Parasitology Research

, Volume 112, Issue 3, pp 1113–1123

Essential oil composition, adult repellency and larvicidal activity of eight Cupressaceae species from Greece against Aedes albopictus (Diptera: Culicidae)

Authors

  • Athanassios Giatropoulos
    • Laboratory of Biological Control of PesticidesBenaki Phytopathological Institute
    • Laboratory of Agricultural Zoology and EntomologyAgricultural University of Athens
  • Danae Pitarokili
    • Department of Pharmacognosy and Chemistry of Natural Products, School of PharmacyUniversity of Athens
  • Fotini Papaioannou
    • Department of Pharmacognosy and Chemistry of Natural Products, School of PharmacyUniversity of Athens
  • Dimitrios P. Papachristos
    • Laboratory of Agricultural Entomology, Department of Entomology and Agricultural ZoologyBenaki Phytopathological Institute
  • George Koliopoulos
    • Laboratory of Biological Control of PesticidesBenaki Phytopathological Institute
  • Nickolaos Emmanouel
    • Laboratory of Agricultural Zoology and EntomologyAgricultural University of Athens
    • Department of Pharmacognosy and Chemistry of Natural Products, School of PharmacyUniversity of Athens
    • Laboratory of Agricultural Entomology, Department of Entomology and Agricultural ZoologyBenaki Phytopathological Institute
Original Paper

DOI: 10.1007/s00436-012-3239-5

Cite this article as:
Giatropoulos, A., Pitarokili, D., Papaioannou, F. et al. Parasitol Res (2013) 112: 1113. doi:10.1007/s00436-012-3239-5

Abstract

The present study evaluated leaf essential oils from eight Cupresaceae species; Cupressus arizonica, Cupressus benthamii, Cupressus macrocarpa, Cupressus sempervirens, Cupressus torulosa, Chamaecyparis lawsoniana, Juniperus phoenicea, and Tetraclinis articulata for their larvicidal and repellent properties against Aedes albopictus, a mosquito of great ecological and medical importance. Based on the LC50 values, C. benthamii essential oil was the most active (LC50 = 37.5 mg/L) while the other tested Cupressaceae essential oils provided rather moderate toxicity against larvae (LC50 = 47.9 to 70.6 mg/L). Under the used laboratory conditions, three of the essential oils (C. benthamii, C. lawsoniana, and C. macrocarpa) provided sufficient protection against mosquito adults, equivalent to the standard repellent “Deet” in the 0.2 mg/cm2 dose, while C. macrocarpa assigned as the superior repellent oil in the 0.08 mg/cm2 dose. Chemical analysis of the essential oils using gas chromatography and gas chromatography–mass spectrometry revealed the presence of 125 components.

Introduction

Aedes (Stegomyia) albopictus (Skuse 1894), the so-called “Asian tiger mosquito,” is listed as one of the “100 of the World’s Worst Invasive Alien Species” (ISSG 2009) and it is considered to be the most invasive mosquito species in the world (Enserink 2008). Native to tropical and subtropical regions of South-East Asia, it has undergone an astonishing expansion of its range, primarily through the international trade of used tires, within the last three decades (Enserink 2008). After its first detection in 2003–2004, Ae. albopictus has been subsequently found in several areas in Greece, while considerably high populations of this species have recently been reported from urban areas of the Greek capital Athens (Giatropoulos et al. 2012a; Giatropoulos et al. 2012b).

Ae. albopictus is a laboratory-competent vector of at least 22 arboviruses, notably Dengue virus, the most important arboviral disease in humans (Gratz 2004). Greece experienced the most serious dengue fever epidemic in Europe during the second half of the 1920s, as more than a million cases were reported with more than 1,500 deaths (Louis 2012). Concern has been raised, nowadays, when two recent cases of dengue fever from autochthonous transmission in France and Croatia (La Ruche et al. 2010; Gjenero-Margan et al. 2011) signed the re-emergence of this disease in continental Europe. Besides, Ae. albopictus was the primary vector involved in outbreaks of Chikungunya virus in Italy in 2007 (Rezza et al. 2007) and in France in 2010 (Grandadam et al. 2011).

Mosquito control and personal protection from mosquito bites are currently the most important measures to control these diseases. Mosquito control is primarily targeted at larval stages in breeding sites using larvicides, since adulticides may reduce the adult population only temporarily (Lim et al. 2011). Repeated use of synthetic larvicides has fostered several health and environmental concerns including widespread development of resistance and undesirable effects on non-target organisms (Isman 2000; Hemingway et al. 2002; Khan et al. 2011). Thus, there is an urgent need to develop new insecticides for controlling mosquitoes, which are more environmentally safe, biodegradable, and target-specific against mosquitoes (Semmler et al. 2009; Kumar et al. 2011a).

