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Biological control of Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain
Journal of Asia-Pacific Entomology 18 (2015) 217–221
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Biological control of Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain Se Jin Lee, Sihyeon Kim, Jeong Seon Yu, Jong Cheol Kim, Yu-Shin Nai ⁎, Jae Su Kim ⁎ Department of Agricultural Biology, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, Republic of Korea
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Article history: Received 31 October 2014 Revised 22 December 2014 Accepted 9 February 2015 Available online 17 February 2015 Keywords: Entomopathogenic fungi Aedes albopictus Metarhizium anisopliae Transformant Granular formulation
a b s t r a c t Mosquitoes have been becoming serious vectors worldwide thus effective and safe control strategies should be established. Entomopathogenic fungi can be alternative controlling agents by substituting chemical insecticides. Herein we assayed 12 soil-borne entomopathogenic fungi against Asian tiger mosquito (Aedes albopictus) larvae in laboratory conditions and tried to establish an effective application method using millet granular formulation (GR). Twelve fungal isolates which belong to 6 genera (Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium and Verticillium) were assayed; M. anisopliae JEF-003 showed the fastest mosquitocidal activity, approximately 73% mortality rate at 2 days post-inoculation (dpi.) and N90% mortality rate at 5 dpi. Conidia of M. anisopliae JEF-003 also showed a dosage dependent activity at 1 × 105, 1 × 106 and 1 × 107 conidia ml−1. Hyphal growth of M. anisopliae JEF-003 in the Ae. albopictus larvae was observed by infection of an M. anisopliae JEF003 EGFP-transformant, which was generated by restriction enzyme-mediated integration (REMI) method. GR of M. anisopliae JEF-003 showed high virulence to Ae. albopictus larvae (N 90% mortality) after 5 days of application. These results suggest that M. anisopliae JEF-003 has a potential to control A. albopictus larvae and GR can be practically used for management of the serious vector in water environment. © 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Introduction Mosquitos (Culicidae) are vectors which carry several serious human diseases from person to person without having the symptoms themselves. Transmission of mosquito-borne diseases may be accelerated by global climate change. Various mosquito species can transmit different diseases, such as malaria (transmitted by the genus Anopheles) and lymphatic filariasis (primarily transmitted by Anopheles spp., Aedes spp., and Culex spp.) (Rozendaal, 1997; Verma and Prakash, 2010). Most of mosquito-borne viral diseases are transmitted by Aedes spp., such as yellow fever, dengue, dengue hemorrhagic fever (Rozendaal, 1997), and chikungunya fever (Powers et al., 2000). Aedes spp. are distributed worldwide. In tropical countries, Ae. aegypti is an important vector of yellow fever, dengue fever, and Chikungunya fever (Rozendaal, 1997; Hochedez et al., 2006); Ae. albopictus is a closely related species that also can transmit viral diseases, such as several types of encephalitis (Savage et al., 1994; Gerhardt et al., 2001), and is known to transmit even several filarial nematodes such as Dirofilaria immitis (Cancrini et al., 2003). Ae. albopictus has become an epidemiologically important vector. ⁎ Corresponding authors at: Department of Agricultural Biology, College of Agriculture and Life Sciences, Chonbuk National University, Republic of Korea. Tel.: + 82 63 270 2525; fax: +82 63 270 2531. E-mail addresses: [email protected] (Y.-S. Nai), [email protected] (J.S. Kim).
To control Aedes vectors, several insecticides have been employed as larvicides that are applied to mosquito breeding sites or adulticides applied to mosquito adults; modes of application include insecticide residual sprays (IRS), space sprayings, treated/impregnated materials (Vontas et al., 2012), and thermal fogging. Mosquitocides are diverse and include organophosphates (e.g. temephos), insect growth regulators (e.g. pyriproxyfen), pyrethroids (Kroeger et al., 2006; World Health Organization, 2011), and bacterial toxins (e.g. Bacillus thuringiensis serovar israelensis) among others. Although insecticidebased interventions have efficiently controlled Aedes mosquito populations, it has been reported that Ae. aegypti shows resistance to all four classes of insecticides (carbamates, organochlorines, organophosphates, and pyrethroids) (Ponlawat et al., 2005; Ranson et al., 2010). Due to the negative effect of insecticides on ecosystems and substantial increase in physiological resistance shown by mosquitoes (Hargreaves et al., 2000), biological control agents which can reduce the drawbacks associated with insecticides (Krischbam, 1985) should be considered. The use of entomopathogenic fungi can be an alternative control method, and some efforts have already been conducted. Fungal pathogens such as Lagenidium, Coelomomyces, and Culicinomyces are known to affect mosquito populations, and have been studied extensively. However, many other fungi infect and kill mosquitoes at the larval and/or adult stage (Scholte et al., 2004), such as Metarhizium anisopliae isolates, Beauveria tenella, and Lagenidium giganteum (Balaramn et al.,
http://dx.doi.org/10.1016/j.aspen.2015.02.003 1226-8615/© 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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1979; Lecy et al., 1988; Scholte et al., 2004; Vyas et al., 2007). Besides, fungi have the distinct advantage of being able to recycle in stagnant water, injecting multiple and overlapping generations of mosquitoes (Legner, 1995). Although there are some efforts to use entomopathogenic fungi in mosquito control (Scholte et al., 2004), however little consideration was given to the other species, such as Beauveria brongniartii, Cordyceps brongniartii, Paecilomyces spp. and Purpureocillium spp. especially against Ae. albopictus larvae. Moreover, the several formulations of fungal conidia, including water- or oil-based suspensions, granules, dusts, and floating formulations (Ramoska et al., 1981; Daoust et al., 1982) have been tested for larval stages of mosquito control, however millet-based fungal granules received little interest in this area. This millet-based formulation has advantages in mass production, application to water, and enhancement of fungal survival after the application. Thus herein, we isolated 12 entomopathogenic fungi from soil samples and assayed their virulence against Asian tiger mosquito (Ae. albopictus) larvae in laboratory conditions. The infectious process of the selected entomopathogenic fungus was also investigated by infecting with the EGFP-fungal mutant, which was generated by restriction enzyme-mediated integration (REMI) method; and also, we tried to apply millet-based fungal granules as a novel approach to control Ae. albopictus larvae in the natural environment.
