Volume 4, Issue 2, July 2011 ISSN 1791-3691 Hellenic Plant Protection Journal
A semiannual scientifi c publication of the BENAKIBEB PHYTOPATHOLOGICAL INSTITUTE The Hellenic Plant Protection Journal (ISSN 1791-3691) is the new scientifi c publication of the Benaki Phytopathological Institute (BPI) replacing the Annals of the Benaki Phyto- pathological Institute (ISSN 1790-1480) which had been published since 1935.
Starting from January 2008, the Benaki Phytopathological Institute is publishing the Hel- lenic Plant Protection Journal semiannually, in January and July each year, and accepts for publication any work related to plant protection in the Mediterranean region regardless of where it was conducted. All aspects of plant protection referring to plant pathogens, pests, weeds (identifi cation, biology, control), pesticides and relevant environmental is- sues are topics covered by the journal.
Articles in the form of either a complete research paper or a short communication (in- cluding new records) may be submitted. Instructions on how to prepare the manuscript are available in BPI’s website (www.bpi.gr). Manuscripts should be submitted in electron- ic form either by e-mail at [email protected] or by post on a disk addressed to the Edito- rial Board, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia (Athens), Greece. Only original articles are considered and published after successful completion Hellenic Plant Protection Journal Protection Hellenic Plant of a review procedure by two competent referees.
EDITORIAL BOARD Editor: Dr F. Karamaouna (Pesticides Control & Phytopharmacy Department, BPI) Associate Editors: Dr A.N. Michaelakis (Entomology & Agric. Zoology Department, BPI) Dr K.M. Kasiotis (Pesticides Control & Phytopharmacy Department, BPI) Dr N.I. Skandalis (Phytopathology Department, BPI) M. Kitsiou (Library Department, BPI) Technical Editor and Secretary: Asteria Karadima (Information Technology Service, BPI)
For subscriptions, exchange agreements, back issues and other publications of the In- stitute contact the Library, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia (Athens), Greece, e-mail: [email protected].
This Journal is indexed by: CAB Abstracts-Plant Protection Database, INIST (Institute for Scientifi c and Technical Information) and SCOPUS.
The olive tree of Plato in Athens is the emblem of the Benaki Phytopathological Institute
Hellenic Plant Protection Journal also available at www.bpi.gr
© Benaki Phytopathological Institute Hellenic Plant Protection Journal 4: 31-34, 2011
SHORT COMMUNICATION
Report of the Geranium Bronze Butterfl y, Cacyreus marshalli for mainland Greece
A.F. Martinou1, D. Papachristos2 and P.G. Milonas1
Summary In July and September 2010, two samples of infested geranium plants (Pelargonium spp.), which were originally collected from Kifi ssia, Attica-Greece, were received at the Laboratory of Bio- logical Control at Benaki Phytopathological Institute, Greece. Larvae were taken from infested plants and kept under laboratory conditions at 25±1°C, 70±5% RH and under a photoperiod of 16L:8D h un- til adults emerged. Adults were identifi ed as the Geranium Bronze Butterfl y, Cacyreus marshalli But- ler (Lepidoptera: Lycaenidae). This species is recorded for the fi rst time for mainland Greece. Cacyreus marshalli is on the EPPO A2 List of pests recommended for the regulation as quarantine pests. Gera- nium Bronze Butterfl y has the potential to establish in Greece and the rest of the Mediterranean ba- sin as climatic conditions can allow this pest to overwinter outdoors and its host plants are common- ly propagated.
Additional keywords: biodiversity, butterfl y, geranium, invasive, Pelargonium spp.
The identifi cation of the species was based on two infested geranium plant samples re- ceived at the Laboratory of Biological Con- trol at Benaki Phytopathological Institute in July and September 2010. The plant samples (leaves and stems) were originally collected from Kifi ssia, Attica-Greece and were kept at 25±1°C and 70±5% RH until adult emer- gence. Based on the adult morphological characteristics, the species was identifi ed as the Geranium Bronze Butterfl y Cacyreus marshalli (Lepidoptera: Lycaenidae) (Figures Figure 1. Adult of Cacyreus marshalli (upperside). 1, 2). Eggs of the Geranium Bronze are whit- ish to light-yellow or brown in colour (Figure 3); 0.5 mm in diameter x 0.3 mm in height (2). Eggs are laid near the fl ower buds or less frequently on the leaves. Caterpillars (Figu- re 4) are found within the fl ower buds or in- side the stem, where they bore through. En- trance holes in buds and stems are easy to detect. Once attacked, the stems turn black-
Laboratories of Biological Control (1) and Agricultural Entomology (2), Department of Entomology and Agri- cultural Zoology, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia (Athens), Greece. Corresponding author: [email protected] Figure 2. Adult of Cacyreus marshalli (underside).