Repellents are also widely used to prevent the transmission of mosquito-borne diseases by minimizing contact between humans and vectors (Fradin and Day 2002). N,N-diethyl-3-methylbenzamide (Deet) remains the gold standard of currently available insect repellents (Fradin 1998). However there have been case reports of Deet toxicity in the literature (Clem et al. 1993; Qiu et al. 1998; Sudakin and Trevathan 2003) causing a growing demand from consumers for natural alternative repellents.

Essential oils (EOs) from plants and their major constituents, monoterpenes and sesquiterpenes, may be an alternative source of mosquito larva control and mosquito repellent agents. They are relatively safe and pose fewer risks to the environment, with minimal impacts to animal and human health, and often act at multiple and novel target sites, thereby reducing the potential for resistance (Perumalsamy et al. 2009; Isman 2006). EOs exhibit biological activity against a wide spectrum of insect pests and may act as fumigants, contact insecticides, repellents, and antifeedants or they can adversely affect the growth rate, reproduction, and behavior of insect pests (Isman 2000; Papachristos and Stamopoulos 2002; Papachristos et al. 2004; Kumar et al. 2011b). A broad spectrum of plant essential oils has been tested as potential mosquito larvicides, while many plant extracts and essential oils repel mosquitoes’ adults (Sukumar et al. 1991; Michaelakis et al. 2009; Maia and Moore 2011; Pitarokili et al. 2011; Evergetis et al. 2012; Regnault-Roger et al. 2012).

According to Flora Europaea, Cupressaceae family is represented in Europe by five genera: Cupressus, Chamaecyparis, Thuja, Tetraclinis, and Juniperus. Cupressaceae species consist of monoecious or dioecious resiniferous trees or shrubs (Moore 1993). Plants of Cupressaceae family are a well-known source of essential oils, exerting different biological actions on humans and insects (Adorjan and Buchbauer 2010). In folk medicine, the use of preparations or EOs from Cupressaceae plants is referred to have many pharmaceutical properties to humans (Newall et al. 1996). Essential oils of plant species belonging to Cupressaceae family, primarily from the genera Juniperus and Cupressus, are reported to display potent repellent and larvicidal activities against the mosquitoes Culex pipiens, Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi (Barnard 1999; Prajapati et al. 2005; Trongtokit et al. 2005; Amer and Mehlhorn 2006a; b; c; Lee 2006; Carroll et al. 2011; Sedaghat et al. 2011; Vourlioti-Arapi et al. 2012). To the best of our knowledge, this is the first report on the larvicidal and repellent activity of Cupressaceae EOs against Ae. albopictus.

Therefore, in the present study, larvicidal and adult repellent effects of EOs from eight Cupressaceae species, namely Cupressus arizonica Greene, Cupressus benthamii Endl., Cupressus macrocarpa Hartw. ex Gordon, Cupressus sempervirens L., Cupressus torulosa D. Don, Chamaecyparis lawsoniana (A. Murray) Parl., Juniperus phoenicea L., and Tetraclinis articulata Mast., were examined against Ae. albopictus. In addition, the chemical composition of these EOs obtained from hydrodistillation was determined by GC and GC-MS analyses.

Materials and methods

Plant materials

Aerial parts of C. arizonica, C. benthamii, C. macrocarpa, C. sempervirens, C. torulosa, C. lawsoniana, J. phoenicea, and T. articulata were collected in June 2011 from J. & A. N. Diomedes Botanic Garden, University of Athens, County Attiki. Voucher specimens have been deposited in the Herbarium of the University of Athens.

Isolation of the essential oils

Fresh leaves were separated from branches and were further cut in small pieces and subjected to hydrodistillation for 3 h, using a modified Clevenger-type apparatus. The oils were obtained using n-pentane as a collecting solvent, and subsequently, they were dried over anhydrous sodium sulfate and stored under N2 atmosphere in amber vials at 4 °C until they were analyzed.

Gas chromatography analysis

Gas chromatography (GC) analysis was carried out using a SRI 8610C GC-FID system, equipped with DB-5 capillary column (30 m × 0.32 mm; film thickness 0.25 μm) and connected to a FID detector. The injector and detector temperature was 280 °C. The carrier gas was He, at flow rate of 1.2 mL/min. The thermal program was 60–280 °C at a rate of 3 °C/min. Two replicates of each oil sample were processed in the same way.

