÀ1 in southeastern Spain palm groves (Gu¨erri-Agullo´etal. (106 treatment) and 104 conidia mlÀ1 (104 treatment). 2011). Some of the field treatment effects could be Sterile distilled water containing 0.2% Tween 80 was assessed by observations of visible differences in dam- used as control. age in treated and untreated palms, but the effects on Palms. Assays were performed on 5-yr-old potted P. larvae hidden inside the palms could not be observed canariensis obtained from an officially inspected nursery directly. (Elche, Alicante, Spain) and certified free of R. ferrugi- Exploring the potential of other methods to assess neus infestation. They were maintained in a greenhouse B. bassiana treatment effects in field environments, we on the University of Alicante campus. Palms were hypothesized that comparisons of the temporal and watered twice weekly and kept inside independent spectral patterns of sounds produced by larvae inside cylindrical, wire cloth cages to avoid accidental addi- trees might reveal significant differences between sig- tional infestation. A 6 by 60-mm screw (PZ3, Standers, nals produced by fungus-treated and untreated larvae. Leroy Merlin, Lezennes, France) was inserted near the Previous laboratory studies reported that fungus- base of the palm for use as a signal waveguide. The treated larvae often produce weaker movements or eat palm then was prepared for artificial infestation by drill- for shorter intervals than untreated larvae (Ekesi 2001, ing two 30-mm-diameter by 6-cm holes into the palm at Nussenbaum and Lecuona 2012). In addition, several opposite ends of a diameter. Three test palms used as previous studies, including Hussein et al. (2010), Guti- no larva controls were prepared for artificial infestation e´rrez et al. (2010),andRach et al. (2013),havedemon- but were not inoculated with larvae. strated that R. ferrugineus larvae can be detected Insects. Adult R. ferrugineus were collected in acoustically. Temporal and spectral pattern analyses of Elche using bucket traps baited with 4-methy-5-nona- sounds produced by insect larvae in trees have been nol and 4-methyl-5 nonanone (Kaakeh et al. 2001). applied to distinguish such sounds from background Insects were maintained in the laboratory in an incuba- noise in laboratory and field environments (Mankin tor at 25 6 0.5C in darkness. Plastic boxes (40 by 30 et al. 2008a,b, 2011). Large insect larvae moving and by 21 cm) were set with a folded piece of moistened fil- feeding in trees produce trains (groups) of brief, broad- ter paper containing thin green apple slices that were band sound impulses separated by intervals <25 ms replaced three times per week. Adults from the stock that can be identified by specialized software. Trains colony were sexed by visual inspection of their snouts with characteristic patterns that contain >6and<200 (Prabhu and Patil 2009). Adults were bred in pairs in impulses (designated as bursts) are known to be reli- individual 100-ml specimen bottles (Deltalab, Barce- able indicators that insects are present within the sen- lona, Spain) using green apple as both food and ovipo- sor detection range (Mankin et al. 2008a). We sition substrate. To maintain the humidity of rearing hypothesized that B. bassiana treatment effects on lar- containers, 20 by 5 cm filter paper (Refe 1510, Filtros vae might be reflected in a slower rate of bursts or a Anoia S. A., Barcelona, Spain) was wet with distilled smaller count of larval impulses per burst. In addition, water and placed into the specimen bottle. treatment effects might be reflected in a smaller count After 2 d, eggs were collected from both apple and of “burst impulses” per recording, where burst impulse paper. The eggs were placed in a sterile 9-cm-diameter refers to impulses occurring only within and not out- plastic Petri dish with an artificial diet substrate (Alar- side a burst. To consider whether sublethal and lethal co´n et al. 2002). Egg hatching was recorded daily for effects might be identifiable, we conducted experi- up to 6 d. Emerged larvae were individually transferred ments with 30-d-old larvae at two different treatment to 20-ml coulter tubes containing 10 ml of artificial levels, about two and four orders of magnitude below diet, and after 15 d, they were transferred into speci- levels known to cause 100% mortality in previous stud- men bottles containing 50 ml of artificial diet, which ies (Dembilio et al. 2010, Rican˜o et al. 2013). was replaced every 15 d. Larvae were inoculated using a dipping technique. With the larval head held by hand, the abdomen was Materials and Methods dipped into the B.