© Benaki Phytopathological Institute 32 Martinou et al.
Figure 3. Egg of Cacyreus marshalli. Figure 4. Caterpillars of Cacyreus marshalli. ish. The fi rst- instar larvae have an average (7, 10). length of 1 mm which increases to 2 mm Pelargonium spp., commonly known as within 8 days. Second, third and fourth- in- geraniums, are the main host plants of this stars grow to 3, 6 and 13 mm, typically in 8, pest but the butterfl y also has the capacity 8 and 9 days, respectively. Their colour var- to infest native Geranium spp. (8). ies, with extremes of yellow and/or green- Cacyreus marshalli is on the EPPO A2 ish shades with or without pink markings List of pests recommended for regula- (2). Pupae are very hairy in shades of green, tion as quarantine pests. Although on the pale-yellow or brown, with brown mottling EPPO A2 List, C. marshalli has not been and an average length of 9 mm (2). Female regulated as a quarantine pest by Euro- adults have a wingspan of 18-27 mm while pean Regional Plant Protection Organiza- male adults have a wingspan of 15-23 mm. tions and it has managed to spread rapid- The two sexes are similar in appearance. ly in islands and mainland Spain and Italy Adults have a bronze-brown colouring at the (4, 9). The potential for natural spread of upper surface with white spots on the fringe this pest is very low as its fl ights are short and highly-patterned undersides. They also in duration with frequent rests (3). The bear substantial tails on their hindwings, most likely means of international disper- along with a nearby eye spot, which diverts sal is the movement of infested plant ma- attacks from birds and other predators away terial. The example of the rapid establish- from the critical body parts. ment of C. marshalli on Mallorca (Balearic The species is indigenous to Southern Islands) and its spread to the Spanish Africa (2). According to the EPPO distribu- mainland shows that the pest has the po- tion maps of quarantine pests, C. marshalli tential to establish in the Mediterranean is present (with no detailed records) in Bo- basin and could pose a threat for the Eu- tswana, Lesotho, Mozambique, South Afri- ropean mainland. Geraniums are exten- ca, Swaziland and Zimbabwe. To date it has sively grown as ornamental plants almost been observed in European countries such throughout Europe, but Spain, France, It- as Belgium and Italy with a few occurrences, aly and Greece, as well as North Africa, France and mainland Spain, where it is re- are at greater risk since their climatic con- corded as present with a restricted distribu- ditions would allow the pest to overwin- tion, and fi nally Germany, Portugal and the ter outdoors. Furthermore, breeding and Balearic and Canary islands, where it is re- propagation of geraniums play an impor- ported as present but with no detailed re- tant role in these regions. Elsewhere in cords. This is the fi rst record for mainland Europe, the pest could establish in glass- Greece while there are previous records for houses (1). the species from the Ionian island of Corfu Cacyreus marshalli has never been re-
© Benaki Phytopathological Institute Geranium Bronze butterfl y report 33 ported as a pest species in its area of ori- We would like to thank Mr Timothy Cowles for gin, probably due to autochthonous para- providing with photos and Mr Rob Parker for sitoids and predators that manage to keep sharing with us his interest on butterfl ies, es- its population under the damage threshold. pecially regarding the Geranium Bronze But- The introduction of the Geranium Bronze terfl y. into Europe is having a great impact on the nursery sector, with a consequent decrease in the demand of geraniums, which are ever Literature cited more often replaced by customers with oth- 1. Baufeld, P. 1993. Pest risk analysis of Cacyreus er ornamental plants. Furthermore, the Ge- marshalli from a phytosanitary point of view. ranium Bronze could cause problems in the Nachrichtenblatt des Deutschen Pfl anzenschutz- mountainous