Gas chromatographymass spectrometry (GC-MS) analysis

Analyses of the oils were performed using a Hewlett Packard 5973-6890 GC-MS system operating in the EI mode at 70 eV, equipped with a split/splitless injector (200 °C). The transfer line temperature was 250 °C. Helium was used as carrier gas (1 mL/min) and the capillary column used was HP-5MS (30 m × 0.25 mm; film thickness 0.25 μm). The temperature program was the same with that used for the GC analysis; split ratio 1:10. The injected volume was 1 μL. Acquisition mass range 40–400 amu.

Identification of components

The identification of the compounds was based on comparison of their retention indices (RI), their retention times (RT), and mass spectra with those obtained from authentic samples (purchased from the Sigma-Aldrich Group) and/or the NIST/NBS, Wiley libraries, and the literature (Adams 2007).

Mosquito rearing

Mosquito larvae were obtained from a laboratory colony of Ae. albopictus which was maintained at 25 ± 2 °C, 80 % relative humidity, and photoperiod of LD 16:8 h, in the laboratory of Benaki Phytopathological Institute, Kifissia, Greece. Adult mosquitoes were kept in wooden framed cage (33 × 33 × 33 cm) covered by a 32 × 32 mesh, with easy access to 10 % sucrose solution through a cotton wick. Females were blood fed from senior author’s forearm, once a fortnight. Larvae were reared in tap water-filled cylindrical enamel pans with diameter of 35 and 10 cm deep covered by fine muslin. Approximately 400 larvae were fed ad libitum with powdered fish food (JBL Novo Tom 10 % Artemia) in each pan until the adults emerged. Adult mosquitoes were often collected using mouth aspirator and transferred to the rearing cage. Plastic beakers with 100 mL water and strips of moistened filter paper were provided in the cage for oviposition. The eggs were kept wet for few days and then placed in the pans for hatching.

Larvicidal bioassays

The larval mortality bioassays were carried out according to the test method of larval susceptibility as suggested by the World Health Organization (2005) with modifications. Sufficient amounts of each compound were transferred to a vial and the residual solvent was removed under high vacuum. Stock solutions of each test compound in dimethyl sulfoxide (DMSO) were prepared with a concentration of 10 % w/v (10 mg of compound in 100 μL DMSO). Twenty late third to early fourth-instar mosquito larvae were placed in 2 % v/v aqueous solution of DMSO (98 mL of tap water plus 2 mL of DMSO), followed by addition of the tested material solution. Gentle shaking to ensure a homogeneous test solution was then performed. Four replicates per dose were made and a treatment with 98 ml of tap water and 2 mL of DMSO was included in each bioassay as control.

Repellent bioassays

For the repellent activity of each EO, the assessment was based on the human landing counts (Coleman et al. 1993; Govere and Durrheim 2006) applying a protocol previously developed (Giatropoulos et al. 2012c). The study was conducted into a cage (33 × 33 × 33 cm) covered by a 32 × 32 mesh and with a 20-cm diameter circular opening fitted with cloth sleeve. Each cage contained 100 adult mosquitoes (sex ratio 1:1), 5–10-day-old, starved for 12 h at 25 ± 2 °C and 70–80 % relative humidity. A plastic glove with an opening measuring 5 × 5 cm was employed for all the bioassays. Different doses (0.05 to 1 mg/cm2) for Deet (provided by Bayer CropScience) were applied and found that the lowest dose, where zero landings were counted, was ≈0.2 mg/cm2. All EOs were applied on paper (Whatman chromatography paper) of 24 cm2 total area and tested at the “high” dose of 0.2 mg essential oil per cm2 of paper taken from 100 μg/μL stock solution of EO in dichloromethane (DCM). EOs that provided total protection (zero landings) were further tested at the “low” dose of 0.08 mg/cm2 of EO in DCM. The paper was placed around the glove opening. Five minutes after treatment with the testing material (to let the solvent evaporate), the treated area (glove with filter paper) was inserted for 5 min through the sleeve into the cage. Control treatments without the EOs and with Deet were also included for the repellency tests as standards (control and positive control, respectively). Each treatment was repeated eight times and four human volunteers were used.

Data analysis

Larvicidal effect for lethal bioassays was recorded 24 h after treatment. Data obtained from each dose–larvicidal bioassay (total mortality per milligram per liter of each concentration in water) were subjected to probit analysis in which probit-transformed mortality was regressed against log10-transformed dose; LC50, LC90 values, and slopes were generated (Finney 1971). Four samples were used in each experiment (n = 4).