Materials and methods Mosquito larvae The Ae. albopictus larvae used in this study were kindly provided by the Insect Physiology Laboratory of Chonbuk National University, Korea. The mosquito larvae were kept at 28 ± 1 °C, 65 ± 5% of relative humidity (RH) with a photoperiod of 16:8 (L:D) and feed with artificial diet in the laboratory (Shin and Lee, 2014).
Isolation and identification of entomopathogenic fungi Entomopathogenic fungal isolates were collected from soil by placing Tenebrio molitor (mealworm) larvae on the soil for two weeks and isolating the out-grown fungi from the surfaces of cadavers. The collection of soil samples are summarized in Table 1. The fungal isolates were identified by sequencing the internal transcribed spacer (ITS) region and examining the morphological characteristics. The newly identified and recorded isolates in Korea were firstly stored at Inset Molecular and Biotechnological Laboratory (IMBL) of Chonbuk National University as conidial suspensions in 20% glycerol at −80 °C (Humber, 1997) and then deposited at the National Institute of Biological Resources (NIBR; www.nibr.go.kr) (Incheon, Korea). Table 1 Fungal species isolated from soil samples in Korea in 2012–2103. Fungal species
Isolate code
Sample locations
Beauveria bassiana B. bassiana B. brongniartii Cordyceps brongniartii Metarhizium anisopliae M. anisopliae Paecilomyces carneus Pa. javanicus Pa. spinulosum Purpureocillium lilacinum Pu. lilacinum Verticillium leptobactrum
JEF-006 JEF-007 KJS-004 KJS-008 JEF-003 JEF-004 KJS-010 KJS-005 KJS-006 KJS-003 KJS-011 KJS-012
Gangwon-do (N 37.29.30.05 E 127.59.05.66) Gangwon-do (N 37.29.30.05 E 127.59.05.66) Jeju island (N 33.21.27.35 E 126.27.47.25) Ulleung island (N 37.30.22.89 E 130.51.25.78) Gangwon-do (N 37.29.30.05 E 127.59.05.66) Gangwon-do (N 37.29.30.05 E 127.59.05.66) Ulleung island (N 37.30.22.89 E 130.51.25.78) Jeju island (N 33.21.27.35 E 126.27.47.25) Jeju island (N 33.21.27.35 E 126.27.47.25) Jeollabuk-do (N 35.50.48.33 E 127.07.45.85) Ulleung island (N 37.30.22.89 E 130.51.25.78) Ulleung island (N 37.30.22.89 E 130.51.25.78)
Solid culture condition The fungal isolates were cultured on quarter-strength Sabouraud dextrose agar (1/4 SDA) (pH 6) in Petri dishes (60-mm diam.) in the dark at 25 ± 1 °C for 10 days (Humber, 2005). Conidia were collected by vortexing the agar blocks (6 mm diam.) in an Eppendorf-tube containing 0.03% siloxane solution (Silwet L-77, Momentive Performance Materials Inc.). The numbers of conidia were counted with a hemocytometer at 400 × magnification (3 times per isolate). Conidia with N90% viability were kept in a refrigerator at 4 °C for 3–4 days before use. Millet-based solid culture The fungal isolates were solid cultured using the millet-based culture system (Bartlett and Jaronski, 1988; Li and Feng, 2005; Kim et al., 2011). To prepare inocula for the solid cultures, a conidial suspension from 3 sporulated agar blocks (6-mm diam.) of M. anisopliae JEF-003 was added to quarter-strength Sabouraud dextrose broth in a 250-ml flask. Flasks were held on a rotary shaker (150 rpm) at 25 ± 2 °C for 3 days. Millet (Panicum miliaceum) grains (200 g) were placed in a polyvinyl bag, soaked in 100 ml of water containing citric acid (0.4 ml l−1) and autoclaved at 120 °C for 15 min. The bag was then cooled to ambient temperature. The liquid culture broth (3 ml) was introduced into the bag and thoroughly mixed. All bags were held for 6 days at 25 ± 1 °C and a 16:8 (L/D) photoperiod. After incubation, mycotized GRs were dried at ambient temperature for 3 days, until the moisture content reached b 5% (determined by a moisture analyzer [Sartorius Omnimark]). All batches of mycotized GRs were assessed for conidial concentration (g−1) with a hemocytometer. Fungal transformation To observe the infection of the selected isolate (M. anisopliae JEF-003) in Ae. albopictus larvae, a transgenic M. anisopliae JEF-003 was generated by a fungal transformation using the pBARKS1-egfp. Briefly, the egfp expression cassette was released from pBluscript II KS (+)-egfp by ClaI and SacI digestion and then cloned into the ClaI/SacIdigested pBARKS1 vector and confirmed by commercial sequencing (MACRO GEN, Korea). The pBARKS1-egfp, has phosphinothricin (PPT) resistant bar fungal selection marker and EGFP genes, both of them are expressed under the control of gpdA promoter. The plasmid, linearized by restriction enzyme digestion, was integrated into the genomic DNA of M. anisopliae JEF-003 by the REMI method based on blastospores (Ying and Feng, 2006; Kim et al., 2013). The M. anisopliae JEF-003 EGFP-transformant was cultured on Czapek's agar supplement with 600 μg ml−1 phosphinothricin. The M. anisopliae JEF-003 EGFPtransformant was subjected to the infection test in the mosquito larvae and observed under fluorescence microscopy. Mosquitocidal assay Virulence of 12 entomopathogenic fungal isolates against Ae. albopictus larvae was investigated as follows. Conidial suspensions were adjusted to 1 × 107 conidia ml−1 using 0.03% siloxane solution in 90-mm-diameter Petri dishes at 10 ml dish−1. Fifteen third-instar larvae were placed in the dish using a disposable dropper. Dishes were covered with lids and kept at 25 ± 1 °C and 16:8 (L/D) photoperiod and examined daily for 8 days. For dosage dependent assay, the conidial concentration of the selected isolate (M. anisopliae JEF-003) was 10-fold diluted from 1 × 105 to 1 × 107 conidia ml−1 and assayed with same petri-dish conditions as described above. This experiment was replicated 3 times. Virulence assay of M. anisopliae JEF-003 GR To investigate the potential control efficacy of GR, mycotized GRs were applied to the water in 90-mm-diam Petri dishes at 1 g dish−1