and hilly habitats where wild dienstes, 45: 257-262. Geranium spp. commonly exist. Adapta- 2. Clark, G.C. and Dickson, C.G.C. 1971. Life histories of the South African lycaenid butterfl ies, pp. 60- tion of C. marshalli in these habitats could 61. Purnell, Cape Town, South Africa. threaten the native fl ora and biodiversity 3. Eitschberger, U. and Stamer, P. 1990. Cacyreus through competition with other species marshalli, a new species of butterfl y for the fau- such as Eumedonia eumedon (Esper) (Lepi- na of Europe? Atalanta, 21: 101-108. doptera: Lycaenidae) and Aricia nicias (Mei- 4. Favilli, L. and Manganelli, G. 2006. Life history of gen) (Lepidoptera: Lycaneidae) (8). Aricia ni- Cacyreus marshalli, a South African species re- cently introduced into Italy. Bollettino della Soci- cias has been characterized as rare for Spain eta Entomologica Italiana, 138: 51–61. (5) while E. eumedon has been characterized 5. Gärdenfors, U. 2005. Rödlistade arter i Sverige as vulnerable in Greece (11). 2005. The 2005 Red List of Swedish Species. 496 No parasitoids or predators were ob- pp. ArtDatabanken, SLU, Uppsala. served in Italy for C. marshalli (4) except of 6. Herrero, S., Borja, M. and Ferre´, J. 2002. Extent a single case where an egg of the pest was of Variation of the Bacillus thuringiensis Tox- parasitized by Trichogramma evanescens in Reservoir: the Case of the Geranium Bronze, Cacyreus marshalli Butler (Lepidoptera: Lycaeni- Westwood. In South Africa, Apanteles spp. dae). Applied and Environmental Microbiology, have been reported to kill third- instar lar- 68: 4090–4094. vae of the pest (2). Products based on Bacil- 7. Parker, R. 2010. Cacyreus mashalli Butler, 1898 lus thuringiensis or difl ubenzuron, fl ufenox- (Lepidoptera, Lycaenidae) newly recorded for uron, hexafl umuron, lambda-cyhalothrin, Corfu, with notes on other butterfl ies on the is- land in September 2008. Entomologist’s Gazette, α-cypermethrin and benfuracarb could be 61: 40-42. used as insecticides for the control of C. 8. Quacchia, A., Ferracini, C., Bonelli, S., Balletto, E. marshalli (9). For the long-term control of and Alma, A. 2008. Can the Geranium Bronze, the Geranium Bronze with B. thuringiensis- Cacyreus marshalli, become a threat for Europe- based insecticides it would be advisable to an biodiversity? Biodiversity and Conservation, 17: 1429-1437. combine Cry1Ab with Cry1Ba (6). It would be wise to start a monitoring programme Web sites for this pest in Pelargonium spp. nurseries as 9. http://dc.eppo.org/ well as in urban resident areas of mainland 10. http://www.pamperis.gr/THE_BUTTERFLIES_OF_ Greece and the islands where these plants GREECE/Nees_anaphores_New_records.html are widely used in gardens and in balconies. 11. http://filotis.itia.ntua.gr/species/d/6642/5. Additionally, surveys could be undertaken Gärdenfors in the monitored areas in order to identify predators or parasitoids that could be used as potential biocontrol agents for this pest. Received: 8 November 2010 ; Accepted: 1 June 2011
© Benaki Phytopathological Institute 34 Martinou et al. ΣΥΝΤΟΜΗ ΑΝΑΚΟΙΝΩΣΗ
Αναφορά του Cacyreus marshalli στην Ηπειρωτική Ελλάδα
Α.Φ. Μαρτίνου, Δ. Παπαχρήστος και Π.Γ. Μυλωνάς
Περίληψη Τους μήνες Ιούλιο και Σεπτέμβριο του 2010, δύο δείγματα από προσβεβλημένα γεράνια (Pelargonium spp.) από την περιοχή της Κηφισιάς εξετάστηκαν στο εργαστήριο Βιολογικής Καταπολέ- μησης στο Μπενάκειο Φυτοπαθολογικό Ινστιτούτο. Οι προνύμφες από τα προσβεβλημένα φυτά διατη- ρήθηκαν σε συνθήκες εργαστηρίου στους 25±1°C, 70±5% σ.υ. και φωτοπερίοδο 16Φ:8Σ μέχρι την εμ- φάνιση των ενηλίκων. Τα ενήλικα αναγνωρίστηκαν ως το είδος Cacyreus marshalli Butler (Lepidoptera: Lycaenidae). Το είδος αυτό καταγράφεται για πρώτη φορά στην Ηπειρωτική Ελλάδα. Το C. marshalli βρίσκεται στην EPPO A2 λίστα οργανισμών καραντίνας. Το λεπιδόπτερο δύναται να εγκατασταθεί στην Ελλάδα και στην υπόλοιπη Μεσογειακή λεκάνη καθώς οι κλιματολογικές συνθήκες ευνοούν τη διαχεί- μαση του ενώ τα φυτά ξενιστές του είναι πολύ κοινά.
