ASPERGILLUS SCLEROTIORUM FUNGUS IS LETHAL TO BOTH WESTERN DRYWOOD (INCISITERMES MINOR) AND WESTERN SUBTERRANEAN (RETICULITERMES HESPERUS) TERMITES. GREGORY M. HANSEN, TYLER S. LAIRD, ERICA WOERTZ, DANIEL OJALA, DARALYNN GLANZER, KELLY RING, AND SARAH M. RICHART* DEPARTMENT OF BIOLOGY AND CHEMISTRY, AZUSA PACIFIC UNIVERSITY, AZUSA CA MANUSCRIPT RECEIVED 21 OCTOBER, 2015; ACCEPTED 16 JANUARY, 2016 Copyright 2016, Fine Focus all rights reserved 24 • FINE FOCUS, VOL. 2 (1) ABSTRACT Termite control costs $1.5 billion per year in the United States alone, and methods for termite control usually consist of chemical pesticides. However, these methods have their drawbacks, which include the development of resistance, environmental pollution, and toxicity to other organisms. Biological termite control, which employs the use of living organisms to combat pests, CORRESPONDING offers an alternative to chemical pesticides. This study highlights the discovery of a fungus, termed “APU AUTHOR strain,” that was hypothesized to be pathogenic to termites. Phylogenetic and morphological analysis *Sarah M. Richart showed that the fungus is a strain of Aspergillus [email protected] sclerotiorum, and experiments showed that both western drywood (Incisitermes minor) and western subterranean (Reticulitermes hesperus) termites die in KEYWORDS a dose-dependent manner exposed to fungal spores of A. sclerotiorum APU strain. In addition, exposure • Entomopathogenic to the A. sclerotiorum Huber strain elicited death in • Phylogenetics a similar manner as the APU strain. The mechanism • Biological control by which the fungus caused termite death is still • Pest management unknown and warrants further investigation. While • Aspergillus sclerotiorum these results support that A. sclerotiorum is a termite • Reticulitermes hesperus pathogen, further studies are needed to determine • Incisitermes minor whether the fungal species has potential as a biological • Termites control agent. INTRODUCTION Termites are a common structural and is also a signifcant challenge in controlling economic pest worldwide. While estimates termite populations (33). Because of such vary considerably, termites are responsible complications, biological termite control for between $500 million and $1 billion of agents that are natural insect pathogens have wood damage and cost $1.5 billion in control been proposed, including parasitoids, viruses, measures per year in the United States alone bacteria, protozoa, nematodes, and fungi (7). (7,32). An array of problems have been associated with the use of chemical pesticides Fungi, especially those that occur naturally to control pests like termites, such as the in termite habitats, are promising candidates development of resistance in target organisms, for use as biological control agents because environmental pollution, and toxicity to they are well-adapted to survive in such non-target organisms, including humans environments (33). Entomopathogenic (25). Avoidance behavior to such chemicals fungal species such as Beauveria bassiana, PATHOGENS AND ANTIMICROBIAL FACTORS • 25 Paecilomyces fumosoroseus, and Metarhizium canals have been described (6, 23). anisopliae have been suggested as potential termite control agents, with the latter being In this current study, we hypothesize that a the best studied and most effective of the novel isolated fungus is entomopathogenic three (17,19,28). There are limitations, however, to termites. The research objectives are to to the effectiveness of fungi as termite control genetically and morphologically identify agents in their native habitats, due to specifc the APU strain, experimentally infect two termite behaviors such as allogrooming (6), different species of termites, Incisitermes spore avoidance (22), and vibrational alarm minor and Reticulitermes hesperus, with APU signals in response to fungal spores (22,29). strain, and determine if dose has any effect on In addition, naturally occurring antimicrobial termite survival. compounds in termite feces and alimentary MATERIALS AND METHODS TERMITE COLLECTION Fungus was repeatedly subcultured on SDA Incisitermes minor and Reticulitermes plates to ensure a pure culture. Aspergillus hesperus termites were obtained either sclerotiorum strain Huber (16892, American from decomposing frewood in Arcadia, Type Culture Collection, Rockville, MD) CA, or from branches found in the foothills was used as a comparison strain. For spores of the San Gabriel Mountains in Glendora, that were used to infect termites, cultures of CA. Termite-containing wood was stored both strains were grown in the dark on SDA in plastic bins in a cool, dark room until plates with penicillin-streptomycin (100 U/ used in experiments. To collect termites, ml penicillin and 100 mg/ml