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and 15 larvae were transferred to each dish. Untreated control served as a negative control. The number of dead mosquito larvae was observed daily. This experiment was replicated 3 times. Data analysis The data on percentage of live Ae. albopictus larvae were analyzed using a general linear model (GLM) followed by Tukey's honest significant difference (HSD) test for multiple comparisons. All analyses were conducted using SPSS ver. 17.1 (SPSS Inc., 2009) at the 0.05 (α) level of significance. Results and discussion Fig. 2. Dosage dependent assay of M. anisopliae JEF-003 against Ae. albopictus larvae.
Isolation and mosquitocidal activity of entomopathogenic fungal isolates From our soil isolations, a total of 12 entomopathogenic fungi were isolated from soil samples in Korea, the sampling details were summarized in Table 1. The results of ITS sequences and fungal morphological identification indicated that these 12 fungal isolates belonged to 6 genera, Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium, and Verticillium. There are more than 13 different fungal genera reported as potential mosquito-pathogenic fungi, such as Lagenidium, Coelomomyces, Entomophthora, Culicinomyces, Beauveria, and Metarhizium (Scholte et al., 2004). Despite many fungi having the potential to be mosquito control agents, a few have been commercialized and applied. New isolates from the domestic area and additional investigation are needed in order to contribute to an expansion of current mosquito control tools which have limited effectiveness. Twelve entomopathogenic fungi were assayed against Ae. albopictus larvae in Petri-dish conditions; in this assay, 11 fungal isolates showed different levels of mosquitocidal activity ranging from 20% to 100% at 8 days post-inoculation (dpi.); Verticillium leptobactrum KJS-012 showed no activity. Among the mosquitocidal fungi, 3 isolates (B. brongniartii KJS-004, and M. anisopliae JEF003 and JEF004) showed high virulence to Ae. albopictus larvae (Fig. 1). Of these 3 most virulent isolates, M. anisopliae JEF-003 showed the fastest virulence, which resulted in approximately 73% mortality rate at 2 dpi. and N 90% mortality rate at 5 dpi., and followed by M. anisopliae JEF004 (~ 47% mortality rate at 2 dpi. and ~ 80% mortality rate at 5 dpi.) (Fig. 1). Additionally, M. anisopliae JEF-003-infected larvae were completely disrupted morphologically (Fig. 1, arrow); fungal conidia
were found on the surface of dead larvae and larvae in the untreated control were healthy. M. anisopliae JEF-003 showed a dosagedependent mosquitocidal activity and had the highest virulence at 1 × 107 conidia ml−1 (Fig. 2). The evaluation of M. anisopliae JEF-003 infection process in mosquito larvae was further evaluated by using the M. anisopliae JEF-003 EGFP-transformant; hyphal growth in the larvae was observed under the fluorescence (Fig. 3). Many fungi that infect and kill mosquitoes at the larval and/or adult stage have been well studied. The spores of entomopathogenic fungus Fusarium pallidoroseum and Chrysosporium tropicum were reported to be effective against adults of female Cx. quinquefasciatus (Mohanty et al., 2008; Verma and Prakash, 2010). Metarhizum anisopliae, Beauveria tenella, Lagenidium giganteum, C. lobatum, and C. tropicum are used against mosquito larvae (Balaramn et al., 1979; Federici, 1981; Lecy et al., 1988; Scholte et al., 2004; Vyas et al., 2007; Mohanty and Prakash, 2008). According to our presented data, this is the first instance of B. brongniartii infecting Ae. albopictus larvae in the laboratory, although the virulence of B. brongniartii was lower than the other two fungal species. This result provides additional impetus to study the applications of entomopathogenic fungi; in particular, the diversity of these fungi should be emphasized. Both B. bassiana and M. anisopliae have been reported as virulent against several Culex spp., Aedes spp., and other mosquito species, primarily larvae (Pinnock et al., 1973; Ramoska et al., 1981; Daoust et al., 1982; Lecy et al., 1988; Ravallec et al., 1989). In this study, we support previous results with new data regarding soil-borne entomopathogenic fungal species.
