Cedrol, a malaria mosquito oviposition attractant is produced by fungi isolated from rhizomes of the grass Cyperus rotundus
© The Author(s) 2016
Received: 31 May 2016
Accepted: 13 September 2016
Published: 17 September 2016
Cedrol, a sesquiterpene alcohol, is the first identified oviposition attractant for African malaria vectors. Finding the natural source of this compound might help to elucidate why Anopheles gambiae and Anopheles arabiensis prefer to lay eggs in habitats containing it. Previous studies suggest that cedrol may be a fungal metabolite and the essential oil of grass rhizomes have been described to contain a high amount of different sesquiterpenes.
Rhizomes of the grass Cyperus rotundus were collected in a natural malaria mosquito breeding site. Two fungi were isolated from an aqueous infusion with these rhizomes. They were identified as Fusarium falciforme and a species in the Fusarium fujikuroi species complex. Volatile compounds were collected from the headspace above fungal cultures on Tenax traps which were analysed by gas chromatography–mass spectrometry (GCMS). Cedrol and a cedrol isomer were detected in the headspace above the F. fujikuroi culture, while only cedrol was detected above the F. falciforme culture.
Cedrol an oviposition attractant for African malaria vectors is produced by two fungi species isolated from grass rhizomes collected from a natural mosquito breeding site.
KeywordsMalaria Oviposition attractant Fungi Rhizome Cedrol
Recently, the sesquiterpene alcohol cedrol was identified as an attractant for gravid female Anopheles gambiae mosquitoes that are vectors of the human malaria parasites . The compound was detected as a volatile from an infusion made with water and soil taken from a natural Anopheles breeding site. A similar soil infusion had previously been shown to mediate an increased oviposition response in cage bioassays when incubated for 6 days compared to 2 and 4 days . In addition, a 6 day old aqueous infusion with soil from the same site was shown to attract An. gambiae females to gravid traps in semi-field settings . Furthermore, cedrol was detected in higher amount from a non-autoclaved soil infusion compared to the same infusion that had been autoclaved . These results suggested a possible microbial link and together with reports about sesquiterpenes as common fungal metabolites [4, 5] indicates that the cedrol released from the soil infusion may be of fungal origin.
Cyperus rotundus grass, also known as nut grass, was found growing in the natural Anopheles breeding site where the soil was collected in the previous studies [1–3]. Furthermore, essential oil from rhizomes of this grass has been reported to contain high amounts of sesquiterpenes  and cedrol has been found in rhizome extract of another grass species in the same genus, Cyperus articulates . Based on these studies it was hypothesized that fungi associated with C. rotundus rhizomes may be the source of the oviposition attractant cedrol. To test this, fungi were isolated from rhizomes of C. rotundus grass collected from the same natural Anopheles breeding site used for the soil infusion studies described above. Furthermore, volatiles were collected from the headspace above cultures of these fungi and analysed on a gas chromatograph coupled to a mass spectrometer.
Fungi isolation and identification
Yeast extract glucose (YEG) agar was prepared by adding 4 g of yeast extract (Applichem: Chemtronica AB, Stockholm Sweden), 4 g of d-glucose (Applichem; Chemtronica AB, Stockholm Sweden) and 15 g agar (Fluka analytical, Sigma, Stockholm Sweden) to 1 L of deionized water. This mixture was sterilized using an autoclave at 121 °C for 20 min. YEG broth was prepared the same way but without the agar.
Single isolates from the plates were transferred to new YEG agar plates and incubated at room temperature for 2–6 days and repeated sub-culturing until single isolates were obtained and confirmed by observation under a microscope. The isolates were sent to CBS-KNAW Fungal Biodiversity Centre for identification (Centraalbureau voor Schimmelcultures, Netherlands).
Headspace collections from fungal cultures
The fungal isolates were inoculated into separate E-flasks (500 ml) with 300 ml YEG broth. Headspace samples were collected on Tenax traps from the headspace above fungal cultures after incubation for 6 days. The E-flask was fitted with a gas wash bottle head (QuickFit joined ware, Staffordshire, United Kingdom) for volatile collections. The traps were made from 25 mg of Tenax® TA polymer (60–80 mesh, Supelco, Bellefonte, PA, USA), placed in GERSTEL-Twister Desorption glass liners (GERSTEL, Muelheim an der Ruhr, Germany). The polymer was held in place with glass wool (Supelco, Bellefonte, PA, USA) on each side. The traps were washed 10 times with 200 µl of methyl tertbutyl ether (MTBE) and sealed with polytetrafluorethylene (PTFE) tape and placed in oven at 40 °C for 4 h before use. Charcoal filtered air was pumped into the E-flasks at a rate of 0.1 l/min and drawn out at the same rate through a Tenax trap. The traps were sealed with PTFE tape after collection and stored at −80 °C. E-flasks containing 300 ml of sterile YEG agar and empty E-flasks were sampled the same way and utilized as controls for culture medium and Tenax trap background volatiles.