Hellenic Plant Protection Journal 4: 31-34, 2011
© Benaki Phytopathological Institute Hellenic Plant Protection Journal 4: 35-43, 2011
Larvicidal evaluation of three Mentha species essential oils and their isolated major components against the West Nile virus mosquito
A. Michaelakis1, D. Papachristos1, A. Kimbaris2 and M. Polissiou3
Summary The larvicidal activity of essential oils derived from three diff erent Mentha species (Lami- aceae) as well as their major aroma p-menthane type components were evaluated against Culex pipi- ens (Diptera: Culicidae). Therefore, pulegone, piperitenone, piperitone, carvone, menthone and men- thol were isolated using column chromatography. The LC50 values revealed that M. pulegium and M. pi- perita oils were the most toxic (46.97 and 40.28 mgl-1 respectively) and pulegone was the most eff ec- tive (27.23 mgl-1) among the major ingredients. The activity of all essential oils is in agreement with the proportion/toxicity rate of their individual major components, apart from the case of M. piperita where -1 the LC50 values of its major ingredients menthone and menthol stand higher than 100 mgl . For the isolated molecules, studies on structure activity relationships revealed that the location of C-C double bond and the presence of the isopropylidene group might be key factors.
Additional keywords: Culex pipiens, essential oil, larvicidal activity, major components isolation, Mentha species
Introduction of 2010, hundreds of people were seriously aff ected from WNV and more than 30 died Control of mosquitoes is crucial due to the in Northern Greece (18). Currently specifi c fact that they are vectors of many virus- treatment and vaccines are not available for es. Culex pipiens serves as both an enzoot- the protection of horses or humans and only ic and an epidemic (i.e. “bridge”) vector of mosquito control measures could reduce the West Nile Virus (WNV) to humans, is a wide risk of the development of serious diseas- spread insect pest with medical importance. es (6). Therefore the use of improved mos- According to Bakonyi et al. (4) WNV emerged quito control measures is strongly empha- in several European countries within the last sized. According to Floore (11) the success 50 years. Outbreaks of WNV encephalitis in of mosquito control relies on product effi ca- humans and horses were reported. Recent- cy and tools that are environmentally friend- ly, new WNV strains were isolated in Central ly. Shaalan et al. (21) stated that “.. the failure Europe from mosquito vectors and from en- to discover a signifi cant new class of insecti- cephalitic cases of vertebrate host (18). Dur- cides has led many researchers back to bio- ing the last decade cases are also reported discovery studies…”. Plant derived pesticides from Russia, Israel, Turkey and other Med- are biodegradable and may be the future ar- iterranean countries (10). During summer senal against mosquitoes but there are many topics that need more investigation. 1 Laboratory of Agricultural Entomology, Department Previous investigations have indicated of Entomology and Agricultural Zoology, Benaki Phy- topathological Institute, 8 St. Delta Str., GR-145 61 Ki- that various Mentha spp. plant extracts dis- fi ssia (Athens), Greece played larvicidal eff ect on Cx. pipiens, Cx. 2 Laboratory of Chemistry & Biochemistry, Department of Agricultural Development, Democritus University quinquefasciatus, Aedes aegypti, Anopheles of Thrace, 193 Pantazidou Str., GR-682 00 Orestiada, stephensi and An. tesselatus (2, 3, 19, 20, 22, 23). Greece Furthermore, pure substances (thymol, men- 3 Chemistry Laboratories, Agricultural University of Athens, 75 Iera Odos Str., GR-118 55 Athens, Greece thone, menthol and pulegone) and menthol Corresponding author: [email protected] derivatives have been tested against mosqui-