Fig. 1. Fungal isolates from soil samples and their pathogenicity. Isolate Metarhizium anisopliae JEF-003 showed fastest pathogenicity against the Aedes albopictus larvae at 2 days after inoculation. The arrow indicates that M. anisopliae JEF-003-infected larvae were completely disrupted morphologically. Scale bar = 0.5 mm.
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Fig. 3. Observation of M. anisopliae JEF-003 EGFP-transformant-infected Ae. albopictus larvae. Mycelial growth in the larvae was observed under fluorescence.
Ae. albopictus larvae prefer temporary water-holding containers such as tree holes, leaf axils, ground pools, and coconut shells. Larvae come to the water surface for respiration and dive to the bottom for feeding or escaping danger. Fungal conidia germinate and penetrate into the respiratory siphon and block the breathing mechanism of mosquito larvae, usually leading to death before significant invasion of the hemocoel in mosquito larvae (Daoust et al., 1982; Lecy et al., 1988). Thus, hyphal formation is not usually observed and cadavers in the aquatic environment are often found with bacterial infection rather than mycelial infection; typically no new conidia are produced (Scholte et al., 2004). According to our results, hyphae and conidia
of M. anisopliae JEF-003 were found on the surface of dead larvae; moreover, according to our preliminarily results, water depth ranging from 3 cm to 15 cm caused no significant influence on the virulence of this fungus (data not shown). These results indicate a fungal strain with an increased possibility of infecting mosquito larvae, and provide new insight on the mosquito larvae control in water environment. Potential of millet-based M. anisopliae JEF-003 GR M. anisopliae JEF-003 GRs were applied to the water in Petri dishes. Some fungal conidia detached from the millet granules when GRs
Fig. 4. Application of M. anisopliae JEF-003 GR to water Petri dishes. Virulence of GRs showed high mortality (N90%) after 5 days of treatment and untreated control showed less than 10% mortality.
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were submerged in water for 2 min. GR virulence revealed N 90% mortality after 5 days of treatment and the untreated control showed less than 10% mortality (Fig. 4). Several types of conidia formulations have been developed and tested for mosquito larval control including granulars, dusts, and wettable suspensions. Among these formulations, M. anisopliae conidia had decreased virulence against Culex pipiens larvae when in aqueous suspensions containing a surfactant or formulations of granular carriers or dust diluents, but significantly enhanced conidial virulence was achieved with a dried castor oil formulation (Daoust et al., 1982). Using GRs, we found virulence as high and stable in the water condition (N 90% mortality). This finding provides the advantage that millet in GR application could also serve a nutritional source for fungal colonization in the natural environment. Moreover, it has been shown that conidia, stored under dry conditions showed higher germination rates than conidia formulated in solution formulation (e.g. paraffin oil) (MorleyDavies et al., 1995). Entomopathogenic fungi are virulent to most insects and cause fungal diseases. Different from many other pathogens, fungi directly infect the host insect by penetrating into the cuticle, making ingestion by the insect unnecessary (Vega and Kaya, 2012). There are several advantages for fungal control of mosquito larvae, for example: easy colonization of the water environment, conidia produced on the larval surface could be transferred to other mosquito populations, and inputs of harmful synthetic chemical pesticides in agriculture, horticultural, and forest systems could be reduced (Strasser et al., 2000). The millet-based fungal GR formulation could increase the possibility of fungus survival in the water condition. However, several application aspects should be still considered and further investigated in the near future (e.g. time for preparing the GR formulation and spread methods). Acknowledgments We thank the Insect Physiology Laboratory in Chonbuk National University for providing the Ae. albopictus larvae. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Project No: 2013R1A1A1057946) and the Research Service Task (Collection of Fungi) from the National Institute of Biological Resources, 2013–2014. References Balaramn, K., Bheema, R.U.S., Rajagopalan, P.K., 1979. Isolation of Metarhizium anisopliae, Beauveria tenella and Fusarium oxysporum (Deuteromycetes) and their pathogenicity to Culex fatigans and Anopheles stephensi. Indian J. Med. Res. 70, 718–722. Bartlett, M.C., Jaronski, S.T., 1988. Mass production of entomopathogenous fungi for biological control of insects. In: Burge, M.N. (Ed.), Fungi in Biological Control Systems. Manchester University Press, Manchester, pp. 61–85. Cancrini, G., di Regalbono, A.F., Ricci, I., Tessarin, C., Gabrielli, S., Pietrobelli, M., 2003. Aedes albopictus is a natural vector of Dirofilaria immitis in Italy. Vet. Parasitol. 118, 195–202. Daoust, R.A., Ward, M.G., Roberts, D.W., 1982. Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae. J. Invertebr. Pathol. 40, 228–236. Federici, B.A., 1981. Mosquito control by fungi Culicinomyces, Lagenidium and Coelomomyces, in: Microbial Control of Pests & Plant Diseases 1970–1980 (Ed.). Academic, New York, pp. 555–572. Gerhardt, R.R., Gottfried, K.L., Apperson, C.S., Davis, B.S., Erwin, P.C., Smith, A.B., Panella, N.A., Powell, E.E., Nasci, R.S., 2001. First isolation of La Crosse virus from naturally infected Aedes albopictus. Emerg. Infect. Dis. 7, 807–811. Hargreaves, K., Koekemoer, L.L., Brooke, B.D., Hunt, R.H., Methembe, J., Coetzee, M., 2000. Anopheles funestus resistant to pyrethroid insecticides in South Africa. Med. Vet. Entomol. 2, 181–189. Hochedez, P., Jaureguiberry, S., Debruyne, M., Bossi, P., Hausfater, P., Brucker, G., Bricaire, F., Caumes, E., 2006. Chikungunya infection in travelers. Emerg. Infect. Dis. 12 (10).