Gas chromatography–mass spectrometry (GC–MS) analysis
Tenax traps with fungal headspace collections were analysed on an Agilent 7890A gas chromatograph connected to an Agilent 5975C inert MSD with Triple Axis detector mass spectrometer (Agilent, Santa Clara CA, USA). The GCMS system was fitted with a GERSTEL Multi-Purpose Sampler (MPS: Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany). The GC capillary column utilized was an Agilent’s HP-5MS column (5 % phenyl and 95 % dimethyl polysiloxane, 30 m, 250 µm internal diameter and 0.25 µm film thickness). Prior to analysis one microliter heptyl acetate (3.16 ng/µl) was added to the Tenax trap in the GERSTEL thermal desorption unit (TDU) followed by thermal desorption of traps in splitless mode at an initial temperature of 40 °C which was then increased by 120 °C/min to 270 °C, the end temperature was held for 5 min. The desorbed volatiles were focused in a GERSTEL CIS inlet at 10 °C. The CIS inlet operated in splitless mode was then heated at a rate of 12 °C/s to 280 °C during which the volatiles were transferred to the column. The MS at full scan identified mass ranges from 30 to 400 m/z with electron ionization at 70 eV and ion source temperature at 230 °C.
The GC–MS data were analysed with Agilent’s enhanced Chemstation software version E.02.01.1177. The data were screened for two of the main MS ions of cedrol (95, 150). The identified peak was confirmed to be cedrol by comparing the retention time and mass spectra to a sample of an authentic standard of cedrol [(+)-cedrol, ≥99.0 % sum of enantiomers, GC, optical activity α D 20 +10.5 ± 1°, Sigma-Aldrich Sweden AB, Stockholm, Sweden] analysed using the same GCMS settings as described above. Epi-cedrol and α-acorenol were identified based on comparison of the mass spectra to mass spectra in the NIST 08 library and in Brock et al. .
Results and discussion
Other fungi such as Beauveria bassiana have been shown to produce spores that are attractive to host seeking Anopheles stephensi mosquitoes . This agrees with this study that fungi can produce cues that attract mosquitoes. In fact, fungal volatiles and insect orientation may be a common ecological phenomenon, for instance it has been observed that volatiles like 2-phenylethanol and 2-methyl-1-butanol from Aureobasidium pullulans are attractive to different insect taxa . Furthermore, volatiles from several fungi were tested as oviposition attractants of female yellow peach moths and the result suggested that the compounds can be used in gravid traps to monitor oviposition .
Cedrol, the first identified oviposition attractant for An. gambiae s.s. and An. arabiensis females was identified as a metabolite of two fungi species in the Fusarium genus. These fungi were isolated from C. rotundus rhizomes collected from a natural Anopheles oviposition site. This finding may be utilized in future studies to understand why malaria mosquitoes utilize cedrol as an attractant and how the compound and possibly the grass species may influence malaria mosquito ecology. Such knowledge could be utilized in development of novel control strategies targeting gravid females.
LKE conceived and designed the experiments. JL and GKR revised the experimental design. LKE and HS performed the experiments. LKE, HS, JL, GKR, AKBK analysed the data. LKE wrote the first draft of the paper; all authors contributed to the final draft. All authors read and approved the final manuscript.