Data concerning the repellency of ΕΟs (mosquito landings) were analyzed using Kruskal–Wallis test. When significant differences were detected, Mann–Whitney U tests were carried out for pair-wise comparison. Bonferroni correction was applied to correct for pair-wise comparisons leading to an adjusted a = 0.0011 and 0.0033 for high and low dose applied.

All analyses were conducted using the statistical package SPSS 14.0 (SPSS Inc., Chicago, IL, 2004).

Results and discussion

Chemical analysis

A total of 125 components were identified, constituting the 95.7–99.9 % of the total oils. The composition of the studied essential oils is shown in Table 1 in order of their elution on the HP-5 MS column.
Table 1

Chemical composition of the studied essential oils

Constituents

RI

CARL

CBEL

CMAL

CSEL

CTOL

CLAL

JPHL

TARL

Tricyclene

924

tr

1.7

a-Thujene

928

tr

2.6

2.1

tr

tr

0.8

a-Pinene

937

17.1

9.1

19.5

54.1

27.9

2.9

31.3

26.4

a-Fenchene

950

tr

tr

1.2

0.8

Camphene

952

tr

1.6

0.6

tr

0.3

tr

0.4

1.9

Thuja-2,4(10)-diene

965

tr

tr

tr

0.1

Verbenene

965

tr

tr

tr

Sabinene

973

2.8

6.5

21.8

2.4

tr

5.4

tr

0.4

β-Pinene

977

tr

0.8

2.7

2.5

1.9

tr

1.6

0.8

Myrcene

988

1.6

3.0

4.1

4.6

3.3

1.6

4.8

3.3

α-Phellandrene

1,000

tr

tr

tr

δ-3-Carene

1,009

 

9.5

tr

10.8

21.4

12.5

tr

a-Terpinene

1,015

tr

1.7

5.7

tr

tr

tr

0.3

tr

p-Cymene

1,022

1.5

tr

tr

tr

0.6

1.0

tr

Limonene

1,027

7.4

13.0

1.7

3.2

3.1

18.5

8.7

β-Phellandrene

1,028

1.0

0.9

tr

0.4

13.0

(Ζ)-β-Ocimene

1,035

0.9

(E)-β-Ocimene

1,048

 

tr

tr

tr

tr

tr

γ-Terpinene

1,057

1.5

2.2

7.9

1.0

0.4

1.7

0.7

0.4

cis-Sabinene hydrate

1,068

tr

tr

0.4

tr

Fenchone

1,084

tr

tr

Terpinolene

1,086

tr

1.6

2.8

4.1

4.3

0.8

1.9

0.6

p-Cymenene

1,089

0.5

tr

tr

tr

tr

tr

Linalool

1,094

tr

0.8

tr

tr

tr

tr

trans-Sabinene hydrate

1,096

tr

tr

0.5

n-Nonanal

1,098

tr

tr

tr

 