streptomycin, wood was broken apart and termites were diluted from 100X stock solution, HyClone picked up with lightweight forceps or Laboratories, Logan, UT) at 25°C for paintbrushes, and placed in Petri dishes approximately 3 weeks, or until a confuent with a brown paper towel as food, with layer of yellow spores was present. Each additional moisture for R. hesperus. Termites plate was then either used at 3 weeks or later were left two or more days in Petri dishes to infect termites directly. after collection and before the start of an experiment to ensure that healthy, uninjured To collect spores, confuent plates were inverted insects were used. and placed atop a 0.45 mm sterile vacuum flter, then gently and repeatedly tapped to FUNGAL CULTURES, DRY dislodge spores. Spores were then left in the flter apparatus under vacuum to dry for SPORE COLLECTION, AND approximately 3 hours. The resulting dry spore QUANTIFICATION. powder was quantifed by resuspending in a A strain of fungus believed to have solution of 0.1% (v/v) Tween-20 and counting eliminated an I. minor colony at Azusa Pacifc in a hemocytometer. Live spore quantifcation University in 2009 (called “APU strain”) was determined by standard plate count on was isolated from a deceased termite and SDA in duplicate or triplicate for each dilution, cultured on Difco Sabouraud Dextrose Agar and fungal colonies were counted on days 2-4, (SDA) (BD Diagnostics, Franklin Lakes, NJ). depending on the growth. 26 • FINE FOCUS, VOL. 2 (1) ITS REGION AND β-TUBULIN used for the alignment. Under the pairwise GENE POLYMERASE alignment and multiple alignment sections, the gap opening penalties were set to 15, CHAIN REACTION (PCR) and the gap extension penalties were set AMPLIFICATION to 6.66. Under the multiple alignment The species identifcation of the strain of section, the gap opening penalty was set to fungus that was presumed to kill a colony 15 and the gap extension penalty to 6.66. of I. minor termites in 2009 (called “APU The DNA weight matrix was set to IUB. strain”) was initially performed by PCR The transition weight was 0.5. The use of a amplifcation of the internal transcribed negative matrix was turned off. The delay spacer region (ITS) of fungal ribosomal of divergent cutoff was set to 30%. After DNA isolated using ZR Fungal/Bacterial alignment, the sequences were trimmed DNA miniprep kit (Zymo, Irvine, CA) to 562bp, and used to construct maximum according to the manufacturer’s protocol, likelihood phylogenetic trees using MEGA6. with the following additional step: prior to The Tamura 3-parameter model was selected, DNA extraction, fungal samples suspended as well as gamma distributed with invariant in water were frozen at -20°C. The primer sites (G + I), as done previously (27). The pair made for amplifcation of the fungal number of discrete gamma categories was ITS region was ITS1 and ITS4 (sequences 5. Gaps and missing data were subject to as previously described) which amplifes complete deletion. The ML Heuristic method a region that includes the entire 5.8S was set to Nearest-Neighbor-Interchange, rRNA gene (31). PCR was also performed and the initial tree was set to Default - NJ/ using primers (Bt2a and Bt2b), designed to BioNJ. The branch swap flter was set to amplify Aspergillus β-tubulin, as previously very strong. The number of threads selected described (10). PCR products were purifed was 1. Bootstrap analysis of 1000 replicates using the Wizard SV Gel and PCR Clean-up was performed. kit (Promega, Madison, WI), and sequenced at the DNA Sequencing Core Lab (City of The sequence of a region of the β-tubulin Hope, Duarte, CA). gene, a commonly sequenced gene in fungi, was also subjected to a megablast PHYLOGENETIC ANALYSIS using BLAST. The frst 14 strains of unique OF ITS AND β-TUBULIN species similar to APU strain were chosen GENE REGIONS for phylogenetic tree construction. The Aspergillus tanneri β-tubulin sequence was OF APU STRAIN acquired from NCBI as an outgroup, as The APU strain ITS sequence was subjected done previously (27). A total of 15 sequences to a megablast search using the fungi of β-tubulin regions were acquired and RefSeq ITS database in BLAST (National aligned along with the APU strain sequence Center for Biotechnology Information). The in MEGA6 using the same parameters as top eight sequences ranked by maximum mentioned above for the ITS region. After score were used. Aspergillus tanneri, the 8th alignment, the sequences were trimmed to sequence, was used as an outgroup, similar 579bp. A maximum likelihood phylogenetic to a previous phylogenetic study (27). For tree was constructed in MEGA6. All selected ITS, sequences were aligned along with options were the same as done for the ITS the APU strain sequence using ClustalW region