Authors thank Paul Ouma for technical assistance and the first observation of the presence of grass rhizomes in the soil samples. The project was funded by Swedish Research Council grant no SWE-2010-129 (AKBK, GKR, LKE) and the National Institute of Health (NIH) grant no. R01AI082537 (AKBK, JL). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting this paper will be made available upon request.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Lindh J, Okal M, Herrera-Varela M, Borg-Karlson AK, Torto B, Lindsay S, et al. Discovery of an oviposition attractant for gravid malaria vectors of the Anopheles gambiae species complex. Malar J. 2015;14:1.View ArticleGoogle Scholar
- Herrera-Varela M, Lindh J, Lindsay SW, Fillinger U. Habitat discrimination by gravid Anopheles gambiae sensu lato-a push–pull system. Malar J. 2014;13:133.View ArticlePubMedPubMed CentralGoogle Scholar
- Okal MN, Herrera-Varela M, Ouma P, Torto B, Lindsay SW, Lindh JM, et al. Analysing chemical attraction of gravid Anopheles gambiae sensu stricto with modified BG-sentinel traps. Parasit Vectors. 2015;8:301.View ArticlePubMedPubMed CentralGoogle Scholar
- Kramer R, Abraham W-R. Volatile sesquiterpenes from fungi: what are they good for? Phytochem Rev. 2012;11:15–37.View ArticleGoogle Scholar
- Brasch J, Horter F, Fritsch D, Beck-Jendroschek V, Tröger A, Francke W. Acyclic sesquiterpenes released by Candida albicans inhibit growth of dermatophytes. Med Mycol. 2014;52:46–55.PubMedGoogle Scholar
- Kilani S, Ledauphin J, Bouhlel I, Ben Sghaier M, Boubaker J, Skandrani I, et al. Comparative study of Cyperus rotundus essential oil by a modified GC/MS analysis method. Evaluation of its antioxidant, cytotoxic, and apoptotic effects. Chem Biodivers. 2008;5:729–42.View ArticlePubMedGoogle Scholar
- Olawore NO, Usman LA, Ogunwande IA, Adeleke KA. Constituents of rhizome essential oils of two types of Cyperus articulatus L. grown in Nigeria. J Essent Oil Res. 2006;18:604–6.View ArticleGoogle Scholar
- Brock NL, Huss K, Tudzynski B, Dickschat JS. Genetic dissection of sesquiterpene biosynthesis by Fusarium fujikuroi. ChemBioChem. 2013;14:311–5.View ArticlePubMedGoogle Scholar
- Wulff EG, Sørensen JL, Lübeck M, Nielsen KF, Thrane U, Torp J. Fusarium spp. associated with rice Bakanae: ecology, genetic diversity, pathogenicity and toxigenicity. Environ Microbiol. 2010;12:649–57.View ArticlePubMedGoogle Scholar
- Brock NL, Tudzynski B, Dickschat JS. Biosynthesis of sesqui- and diterpenes by the gibberellin producer Fusarium fujikuroi. ChemBioChem. 2011;12:2667–76.View ArticlePubMedGoogle Scholar
- Okal MN, Lindh JM, Torr SJ, Masinde E, Orindi B, Lindsay SW, et al. Analysing the oviposition behaviour of malaria mosquitoes: design considerations for improving two-choice egg count experiments. Malar J. 2015;14:250.View ArticlePubMedPubMed CentralGoogle Scholar
- Khwatenge IL. Isolation, identification and biodiversity of air-borne organic volatile semiochemicals for Chilo partellus for Zea mays Sorghum bicolor seedlings. Nairobi: University of Nairobi Jomo Kenyatta Memorial Library: University of Nairobi; 1999. p. 124.Google Scholar
- Mercke P, Crock J, Croteau R, Brodelius PE. Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L. Arch Biochem Biophys. 1999;369:213–22.View ArticlePubMedGoogle Scholar
- Luczkiewicz M, Jesionek A, Kokotkiewicz A, Migas P, Mardarowicz M, Szreniawa-Sztajnert A, et al. Production of essential oils from in vitro cultures of Caryopteris species and comparison of their concentrations with in vivo plants. Acta Physiol Plant. 2015;37:1–11.View ArticleGoogle Scholar
- George J, Jenkins NE, Blanford S, Thomas MB, Baker TC. Malaria mosquitoes attracted by fatal fungus. PLoS ONE. 2013;8:e62632.View ArticlePubMedPubMed CentralGoogle Scholar
- Davis TS, Landolt PJ. A survey of insect assemblages responding to volatiles from a ubiquitous fungus in an agricultural landscape. J Chem Ecol. 2013;39:860–8.View ArticlePubMedGoogle Scholar
- Honda H, Ishiwatari T, Matsumoto Y. Fungal volatiles as oviposition attractants for the yellow peach moth Conogethes punctiferalis. J Insect Physiol. 1988;34:211.View ArticleGoogle Scholar