1,3,8-p-Menthatriene

1,108

tr

tr

tr

tr

trans-Thujone

1,112

tr

tr

tr

tr

cis-p-Menth-2-en-1-ol

1,119

tr

0.4

1.3

0.6

0.5

α-Campholenal

1,124

tr

tr

0.2

0.7

trans-Pinocarveol

1,136

0.5

trans-p-Mentha-2-en-1-ol

1,137

0.9

tr

tr

tr

Camphor

1,144

2.8

1.0

tr

tr

9.5

Camphene hydrate

1,147

tr

0.7

Citronellal

1,151

0.6

Pinocarvone

1,162

tr

tr

tr

tr

Borneol

1,167

tr

tr

4.7

p-Mentha-1,5-dien-8-ol

1,168

0.8

tr

Umbellulone

1,169

8.0

15.9

tr

cis-Pinocamphone

1,173

tr

Terpinen-4-ol

1,175

3.5

4.1

18.9

1.5

tr

6.9

0.3

1.0

p-Cymen-8-ol

1,181

tr

0.7

tr

tr

tr

α-Terpineol

1,186

tr

0.7

2.0

tr

tr

0.6

0.9

1.0

(4Z)-Decenal

1,191

1.0

cis-Piperitol

1,193

tr

tr

tr

Verbenone

1,203

tr

tr

tr

trans-Piperitol

1,206

0.6

0.5

tr

tr

trans-Carveol

1,214

tr

tr

tr

endo-Fenchyl acetate

1,218

tr

tr

tr

Citronellol

1,223

1.1

tr

tr

Thymol, methyl ether

1,233

tr

1.1

tr

tr

0.4

tr

tr

Carvone

1,241

tr

tr

tr

tr

Car-3-en-2-one

1,246

tr

Piperitone

1,250

tr

tr

tr

0.9

(4Z)-Decen-1-ol

1,257

0.7

Perilla aldehyde

1,269

0.6

Isopulegyl acetate

1,275

0.8

Isobornyl acetate

1,283

tr

tr

tr

Bornyl acetate

1,286

tr

0.9

tr

0.8

25.3

Thymol

1,288

tr

2.6

3-Thujanol acetate

1,292

0.4

Methyl myrtenate

1,293

9.7

Carvacrol

1,297

tr

tr

tr

α-Terpinyl acetate

1,347

1.4

5.0

5.5

5.9

2.7

12.5

1.3

α-Copaene

1,375

tr

tr

tr

tr

Geranyl acetate

1,378

tr

tr

β-Elemene

1,387

tr

0.4

tr

Sibirene

1,397

0.4

(E)-Caryophyllene

1,416

tr

tr

tr

tr

0.6

1.4

2,5-Dimethoxy-p-cymene

1,423

0.4

cis-Muurola-3,5-diene

1,447

6.5

tr

0.7

tr

trans-Muurola-3,5-diene

1,450

15.5

0.3

a-Humulene

1,451

tr

tr

tr

tr

tr

0.6

0.4

cis-Cadina-1(6),4-diene

1,460

tr

1.6

tr

trans-Cadina-1(6),4-diene

1,473

tr

0.9

γ-Muurolene

1,476

tr

tr

tr

tr

tr

Germacrene D

1,482

tr

2.4

tr

1.3

0.6

β-Selinene

1,487

tr

tr

tr

trans-Muurola-4(14),5-diene

1,490

0.7

tr

epi-Cubebol

1,492

0.6

tr

α-Muurolene

1,498

5.6

tr

tr

tr

tr

Epizonarene

1,499

0.5

γ-Cadinene

1,510

tr

tr

tr

tr

tr

Cubebol

1,512

tr

1.5

trans-Calamene

1,519

5.2

δ-Cadinene

1,520

tr

0.7

tr

tr

1.0

1.0

0.4

10-epi-Cubebol

1,532

0.7

 

a-Cadinene

1,536

tr

tr

tr

tr

a-Calacorene

1,542

tr

tr

tr

tr

tr

tr

cis-Muurol-5-en-4-β-ol

1,549

1.8

tr

cis-Muurol-5-en-4-α-ol

1,557

1.2

 

tr

Germacrene B

1,558

tr

tr

0.7

(E)-Nerolidol

1,561

0.4

tr

β-Calacorene

1,562

tr

tr

tr

tr

tr

Spathulenol

1,575

tr

tr

tr

tr

tr

Caryophyllene oxide

1,580

0.9

tr

tr

0.3

1.6

Cedrol

1,597

tr

tr

4.9

12.5

β-Oplopenone

1,604

tr

tr

2.4

tr

tr

Humulene epoxide II

1,605

tr

tr

0.4

1,10-di-epi-Cubenol

1,616

tr

0.4

tr

1-epi-Cubenol

1,626

2.4

3.3

a-Acorenol

1,630

3.3

tr

tr

β-Acorenol

1,634

tr

tr

tr

epi-α-Cadinol

1,637

tr

tr

tr

tr

0.8

epi-α-Muurolol

1,639

tr

tr

tr

tr

tr

tr

0.2

a-Muurolol

1,642

tr

tr

tr

tr

Cubenol

1,643

 

0.2

a-Cadinol

1,651

2.0

1.2

tr

tr

0.7

1.0

0.7

Germacra-4(15),5,10(14)-trien-1-α-ol

1,682

tr

0.5

a-Bisabolol

1,683

tr

tr

tr

cis-14-nor-Muurol-5-en-4-one

1,685

7.2

tr

tr

Shyobunol

1,686

1.0

(Z)-Nuciferol

1,721

0.6

Oplopanonyl acetate

1,883

0.5

15.9

Beyerene

1,927

17.1

Pimaradiene

1,945

tr

tr

2.9

1.0

tr

tr

Isophyllocladene

1,963

tr

1.6

Manool oxide

1,983

tr

0.6

1.0

0.5

Abietatriene

2,052

tr

0.7

tr

tr

4.5

tr

tr

tr

Abietadiene

2,083

3.0

3.5

tr

Nezukol

2,129

1.3

Abienol

2,145

tr

tr

1.5

Phyllocladanol

2,206

tr

0.8

Sempervirol

2,279

tr

tr

0.7

trans-Totarol

2,310

tr

3.4

tr

tr

Total

 