© Benaki Phytopathological Institute 36 Michaelakis et al. to adults, while menthone, thymol and car- hexanone were bought from Jansen Chimi- vacrol against Culex species for larvicidal ac- ca (Beerse, Belgium). Terpinolene, iso-men- tivity (20, 23). These substances are known thone and piperitone were purchased from active ingredients not only of Mentha species Extra Synthese (Genay, France). Diethyl ether essential oils but also of many other plants, in (BHT free) was purchased from SDS (Cedex, which their concentrations vary (7). France). Pentane was bought from Lab-Scan Main objectives of this study were: a) to (Doublin, Ireland). Piperitenone was pre- evaluate the effi cacy of essential oils derived pared according to literature synthetic pro- from three Mentha species, M. pulegium cedure (5), due to commercial unavailability. (three populations), M. piperita and M. spi- Purity of the isolated product (97.3%) was es- cata (spearmint) against Cx. pipiens larvae, timated according to GC-MS analytical con- b) to isolate and evaluate essential oil major ditions given bellow. Structural characteriza- components, c) to correlate larvicidal activi- tion that was accomplished by mass spectral ty of isolated components with their precur- analysis and NMR experiments was in agree- sor oils and d) to study the structure activity ment to literature data (5). NMR spectra were relationships for the isolated molecules. recorded on Brucer Avance DRX-500 instru- ment. Potassium hydroxide, tetrahydrofuran (THF), silica gel 60G and TLC plates (silica gel Materials and Methods 60, F254) were purchased from Merck (Darm- stadt, Germany). Plant Materials Three diff erent species of the genus Men- Isolation of the Essential Oils tha were used: M. pulegium (pennyroyal), M. Aerial parts from PUL1, PUL2, PUL3, piperita (mint) and M. spicata (spearmint). All SP and PIP were powdered in an electrical plant materials were collected from various blender and 1 Kg of each sample submit- natural habitats in Greece by members of ted to hydrodistillation for 4 h in a Cleveng- our team, between July and August 2007 in er-type apparatus. The obtained essential the fl owering stage. Specifi cally, in the case oils, named as EOpul1, EOpul2, EOpul3, EOsp of M. pulegium samples were collected from and EOpip respectively, were dried over an- three populations according to their geo- hydrous magnesium sulfate. After fi ltration graphical origin, the fi rst one from Orestia- their volume were calculated and expressed da (North-East of Greece, named as PUL1), as ml of essential oil/100 g of dry material the second from Karditsa (center of Greece, (Table 1) and fi nally stored in labeled sterile PUL2) and the third from Heraklion (Island of screw capped bottles at -22oC until use. Crete, PUL3). Samples of M. spicata (SP) and M. piperita (PIP) were purchased from local Gas chromatography – Mass spectrome- farmers from the region of Karditsa. All fi ve try (GC-MS) analysis plant materials were air dried and stocked The essential oils were analyzed using a for further use. Hewlett Packard II 5890 gas chromatogra- phy (GC) system, equipped with a FID de- Chemicals tector and HP-5ms capillary column (30 m R-(+)-pulegone, terpinen-4-ol, β-farnesene, x 0.25 mm, fi lm thickness 0.25 μm). Injector α-pinene, β-pinene, mesityl oxide, meth- and detector temperatures were set at 220oC yl vinyl ketone, menthol and menthone and 290oC, respectively. GC oven tempera- were purchased from Aldrich (Steinheim, ture was programmed from 60oC to 240oC at Germany). Eucalyptol, β-myrcene, S-(-)-li- a rate of 3oC /min and held isothermally for monene, γ-terpinene, thymol, carvacrol and 10 min. Helium was the carrier gas at a fl ow β-caryophyllene were purchased from Sig- rate of 1 ml/min. Diluted samples (1/100 in ma (St. Louis, USA). Carvone was bought from diethyl ether, mgl-1) of 1.0 μl were injected Fluka (Steinheim, Germany). 3-methyl cyclo- manually and in the splitless mode. Quan-
© Benaki Phytopathological Institute p-menthane type compounds against mosquito 37 titative data were obtained electronically en framed cages (33x33x33 cm) with a 32x32 from FID area percent data without the use mesh at 25±2oC, 80±2% relative humidi- of correction factors. Qualitative analysis of ty and photoperiod of 14:10 (L:D) h. Cotton the essential oils was performed using the wicks saturated with 10% sucrose solution same conditions with GC, in a Hewlett Pack- are used as food source. Females lay eggs in ard II 5890 gas chromatograph equipped round, plastic containers (10 cm diameter x with Hewlett Packard II 5972 mass selec- 5 cm depth) fi lled with 150 ml of tap water. tive detector in the electron impact mode Egg rafts are removed daily and placed in (70 eV). Most ingredients of the essential cylindrical enamel pans (with diameter of 35 oils were identifi ed on the basis of compari- cm and 10 cm deep), in order to hatch. Lar- son of GC relative retention times and mass vae are reared under the same conditions of spectra with those of pure standards. As for temperature and light and are fed daily with the rest components, tentative identifi ca- baby fi sh food (TetraMin, Baby Fish Food) at tion was based on the comparison of their a concentration of 0.25 gl-1 of water until pu- mass spectra and elution order with those pation. Pupae are then collected and intro- obtained from the NIST 98 and Wiley 275 li- duced into the adult rearing cages. brary data of the GC-MS system and from Adams CD computer library (1). Larvicidal Bioassays Stock solutions were prepared in etha- Isolation of Essential Oils Major Ingredi- nol with a concentration of 1% mgl-1. A se- ents ries of aqueous solutions of the tested ma- Ten (10) g of the extracted crude essential terial, expressed as mgl-1, were made and oils EOpul1, EOpul2, EOpul3, EOpip and EOsp tested under laboratory conditions. Prelim- were fractioned by column chromatography inary experiments (data not shown) were on silica gel and eluted with a gradient of sol- performed to evaluate toxicity and subse- vents of increasing polarity (pentane + dieth- quently diff erent concentrations were em- yl ether: 100 + 0, 97 + 3, 95 + 5, 93 + 7, 90 + ployed ranging from 10 to 200 mgl-1 for each 10 and 80 + 20). Column fractions were moni- tested material. tored by thin layer chromatography (TLC) with The larval mortality bioassays were carried pentane + diethyl ether (9.5 + 0.5). Fractions out according to the test method of larval sus- with similar Rf values on the TLC plates to au- ceptibility as proposed by the World Health thentic co-eluted compounds were pooled. Organization (24). Twenty 3rd to 4th instar lar- Spots were detected under UV lamp and af- vae of the species Cx. pipiens biotype moles- terwards by spraying with a mixture of 1% tus were collected from the colony. They were vanillin and 5% sulfuric acid solution (in eth- placed in glass beaker with 250 ml of aqueous anol) and heating at 120oC and/or PMA solu- suspension of tested material at various con- tion (phosphomolybdic acid 7.5% mgl-1 in eth- centrations and an emulsifi er was added in anol) and charring on a hot plate. The chosen the fi nal test solution (less than 0.05%). Four fractions merged to one and elution solvents replicates were made per concentration and a removed by a rotary fl ash evaporator. The dis- control treatment with tap water and emulsifi - tillation was stopped when the volume of sol- er was also included. Beakers with larvae were vents was reduced to ~10 ml and completed placed at 25±2oC, 80±2% relative humidity by fl ushing through nitrogen. and photoperiod of 14:10 (L:D) h.
Mosquito Rearing Data Analysis The colony of the species Cx. pipiens bi- Larvicidal eff ect was recorded 48 h after otype molestus has been maintained in the treatment. Data obtained from each dose– laboratory of Benaki Phytopathological In- larvicidal bioassay (total mortality, mgl-1 stitute, Kifi ssia, Greece for more than 25 concentration in water) were subjected to years. Adult mosquitoes are kept in wood- probit analysis in which probit–transformed
© Benaki Phytopathological Institute 38 Michaelakis et al.
mortality was regressed against log10–trans- obtained essential oils are shown in Table 1. formed dose; LC50, LC90 values, and slopes Literature data suggests that M. pulegium is were calculated (SPSS 11.0). a chemical polymorph species both in qual- itative and/or quantitative composition. Re- cently, it has been demonstrated the link be- Results tween the oil composition and origin of 38 populations of M. pulegium scattered along Chemical Composition of the Essential Greece (12). Thus, the three diff erent sam- Oils ples resulted in diff erent chemical profi les. Yields and chemical composition of the In detailed, in EOpul1 pulegone (61.1%) was
Table 1. Percentage composition of essential oils isolated from M. pulegium (three popula- tions: EOpul1, EOpul2 and EOpul3), M. spicata (EOsp) and M. piperita (EOpip). Compounds are listed in order of elution from an HP-5 MS column.