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Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Biological control of Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain Se Jin Lee, Sihyeon Kim, Jeong Seon Yu, Jong Cheol Kim, Yu-Shin Nai ⁎, Jae Su Kim ⁎ Department of Agricultural Biology, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, Republic of Korea
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Article history: Received 31 October 2014 Revised 22 December 2014 Accepted 9 February 2015 Available online 17 February 2015 Keywords: Entomopathogenic fungi Aedes albopictus Metarhizium anisopliae Transformant Granular formulation
a b s t r a c t Mosquitoes have been becoming serious vectors worldwide thus effective and safe control strategies should be established. Entomopathogenic fungi can be alternative controlling agents by substituting chemical insecticides. Herein we assayed 12 soil-borne entomopathogenic fungi against Asian tiger mosquito (Aedes albopictus) larvae in laboratory conditions and tried to establish an effective application method using millet granular formulation (GR). Twelve fungal isolates which belong to 6 genera (Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium and Verticillium) were assayed; M. anisopliae JEF-003 showed the fastest mosquitocidal activity, approximately 73% mortality rate at 2 days post-inoculation (dpi.) and N90% mortality rate at 5 dpi. Conidia of M. anisopliae JEF-003 also showed a dosage dependent activity at 1 × 105, 1 × 106 and 1 × 107 conidia ml−1. Hyphal growth of M. anisopliae JEF-003 in the Ae. albopictus larvae was observed by infection of an M. anisopliae JEF003 EGFP-transformant, which was generated by restriction enzyme-mediated integration (REMI) method. GR of M. anisopliae JEF-003 showed high virulence to Ae. albopictus larvae (N 90% mortality) after 5 days of application. These results suggest that M. anisopliae JEF-003 has a potential to control A. albopictus larvae and GR can be practically used for management of the serious vector in water environment. © 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Introduction Mosquitos (Culicidae) are vectors which carry several serious human diseases from person to person without having the symptoms themselves. Transmission of mosquito-borne diseases may be accelerated by global climate change. Various mosquito species can transmit different diseases, such as malaria (transmitted by the genus Anopheles) and lymphatic filariasis (primarily transmitted by Anopheles spp., Aedes spp., and Culex spp.) (Rozendaal, 1997; Verma and Prakash, 2010). Most of mosquito-borne viral diseases are transmitted by Aedes spp., such as yellow fever, dengue, dengue hemorrhagic fever (Rozendaal, 1997), and chikungunya fever (Powers et al., 2000). Aedes spp. are distributed worldwide. In tropical countries, Ae. aegypti is an important vector of yellow fever, dengue fever, and Chikungunya fever (Rozendaal, 1997; Hochedez et al., 2006); Ae. albopictus is a closely related species that also can transmit viral diseases, such as several types of encephalitis (Savage et al., 1994; Gerhardt et al., 2001), and is known to transmit even several filarial nematodes such as Dirofilaria immitis (Cancrini et al., 2003). Ae. albopictus has become an epidemiologically important vector. ⁎ Corresponding authors at: Department of Agricultural Biology, College of Agriculture and Life Sciences, Chonbuk National University, Republic of Korea. Tel.: + 82 63 270 2525; fax: +82 63 270 2531. E-mail addresses: [email protected] (Y.-S. Nai), [email protected] (J.S. Kim).
To control Aedes vectors, several insecticides have been employed as larvicides that are applied to mosquito breeding sites or adulticides applied to mosquito adults; modes of application include insecticide residual sprays (IRS), space sprayings, treated/impregnated materials (Vontas et al., 2012), and thermal fogging. Mosquitocides are diverse and include organophosphates (e.g. temephos), insect growth regulators (e.g. pyriproxyfen), pyrethroids (Kroeger et al., 2006; World Health Organization, 2011), and bacterial toxins (e.g. Bacillus thuringiensis serovar israelensis) among others. Although insecticidebased interventions have efficiently controlled Aedes mosquito populations, it has been reported that Ae. aegypti shows resistance to all four classes of insecticides (carbamates, organochlorines, organophosphates, and pyrethroids) (Ponlawat et al., 2005; Ranson et al., 2010). Due to the negative effect of insecticides on ecosystems and substantial increase in physiological resistance shown by mosquitoes (Hargreaves et al., 2000), biological control agents which can reduce the drawbacks associated with insecticides (Krischbam, 1985) should be considered. The use of entomopathogenic fungi can be an alternative control method, and some efforts have already been conducted. Fungal pathogens such as Lagenidium, Coelomomyces, and Culicinomyces are known to affect mosquito populations, and have been studied extensively. However, many other fungi infect and kill mosquitoes at the larval and/or adult stage (Scholte et al., 2004), such as Metarhizium anisopliae isolates, Beauveria tenella, and Lagenidium giganteum (Balaramn et al.,
http://dx.doi.org/10.1016/j.aspen.2015.02.003 1226-8615/© 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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1979; Lecy et al., 1988; Scholte et al., 2004; Vyas et al., 2007). Besides, fungi have the distinct advantage of being able to recycle in stagnant water, injecting multiple and overlapping generations of mosquitoes (Legner, 1995). Although there are some efforts to use entomopathogenic fungi in mosquito control (Scholte et al., 2004), however little consideration was given to the other species, such as Beauveria brongniartii, Cordyceps brongniartii, Paecilomyces spp. and Purpureocillium spp. especially against Ae. albopictus larvae. Moreover, the several formulations of fungal conidia, including water- or oil-based suspensions, granules, dusts, and floating formulations (Ramoska et al., 1981; Daoust et al., 1982) have been tested for larval stages of mosquito control, however millet-based fungal granules received little interest in this area. This millet-based formulation has advantages in mass production, application to water, and enhancement of fungal survival after the application. Thus herein, we isolated 12 entomopathogenic fungi from soil samples and assayed their virulence against Asian tiger mosquito (Ae. albopictus) larvae in laboratory conditions. The infectious process of the selected entomopathogenic fungus was also investigated by infecting with the EGFP-fungal mutant, which was generated by restriction enzyme-mediated integration (REMI) method; and also, we tried to apply millet-based fungal granules as a novel approach to control Ae. albopictus larvae in the natural environment.