99.9

97.2

99.1

99.9

95.7

97.0

99.1

99.2

Constituents listed in order of elution from a HP-5 MS column

RI retention indices on HP-5 MS column relative to C9-C23 n-alkanes, tr trace (<0.1 %), CARL Cupressus arizonica, CBEL C. benthamii, CMAL C. macrocarpa, CSEL C. sempervirens, CTOL C. torulosa, CLAL Chamaecyparis lawsoniana, JPHL Juniperus phoenicea, TARL Tetraclinis articulata

The oil of C. arizonica leaf was characterized by α-pinene (17.1 %), followed by trans-muurola-3,5-diene (15.5 %), umbellulone (8.0 %), and limonene (7.4 %). There are several reports on the leaf oil constituents of this species (Afsharypuor and Tavakoli 2005; Chéiraff et al. 2007; Emami et al. 2010). Our results are in accordance with the leaf oil of C. arizonica from Iran and Tunisia (Afsharypuor and Tavakoli 2005; Chéiraff et al. 2007, respectively). The major constituents of C. benthamii foliage oil were umbellulone (15.9 %), limonene (13.0 %), δ-3-carene (9.5 %), and α-pinene (9.1 %). Adams et al. (1997) reported as dominated constituents from Mexican C. benthamii leaf oil the diterpenes abietatriene (26 %) and trans-totarol (19.3 %); whereas in the studied oil, these components were present in traces and 0.7 %, respectively. The main constituents of the leaf oil of C. macrocarpa were sabinene (21.8 %), α-pinene (19.5 %), terpinen-4-ol (18.9 %), and γ-terpinene (7.9 %). Malizia et al. (2000) reported an analogous composition on this species leaf oil from Argentina. There are several papers on the chemical composition of essential oils of C. sempervirens (Chanegriha et al. 1997; 1993; Milos et al. 2002; Mazari et al. 2010; Tumen et al. 2010) where α-pinene was the dominating compound. Likewise, the studied oil of C. sempervirens is characterized by the aboundance of α-pinene (54.1 %). δ-3-Carene was the second major component (10.8 %) while all the other constituents were present in lower amounts (<5.5 %). The leaf oil of C. torulosa was characterized by the presence of α-pinene (27.9 %), δ-3-carene (21.4 %), and cedrol (12 %); whereas α-pinene (25.8 %), sabinene (22.3 %), and terpinen-4-ol (9.3 %) were reported from Argentinean leaf oil (Malizia et al. 2000).

The main constituents of the leaf oil of C. lawsoniana were limonene (18.5 %), beyerenne (17.1 %), oplopanonyl acetate (15.9 %), and methyl myrtenate (9.7 %). To the best of our knowledge, this is the first report on the chemical composition of C. lawsoniana leaf oil. Emami et al. (2009) have reported on the oil analysis from aerial parts of C. lawsoniana, where terpinen-4-ol (22.0 %), sabinene (21.0 %), camphor (7.8 %), and citronellol (7.3 %) were the major compounds.

There are several references on J. phoenicea leaf oil (Adams et al. 1996; Angioni et al. 2003; Barrero et al. 2006; Dob et al. 2008; Derwich et al. 2010; Mazari et al. 2010; Vourlioti-Arapi et al. 2012). The studied oil of J. phoenicea leaves possessed a high amount of α-pinene (31.3 %), followed by δ-3 carene (12.5 %), β-phellandrene (13.0 %), and α-terpinyl acetate (12.5 %) as main constituents. Similar qualitative but different quantitative composition was reported from Greek J. phoenicea leaf oil by Adams et al. (1996) and Vourlioti-Arapi et al. (2012) from three different locations (Epidavros, Marathonas beach and Antikyra beach). From the literature study, it can be concluded that all reported oils were dominated by α-pinene with the exception of the Greek leaf oil from Marathonas beach.

The leaf oil of T. articulata was mainly characterized by α-pinene (26.4 %) and bornyl acetate (25.3 %) followed by camphor (9.5 %) and limonene (8.7 %). Our results are in accordance with Achak et al. (2009) who reported the dominance of α-pinene (41.2 %) and bornyl acetate (20.6-36.4 %) from Moroccan leaf oil; whereas Barrero et al. (2005) obtained the oxygenated terpenes camphor (19.1 %) and bornyl acetate (16.5 %) as main compounds.