Composition (%) Components EOpul1c EOpul2c EOpul3 EOsp EOpipc 3-methyl cyclohexanonea 0.4 α-pinenea 0.2 0.4 0.7 β-pinenea 0.2 0.2 1.2 3-octanola 1.4 0.4 0.7 0.2 limonenea 1.2 2.7 1.8 eucalyptola 9.0 6.9 γ-terpinenea 0.4 0.7 menthonea* 4.3 1.8 1.6 39.0 isomenthonea* 13.0 0.3 24.8 9.9 menthola* 25.9 terpinen-4-ola 0.7 cis-dihydro carvoneb 1.0 isopulegoneb 2.9 trans-carveolb 0.8 pulegonea* 61.1 2.0 carvonea* 71.8 piperitonea* 1.1 92.6 69.3 1.3 piperitenonea* 4.0 0.1 0.4 piperitenone oxideb 1.8 β-bourboneneb 0.1 0.8 0.2 1.5 0.4 β-elemeneb 1.1 0.2 β-caryophyllenea 2.2 3.5 β-farnesenea 0.1 0.6 germacrene Db 0.1 1.3 2.9 bicyclogermacreneb 0.8 0.5 caryophyllene oxidea 0.2 Total 91.4 98.7 99.2 91.7 95.7 oil yield (ml/100 dry wt) 2.2 2.1 2.1 1.8 3.2 a Comparison with pure standards. b Tentative identifi cation based on data obtained from NIST 98 and Wiley 275 library of the GC-MS system and from Adams CD computer library (16). c For these essential oils the phytochemical analysis for major ingredients (>1%) has already been published (14). * Components that were isolated and applied in bioassays.
© Benaki Phytopathological Institute p-menthane type compounds against mosquito 39 the most abundant ingredient followed by purity) from EOpip and menthone (3.16 g, isomenthone (13.0%), menthone (4.3%), pi- 99.2% purity) and menthol (2.17 g, 99.0% pu- peritenone (4.0%), isopulegone (2.9%) and rity) from EOsp. Pulegone, piperitenone, pip- piperitenone oxide (1.8%). EOpul2 charac- eritone, isomenthone, carvone, menthone terized by the dominant occurrence of pi- and menthol have been isolated in high pu- peritone (92.6%) and the minor presence of rities and their chemical structures are giv- limonene (2.7%) and menthone (1.8%). Final- en in Figure 1. Although piperitenone does ly, EOpul3 consisted of piperitone (69.3%), not constitute a major ingredient, it has also isomenthone (24.8%), limonene (1.8%) and been isolated due to the fact that it is struc- menthone (1.6%). Concerning the other two turally similar to pulegone and piperitone. plants, the major ingredient of EOsp was car- vone (71.8%) followed by eucalyptol (9.0%) Larvicidal Assays and β-caryophyllene (2.2%) among others, Essential oils and their major ingredi- while EOpip consisted of menthone (39.0%) ents were tested for their larvicidal activity and menthol (25.9%) with minor compo- against Cx. pipiens 3 rd-4th instar larvae. EOpul1 nents isomenthone (9.9%), eucalyptol (6.9%) was the most active essential oil as well as its and β-caryophyllene (3.5%) among other major component pulegone among the es- less abundant ingredients. sential oil major ingredients (based on non- overlapping confi dence intervals) (see Table
Purity of the isolated major ingredients 2 for LC50 and LC90 values). The essential oils The purity of the isolated components EOpul2 and EOsp and their dominated com- was determined by GC-MS analysis and is pounds, piperitone and carvone respective- given as follows: pulegone (5.45 g, 99.2% pu- ly, presented a rather medium activity (Table rity) and piperitenone (0.38 g, 98.6% purity) 2). EOpul3 was the only inactive essential oil from EOpul1, piperitone (8.63 g, 99.5% pu- showing no larvicidal eff ect. The larvicidal rity) from EOpul2, piperitone (6.21 g, 99.4% pattern of the EOpip was quite diff erent: LC purity) and iso-menthone (1.86 g, 98.9% pu- values were high and almost the same with -1 rity) from EOpul3, carvone (6.05 g, 99.6% the EOpul1 (LC50 value 40.28 and 46.97 mgl
Figure 1. Chemical structures of essential oils’ isolated components.