Materials and methods Mosquito larvae The Ae. albopictus larvae used in this study were kindly provided by the Insect Physiology Laboratory of Chonbuk National University, Korea. The mosquito larvae were kept at 28 ± 1 °C, 65 ± 5% of relative humidity (RH) with a photoperiod of 16:8 (L:D) and feed with artificial diet in the laboratory (Shin and Lee, 2014).
Isolation and identification of entomopathogenic fungi Entomopathogenic fungal isolates were collected from soil by placing Tenebrio molitor (mealworm) larvae on the soil for two weeks and isolating the out-grown fungi from the surfaces of cadavers. The collection of soil samples are summarized in Table 1. The fungal isolates were identified by sequencing the internal transcribed spacer (ITS) region and examining the morphological characteristics. The newly identified and recorded isolates in Korea were firstly stored at Inset Molecular and Biotechnological Laboratory (IMBL) of Chonbuk National University as conidial suspensions in 20% glycerol at −80 °C (Humber, 1997) and then deposited at the National Institute of Biological Resources (NIBR; www.nibr.go.kr) (Incheon, Korea). Table 1 Fungal species isolated from soil samples in Korea in 2012–2103. Fungal species
Isolate code
Sample locations
Beauveria bassiana B. bassiana B. brongniartii Cordyceps brongniartii Metarhizium anisopliae M. anisopliae Paecilomyces carneus Pa. javanicus Pa. spinulosum Purpureocillium lilacinum Pu. lilacinum Verticillium leptobactrum
JEF-006 JEF-007 KJS-004 KJS-008 JEF-003 JEF-004 KJS-010 KJS-005 KJS-006 KJS-003 KJS-011 KJS-012
Gangwon-do (N 37.29.30.05 E 127.59.05.66) Gangwon-do (N 37.29.30.05 E 127.59.05.66) Jeju island (N 33.21.27.35 E 126.27.47.25) Ulleung island (N 37.30.22.89 E 130.51.25.78) Gangwon-do (N 37.29.30.05 E 127.59.05.66) Gangwon-do (N 37.29.30.05 E 127.59.05.66) Ulleung island (N 37.30.22.89 E 130.51.25.78) Jeju island (N 33.21.27.35 E 126.27.47.25) Jeju island (N 33.21.27.35 E 126.27.47.25) Jeollabuk-do (N 35.50.48.33 E 127.07.45.85) Ulleung island (N 37.30.22.89 E 130.51.25.78) Ulleung island (N 37.30.22.89 E 130.51.25.78)
Solid culture condition The fungal isolates were cultured on quarter-strength Sabouraud dextrose agar (1/4 SDA) (pH 6) in Petri dishes (60-mm diam.) in the dark at 25 ± 1 °C for 10 days (Humber, 2005). Conidia were collected by vortexing the agar blocks (6 mm diam.) in an Eppendorf-tube containing 0.03% siloxane solution (Silwet L-77, Momentive Performance Materials Inc.). The numbers of conidia were counted with a hemocytometer at 400 × magnification (3 times per isolate). Conidia with N90% viability were kept in a refrigerator at 4 °C for 3–4 days before use. Millet-based solid culture The fungal isolates were solid cultured using the millet-based culture system (Bartlett and Jaronski, 1988; Li and Feng, 2005; Kim et al., 2011). To prepare inocula for the solid cultures, a conidial suspension from 3 sporulated agar blocks (6-mm diam.) of M. anisopliae JEF-003 was added to quarter-strength Sabouraud dextrose broth in a 250-ml flask. Flasks were held on a rotary shaker (150 rpm) at 25 ± 2 °C for 3 days. Millet (Panicum miliaceum) grains (200 g) were placed in a polyvinyl bag, soaked in 100 ml of water containing citric acid (0.4 ml l−1) and autoclaved at 120 °C for 15 min. The bag was then cooled to ambient temperature. The liquid culture broth (3 ml) was introduced into the bag and thoroughly mixed. All bags were held for 6 days at 25 ± 1 °C and a 16:8 (L/D) photoperiod. After incubation, mycotized GRs were dried at ambient temperature for 3 days, until the moisture content reached b 5% (determined by a moisture analyzer [Sartorius Omnimark]). All batches of mycotized GRs were assessed for conidial concentration (g−1) with a hemocytometer. Fungal transformation To observe the infection of the selected isolate (M. anisopliae JEF-003) in Ae. albopictus larvae, a transgenic M. anisopliae JEF-003 was generated by a fungal transformation using the pBARKS1-egfp. Briefly, the egfp expression cassette was released from pBluscript II KS (+)-egfp by ClaI and SacI digestion and then cloned into the ClaI/SacIdigested pBARKS1 vector and confirmed by commercial sequencing (MACRO GEN, Korea). The pBARKS1-egfp, has phosphinothricin (PPT) resistant bar fungal selection marker and EGFP genes, both of them are expressed under the control of gpdA promoter. The plasmid, linearized by restriction enzyme digestion, was integrated into the genomic DNA of M. anisopliae JEF-003 by the REMI method based on blastospores (Ying and Feng, 2006; Kim et al., 2013). The M. anisopliae JEF-003 EGFP-transformant was cultured on Czapek's agar supplement with 600 μg ml−1 phosphinothricin. The M. anisopliae JEF-003 EGFPtransformant was subjected to the infection test in the mosquito larvae and observed under fluorescence microscopy. Mosquitocidal assay Virulence of 12 entomopathogenic fungal isolates against Ae. albopictus larvae was investigated as follows. Conidial suspensions were adjusted to 1 × 107 conidia ml−1 using 0.03% siloxane solution in 90-mm-diameter Petri dishes at 10 ml dish−1. Fifteen third-instar larvae were placed in the dish using a disposable dropper. Dishes were covered with lids and kept at 25 ± 1 °C and 16:8 (L/D) photoperiod and examined daily for 8 days. For dosage dependent assay, the conidial concentration of the selected isolate (M. anisopliae JEF-003) was 10-fold diluted from 1 × 105 to 1 × 107 conidia ml−1 and assayed with same petri-dish conditions as described above. This experiment was replicated 3 times. Virulence assay of M. anisopliae JEF-003 GR To investigate the potential control efficacy of GR, mycotized GRs were applied to the water in 90-mm-diam Petri dishes at 1 g dish−1