Larvicidal activity

Mosquito larvicidal activities of EOs from the eight Cupressaceae species are presented in Table 2. All tested materials showed increasing mortality with increasing concentration against third to fourth larval stages of Ae. albopictus. Among the EOs tested for larvicidal action, the most potent one derived from C. benthamii, which exhibited considerably low LC50 value of 37.5 mg/L. On the contrary, the poorer lethal effect was observed with the T. articulata oil, which resulted in LC50 of 70.6 mg/L and LC90 near 100 mg/L. The application of EOs from the other plant species exhibited rather moderate toxicity on mosquito larvae with LC50 values ranging from 47.9 to 64.8 mg/L.
Table 2

LC50 and LC90 values for the eight leave essential oils from Cupressaceae plants against third to fourth instar larvae of Aedes albopictus

Essential oil

Slope (±SE)

LC50 (95 % CL)a

LC90 (95 % CL)a

χ2b

df

C. arizonica

6.8 ± 0.6

64.8 (62.9–66.6)

78.2 (75.4–82.0)

16.782

16

C. macrocarpa

6.9 ± 0.6

54.6 (52.2–57.0)

84.0 (78.6–91.7)

27.685

25

C. sempervirens

3.6 ± 0.4

54.7 (58.9–65.0)

78.1 (73.4–84.9)

24.771

16

C. lawsoniana

10.2 ± 0.9

47.9 (46.0–49.7)

64.0 (61.0–68.1)

10.895

19

C. torulosa

7.8 ± 0.8

57.1 (54.1–59.9)

83.5 (78.1–91.6)

22.844

16

C. benthamii

14.9 ± 1.4

37.5 (36.5–38.7)

45.8 (43.8–48.5)

16.740

13

J. phoenicea

9.0 ± 0.8

55.5 (53.0–58.0)

77.0 (72.6–83.1)

15.053

16

T. articulata

3.8 ± 0.4

70.6 (67.4–73.8)

99.0 (93.1–107.7)

20.516

16

aLC values are expressed in mg/L and they are considered significantly different when 95 % CL fail to overlap

bSince goodness-of-fit test is significant (P < 0.05), a heterogeneity factor is used in the calculation of confidence limits (CL)

The range of EOs’ toxicity to Ae. albopictus larvae (LC50 = 37.5 – 70.6 mg/L), in the current screening bioassays, is generally in accordance with relevant studies on other mosquito species. Fourteen EOs derived from Juniperus species exerted significant larvicidal potential with lethal concentration of 50 % on Cx. pipiens, ranging between 26.5 and 96.7 mg/L. The most active EO was derived from the wood of Juniperus drupacea, whereas the least effective was isolated from J. phoenicea berries (Vourlioti-Arapi et al. 2012). Moreover, Sedaghat et al. (2011) determined that the LC50 of leaf EO from C. arizonica on An. stephensi larvae was 79.3 ppm.

Larvicidal activity of the studied EOs could be attributed to the mosquitocidal action of some of the terpenes. Such potent larvicidal action has been demonstrated through bioassays on one or more species of mosquitoes for many components, frequently found in EOs like a-pinene, 3-carene, (R)-(+)-limonene, myrcene, and terpinen-4-ol (Kassir et al. 1989; Chantraine et al. 1998; Perumalsamy et al. 2009; Pohlit et al. 2011; Santos et al. 2011). Cheng et al. (2009a, 2009b) reported strong mosquitocidal effect on Ae. aegypti and Ae. albopictus larvae for limonene, β-myrcene, 3-carene, γ-terpinene, and terpinolene (LC50 = 15–32 mg/L); but a lower one for α-terpinyl acetate, α-pinene, and (−)-terpinen-4-ol (LC50 values > 50 mg/L). According to Govindarajan et al. (2011), sabinene appeared to be highly potent (LC50 = 20–25 mg/L) against Cx. quinquefasciatus, Ae. aegypti, and An. stephensi larvae. Similarly, γ-terpinene was proved to be highly toxic (LC50 = 20 mg/L) against Ae. albopictus larvae (Giatropoulos et al. 2012c).

Repellent activity

In repellent bioassays, the number of landings differed significantly among studied essential oils at both applied doses (X2 = 70.888; df = 9, P < 0.001; X2 = 36.391, df = 5, P < 0.001; for “low” and “high” dose, respectively; Fig. 1). The untreated control provided considerably high numbers of landings (28.8 landings), whereas Deet offered full protection (zero landings) at “high” dose (≈ 0.2 mg/cm2) and weaker repellence (3.8 landings) at the supplementary “low” dose (≈ 0.08 mg/cm2). At “high” dose, the screening test revealed a complete repellence activity (zero landings) of EOs from C. benthamii, C. lawsoniana, and C. macrocarpa, and a strong landing inhibition effect for T. articulata and J. phoenicea oils. EOs derived from C. arizonica, C. sempervirens, and C. torulosa resulted in considerably weaker, but still sufficient, mosquito inhibition landing rate (3.5–6 landings) compared to Deet and to the untreated control. Subsequently, the application of about 2.5 fold less dose rate (“low” dose), assigned C. macrocarpa as the best repellent oil, followed by C. lawsoniana, T. articulate, and C. benthamii oils (0, 1, 4, and 7 landings, respectively).
https://static-content.springer.com/image/art%3A10.1007%2Fs00436-012-3239-5/MediaObjects/436_2012_3239_Fig1_HTML.gif
Fig. 1

