The ISME Journal (2015) 9, 1793–1801 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Farming of a defensive fungal mutualist by an attelabid weevil Lin Wang1,5, Yu Feng2,5, Jianqing Tian1, Meichun Xiang1, Jingzu Sun1, Jianqing Ding3, Wen-Bing Yin1, Marc Stadler4, Yongsheng Che2 and Xingzhong Liu1 1State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; 2State Key Laboratory of Toxicology and Medical Counter Measures, Beijing Institute of Pharmacology and Toxicology, Beijing, China; 3Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, China and 4Department of Microbial Drugs, Helmholtz Centre for Infection Research, Braunschweig, Germany The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called ‘proto-fungiculture’, involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic ( þ )-scleroder- olide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial ‘weeds’ and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in ‘proto-fungiculture’ animals. The ISME Journal (2015) 9, 1793–1801; doi:10.1038/ismej.2014.263; published online 6 February 2015 Introduction dependency (Mueller et al., 2005). Instances of advanced fungiculture have been extensively docu- Insects developed the capacity to cultivate fungi as a mented and studied, including fungus-farming ants, food source approximately 40–60 million years ago, termites and ambrosia beetles (Farrell et al., 2001; and agricultural life ultimately enabled all of these Aanen et al.,2002;Currieet al., 2003a). The farming insect farmers to rise to major ecological importance strategies of these insect farmers include evolved (Mueller and Gerardo, 2002; Mueller et al., 2005). mechanisms for substrate preparation, inoculation The capacity to grow fungus has not been restricted with crop propagules, optimization of fungal growth to insect lineages; also snails have been discovered through regular activities, protection of the crop to be nutritionally dependent on external fungal against parasites or diseases and harvest and con- symbionts (Silliman and Newell, 2003). sumption of fungi (Schultz et al.,2004;Muelleret al., Insect fungiculture shares some defining features 2005). The insect farmers have developed task- of agriculture, including habitual planting (‘inocula- partitioned societies cooperating in their fungiculture tion’), cultivation, harvesting and nutritional biology (Biedermann and Taborsky, 2011; Nobre et al., 2011). In ant and termite farmers, agriculture tasks are partitioned between different castes, each specialized Correspondence: Y Che, State Key Laboratory of Toxicology & on one main task (Hart et al., 2002; Nobre et al., 2011). Medical Countermeasures, Beijing Institute of Pharmacology & In ambrosia beetle (Xyleborinus saxesenii), division of Toxicology 27 Taiping Road, Beijing 100850, China. E-mail: [email protected] labor is shown between larval and adult colony or X Liu, State Key Laboratory of Mycology, Institute of members (Biedermann and Taborsky, 2011). Microbiology, Chinese Academy of Sciences, No 3, 1st Beichen Proto-fungiculture mutualism cases have been West Road, Chaoyang District, Beijing 100101, China. reported from non-social organisms, and the insects E-mail: [email protected] 5These authors contributed equally to the work. show no division of labor. Outside the class Insecta, Received 25 August 2014; revised 30 November 2014; accepted 10 marine snails farming intertidal fungi provide an December 2014; published online 6 February 2015 example in the marine environment. Littoria snails Attelabid weevil and mycangial fungus mutualism L Wang et al 1794 (Littoraria irrorata) ‘protofarm’ fungi by creating collected in four locations in Jiangxi and Hubei plant wounds that become infected with fungal provinces, China. The collected host plant leaves mycelia that are part of the snails’ diet, but the snails and weevils were placed in separate containers. The do not actively inoculate plant wounds or otherwise leaf-rolls were maintained individually on sterilized garden the fungi (Silliman and Newell, 2003). The moist soil to permit the weevils to undergo complete fungus-growing lizard beetle Doubledaya buccu- metamorphosis. lenta transmits symbiotic yeast into bamboo inter- node cavities and disperses the yeast in the cavities Scanning electron microscopy (SEM) to make gardens (Toki et al., 2013). These proto- Dissected mycangia and leaf-rolls were used for farming phenomena have also been reported in SEM screening. The mycangia of 20 adult female some species of gall midges and wood wasps weevils were dissected. After the eggs and larvae (Kukor and Martin, 1983; Heath and Stireman, were removed, 1.5 cm2 pieces were cut from 20 leaf- 2010; Pazoutova et al., 2010). rolls. These specimens (20 each of mycangia and A seldom-studied example of proto-fungiculture leaf-rolls without insects) were fixed in glutaralde- is exhibited by attelabid weevils. Among the sub- hyde and processed according to previously pub- family Attelabinae, species in the genus Euops have lished methods (Yuceer et al., 2011). The specimens a symbiotic association with fungi: the female were subsequently examined using a Quanta 200 weevils roll leaf pieces and inoculate them with a scanning electron microscope (FEI, Czech). fungal symbiont stored in their mycangium. They lay one or a few eggs in the leaf cradles (leaf-rolls), Isolation of microorganisms from mycangia and then the larvae develop by feeding on the inside A total of 89 E. chinensis females were collected layers of the leaf cradles (Sakurai, 1985; Wang et al., from the host shrub F. japonica in Jiangxi and Hubei 2010). Inoculation with the fungal symbiont provinces, China. Spores stored in the mycangia of decreases the cellulose content of the host plant’s these females were removed with a sterile needle leaves. Experimental removal of the cultivated and placed on 1/4-strength potato dextrose agar fungus results in increased larval mortality (Li (PDA) plates at room temperature. After the spores et al., 2012). The mycangial fungi have been germinated, the colonies were transferred to full- identified as species of the genus Penicillium strength PDA plates. Sporulating colonies were used (Kobayashi et al., 2008; Li et al., 2012). to identify the fungi. Nutrient agar (NA; per liter: 5 g Morphological and behavioral adaptations for a peptone, 3 g beef extract, and 15 g agar) and chitin ‘fungiculture lifestyle’ have been observed in attelabid medium (Hsu and Lockwood, 1975) were used to weevils. These adaptations include the female’s mycan- isolate bacteria (including Actinobacteria) from gium, the comb plates on her abdomen, and the female weevils’ mycangia. The plates were incu- characteristic posture she assumes when depositing bated in the dark at 25 1C for fungal isolation fungal spores (Sakurai, 1985). Recent studies suggested (including Actinobacteria) and at 30 1C for bacterial that the fungal cultivar tissues have only a minor isolation. nutritional value (Kobayashi et al., 2008; Li et al., 2012). Our study of the attelabid weevil, E. chinensis,and its symbiotic fungus Penicillium herquei have indi- Isolation of microorganisms from non-rolled host plant cated that important aspects of this symbiosis have leaves and leaf-rolls not been previously described. Based on both video The un-cut parts of the leaves left by weevils were recordings of living specimens in the field and used as the non-rolled leaf samples. Leaf-rolls detailed laboratory investigations, we describe new were unrolled, and the eggs or larvae inside were specialized structures and behaviors that E. chinensis removed. The non-rolled leaf parts and the leaf-rolls females use for leaf-rolling and fungus inoculation. were processed separately. The leaf tissues were We also report that P. herquei produces an antibiotic, washed twice with sterile 0.1% Tween 20. The ( þ )-scleroderolide, that protects the fungal garden samples were then homogenized in 10 ml of sterile from ‘weed microorganisms’. The characteristics of deionized water, and the homogenate was serially À 1 À 4 E. chinensis–P. herquei mutualism include the leaf- diluted from 10 to 10 . Each dilution was roll habitat, non-social nature and the highly evolved divided