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and 15 larvae were transferred to each dish. Untreated control served as a negative control. The number of dead mosquito larvae was observed daily. This experiment was replicated 3 times. Data analysis The data on percentage of live Ae. albopictus larvae were analyzed using a general linear model (GLM) followed by Tukey's honest significant difference (HSD) test for multiple comparisons. All analyses were conducted using SPSS ver. 17.1 (SPSS Inc., 2009) at the 0.05 (α) level of significance. Results and discussion Fig. 2. Dosage dependent assay of M. anisopliae JEF-003 against Ae. albopictus larvae.
Isolation and mosquitocidal activity of entomopathogenic fungal isolates From our soil isolations, a total of 12 entomopathogenic fungi were isolated from soil samples in Korea, the sampling details were summarized in Table 1. The results of ITS sequences and fungal morphological identification indicated that these 12 fungal isolates belonged to 6 genera, Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium, and Verticillium. There are more than 13 different fungal genera reported as potential mosquito-pathogenic fungi, such as Lagenidium, Coelomomyces, Entomophthora, Culicinomyces, Beauveria, and Metarhizium (Scholte et al., 2004). Despite many fungi having the potential to be mosquito control agents, a few have been commercialized and applied. New isolates from the domestic area and additional investigation are needed in order to contribute to an expansion of current mosquito control tools which have limited effectiveness. Twelve entomopathogenic fungi were assayed against Ae. albopictus larvae in Petri-dish conditions; in this assay, 11 fungal isolates showed different levels of mosquitocidal activity ranging from 20% to 100% at 8 days post-inoculation (dpi.); Verticillium leptobactrum KJS-012 showed no activity. Among the mosquitocidal fungi, 3 isolates (B. brongniartii KJS-004, and M. anisopliae JEF003 and JEF004) showed high virulence to Ae. albopictus larvae (Fig. 1). Of these 3 most virulent isolates, M. anisopliae JEF-003 showed the fastest virulence, which resulted in approximately 73% mortality rate at 2 dpi. and N 90% mortality rate at 5 dpi., and followed by M. anisopliae JEF004 (~ 47% mortality rate at 2 dpi. and ~ 80% mortality rate at 5 dpi.) (Fig. 1). Additionally, M. anisopliae JEF-003-infected larvae were completely disrupted morphologically (Fig. 1, arrow); fungal conidia
were found on the surface of dead larvae and larvae in the untreated control were healthy. M. anisopliae JEF-003 showed a dosagedependent mosquitocidal activity and had the highest virulence at 1 × 107 conidia ml−1 (Fig. 2). The evaluation of M. anisopliae JEF-003 infection process in mosquito larvae was further evaluated by using the M. anisopliae JEF-003 EGFP-transformant; hyphal growth in the larvae was observed under the fluorescence (Fig. 3). Many fungi that infect and kill mosquitoes at the larval and/or adult stage have been well studied. The spores of entomopathogenic fungus Fusarium pallidoroseum and Chrysosporium tropicum were reported to be effective against adults of female Cx. quinquefasciatus (Mohanty et al., 2008; Verma and Prakash, 2010). Metarhizum anisopliae, Beauveria tenella, Lagenidium giganteum, C. lobatum, and C. tropicum are used against mosquito larvae (Balaramn et al., 1979; Federici, 1981; Lecy et al., 1988; Scholte et al., 2004; Vyas et al., 2007; Mohanty and Prakash, 2008). According to our presented data, this is the first instance of B. brongniartii infecting Ae. albopictus larvae in the laboratory, although the virulence of B. brongniartii was lower than the other two fungal species. This result provides additional impetus to study the applications of entomopathogenic fungi; in particular, the diversity of these fungi should be emphasized. Both B. bassiana and M. anisopliae have been reported as virulent against several Culex spp., Aedes spp., and other mosquito species, primarily larvae (Pinnock et al., 1973; Ramoska et al., 1981; Daoust et al., 1982; Lecy et al., 1988; Ravallec et al., 1989). In this study, we support previous results with new data regarding soil-borne entomopathogenic fungal species.
Fig. 1. Fungal isolates from soil samples and their pathogenicity. Isolate Metarhizium anisopliae JEF-003 showed fastest pathogenicity against the Aedes albopictus larvae at 2 days after inoculation. The arrow indicates that M. anisopliae JEF-003-infected larvae were completely disrupted morphologically. Scale bar = 0.5 mm.