Repellent activity of tested materials (essential oils plus Deet) on Ae. albopictus adults at “high” dose of 0.2 mg/cm2 (a) and “low” dose of 0.08 mg/cm2 (b) of each testing material. Mean number of landings per 5 min exposure. Means in a column followed by the same letter are not significantly different (P ≥ 0.05), Mann–Whitney U test with Bonferroni correction (adjusted a = 0.0011 and 0.0033 for high and low dose, respectively)

Mosquito repellent potential of Cupressaceae EOs has been investigated giving varying results. Neither the oil of Cupressus funebris nor that of Juniperus chinensis consistently repelled female A. aegypti mosquitoes from biting through cloth treated at the highest dosage tested (1.5 mg/cm2); whereas, the oil of Juniperus communis had a minimum effective dose of 0.06 mg/cm2 (Carroll et al. 2011). In tests with cedarwood oil against Anopheles albimanus, Curtis et al. (1987) determined the ED90 (effective dose) to be 0.00618 mg/cm2 and concluded that this oil was repellent to mosquitoes, whereas according to Barnard (1999) it was ineffective as a mosquito repellent against Ae. aegypti and An. albimanus. Amer and Mehlhorn (2006b) found that EOs from cedarwood (Juniperus virginiana) and Juniper (J. communis) in a 20 % solution in a complex formulation, against three mosquito species (Ae. aegypti, An. stephensi, and Cx. quinquefasciatus) provided total human skin protection (0 % landing and biting rate after 2 min exposure) against Cx. quinquefasciatus and weaker repellency properties against the other two mosquito species where rates of landing and biting mosquitoes ranged from 0–6.8 %. EO from J. macropoda showed no repellent activities towards the three abovementioned mosquito species (Prajapati et al. 2005), while EO from C. funebris offered complete repellency for 10 min in its undiluted form against Ae. aegypti adults (Trongtokit et al. 2005).

Essential oils consist of volatile, natural complex compounds and their repellent activity has been associated to the presence of sesquiterpenes and monoterpenes (Nerio et al. 2010). Conti et al. (2012) indicated that EO from leaves of Hyptis suaveolens (Lamiaceae) that contained high percentages of sabinene (21.9 %), β-caryophyllene (16.1 %), terpinolene (9.6 %), and 4-terpineol (7.3 %), had a significant repellent activity against Ae. albopictus adults, repelling 50 % of mosquitoes at the dose of 0.00035 μg/cm2. Gu et al. (2009) evaluated the repellent activities of 3-carene, α-terpinene, limonene, γ-terpinene, terpinolene, and (−)-terpinen-4-ol from leaf essential oil of Cryptomeria japonica (Sugi) (Pinaceae) and found that (−)-terpinen-4-ol exhibited the best repellent activity against Ae. aegypti and Ae. albopictus adults. Similarly, the aforementioned terpenes were identified among others in Cupressaceae EOs of the present study.

Conclusion

As natural products are generally preferred in vector control due to their low-risk profile for environment and humans, it is worthwhile to investigate the bioefficacy of EOs from plants against mosquitoes. Accordingly, in the present study, we investigated the larvicidal and repellent action of EOs from leaves of eight Cupressaceae species against the dengue vector Ae. albopictus. Our results indicated that the EOs tested might be used in mosquito larval control and have the potential to be applied as repellents against this species. In the current study a plethora of components was identified for EOs derived from Cupressaceae plants, which could be responsible for their bioefficacy on Ae. albopictus. Further research on the efficacy of the EOs’ main components is required along with other issues concerning their formulation and safety properties.

Acknowledgments

The authors would like to thank Prof. B. Galatis (J. & A. N. Diomedes Botanic Garden, University of Athens) for providing the plant material and Dr. I. Vallianatou (J. & A. N. Diomedes Botanic Garden, University of Athens) for the collection and plant identification.

Copyright information

© Springer-Verlag Berlin Heidelberg 2012