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Fig. 3. Observation of M. anisopliae JEF-003 EGFP-transformant-infected Ae. albopictus larvae. Mycelial growth in the larvae was observed under fluorescence.
Ae. albopictus larvae prefer temporary water-holding containers such as tree holes, leaf axils, ground pools, and coconut shells. Larvae come to the water surface for respiration and dive to the bottom for feeding or escaping danger. Fungal conidia germinate and penetrate into the respiratory siphon and block the breathing mechanism of mosquito larvae, usually leading to death before significant invasion of the hemocoel in mosquito larvae (Daoust et al., 1982; Lecy et al., 1988). Thus, hyphal formation is not usually observed and cadavers in the aquatic environment are often found with bacterial infection rather than mycelial infection; typically no new conidia are produced (Scholte et al., 2004). According to our results, hyphae and conidia
of M. anisopliae JEF-003 were found on the surface of dead larvae; moreover, according to our preliminarily results, water depth ranging from 3 cm to 15 cm caused no significant influence on the virulence of this fungus (data not shown). These results indicate a fungal strain with an increased possibility of infecting mosquito larvae, and provide new insight on the mosquito larvae control in water environment. Potential of millet-based M. anisopliae JEF-003 GR M. anisopliae JEF-003 GRs were applied to the water in Petri dishes. Some fungal conidia detached from the millet granules when GRs
Fig. 4. Application of M. anisopliae JEF-003 GR to water Petri dishes. Virulence of GRs showed high mortality (N90%) after 5 days of treatment and untreated control showed less than 10% mortality.
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were submerged in water for 2 min. GR virulence revealed N 90% mortality after 5 days of treatment and the untreated control showed less than 10% mortality (Fig. 4). Several types of conidia formulations have been developed and tested for mosquito larval control including granulars, dusts, and wettable suspensions. Among these formulations, M. anisopliae conidia had decreased virulence against Culex pipiens larvae when in aqueous suspensions containing a surfactant or formulations of granular carriers or dust diluents, but significantly enhanced conidial virulence was achieved with a dried castor oil formulation (Daoust et al., 1982). Using GRs, we found virulence as high and stable in the water condition (N 90% mortality). This finding provides the advantage that millet in GR application could also serve a nutritional source for fungal colonization in the natural environment. Moreover, it has been shown that conidia, stored under dry conditions showed higher germination rates than conidia formulated in solution formulation (e.g. paraffin oil) (MorleyDavies et al., 1995). Entomopathogenic fungi are virulent to most insects and cause fungal diseases. Different from many other pathogens, fungi directly infect the host insect by penetrating into the cuticle, making ingestion by the insect unnecessary (Vega and Kaya, 2012). There are several advantages for fungal control of mosquito larvae, for example: easy colonization of the water environment, conidia produced on the larval surface could be transferred to other mosquito populations, and inputs of harmful synthetic chemical pesticides in agriculture, horticultural, and forest systems could be reduced (Strasser et al., 2000). The millet-based fungal GR formulation could increase the possibility of fungus survival in the water condition. However, several application aspects should be still considered and further investigated in the near future (e.g. time for preparing the GR formulation and spread methods). Acknowledgments We thank the Insect Physiology Laboratory in Chonbuk National University for providing the Ae. albopictus larvae. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Project No: 2013R1A1A1057946) and the Research Service Task (Collection of Fungi) from the National Institute of Biological Resources, 2013–2014. References Balaramn, K., Bheema, R.U.S., Rajagopalan, P.K., 1979. Isolation of Metarhizium anisopliae, Beauveria tenella and Fusarium oxysporum (Deuteromycetes) and their pathogenicity to Culex fatigans and Anopheles stephensi. Indian J. Med. Res. 70, 718–722. Bartlett, M.C., Jaronski, S.T., 1988. Mass production of entomopathogenous fungi for biological control of insects. In: Burge, M.N. (Ed.), Fungi in Biological Control Systems. Manchester University Press, Manchester, pp. 61–85. Cancrini, G., di Regalbono, A.F., Ricci, I., Tessarin, C., Gabrielli, S., Pietrobelli, M., 2003. Aedes albopictus is a natural vector of Dirofilaria immitis in Italy. Vet. Parasitol. 118, 195–202. Daoust, R.A., Ward, M.G., Roberts, D.W., 1982. Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae. J. Invertebr. Pathol. 40, 228–236. Federici, B.A., 1981. Mosquito control by fungi Culicinomyces, Lagenidium and Coelomomyces, in: Microbial Control of Pests & Plant Diseases 1970–1980 (Ed.). Academic, New York, pp. 555–572. Gerhardt, R.R., Gottfried, K.L., Apperson, C.S., Davis, B.S., Erwin, P.C., Smith, A.B., Panella, N.A., Powell, E.E., Nasci, R.S., 2001. First isolation of La Crosse virus from naturally infected Aedes albopictus. Emerg. Infect. Dis. 7, 807–811. Hargreaves, K., Koekemoer, L.L., Brooke, B.D., Hunt, R.H., Methembe, J., Coetzee, M., 2000. Anopheles funestus resistant to pyrethroid insecticides in South Africa. Med. Vet. Entomol. 2, 181–189. Hochedez, P., Jaureguiberry, S., Debruyne, M., Bossi, P., Hausfater, P., Brucker, G., Bricaire, F., Caumes, E., 2006. Chikungunya infection in travelers. Emerg. Infect. Dis. 12 (10).
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