© Benaki Phytopathological Institute 40 Michaelakis et al. for EOpip and EOpul1 respectively). three Mentha species, M. pulegium (three populations), M. piperita and M. spicata, was based on obtaining essential oils that pos- Discussion sessed diff erent chemical profi les. The iso- lated essential oils can be characterized as In this study, essential oils of Mentha spe- pulegone type (EOpul1), piperitone type cies and their major ingredients were tested (EOpul2), piperitone-isomenthone type with respect to their larvicidal eff ect against (EOpul3), menthone-menthol type (EOpip) Cx. pipiens mosquitoes. The selection of the and carvone type (EOsp). These results of
Table 2. LC50 and LC90 values for essential oils and their isolated major components against larvae of Culex pipiens biotype molestus. Major components percentage composition, in re- lation to their precursor essential oils, is also given in parenthesis.
c LC50 LC90 2 n Slope (±SE) a a χ df Essential oil / Major components (95% CL) (95% CL) (% composition in essential oil)
46.97 85.42 EOpul1 400 4.93±0.53 15.440 18 (43.92-50.73) (74.51-104.51)
27.23 49.91 pulegone (61.1%) 400 4.87±0.52 12.402 15 (24.39-29.7) (45.4-56.61)
75.91 97.31 piperitenone (4.0%) 320 5.16±0.55 5.909 10 (72.54-79.2) (92.16-104.84)
168.59 235.44 EOpul2 480 8.84±0.94 37.275b 16 (157.76-182.99) (209.67-293.82)
131.91 186.84 Piperitone (92.6%) 320 8.47±0.9 47.024b 10 (112.92-155.81) (157.64-287.79)
EOpul3 320 –– >200 –– –– ––
129.51 178.98 piperitone (69.3%) 400 8.32±0.83 35.466b 13 (118.78-142.17) (159.16-223.33)
isomenthone (24.8%) 320 –– > 200 –– –– ––
95.9 126.46 EOsp 320 10.66±1.19 43.362b 10 (80.44-107.13) (112.57-162.87)
95.31 162.31 carvone (71.8%) 400 5.54±0.68 26.735 13 (84.58-106.77) (137.02-228.97)
40.28 146.17 EOpip 480 22.29±0.22 44.197b 19 (30.9-50.56) (105.4-252.54)
111.11 152.63 menthone (39.0%) 480 9.29±0.92 18.942 16 (104.54-116.67) (145.23-162.51)
120.97 170.45 menthol (25.9%) 400 8.6±1.31 20.977 13 (105.35-130.02) (157.72-199.31) a LC values are expressed in mgl-1 and they are considered signifi cantly diff erent when 95% CL fail to overlap. b Since goodness–of–fi t test is signifi cant (P<0.05), a heterogeneity factor is used in the calculation of confi dence limits (CL). c Total number of larvae tested.
© Benaki Phytopathological Institute p-menthane type compounds against mosquito 41 phytochemical analysis are in agreement Particularly, it combines all of the former with literature data (13, 17). molecules characteristics to one structure Essential oils are a mixture of diff erent as it bares two double bonds, where the fi rst ingredients, mostly terpenes, which in the one is internal at the cyclohexane ring (re- analysed Mentha spp. essential oils includ- sembling to piperitone) and the second one ed pulegone, piperitone, isomenthone, car- external (resembling to pulegone). There- vone, menthone and menthol and they are fore, two arguments can be made: either comparable to fi ndings in the literature (8, piperitenone’s internal double bond reduc- 13). Besides menthone, the rest isolated in- es the total toxicity or its external double gredients were evaluated for the fi rst time bond increases the toxicity. In any case, tox- against Cx. pipiens larvae. Among them, icity seems to be in direct connection to car- pulegone demonstrated the strongest larv- bon-carbon double bond’s position. icidal activity. The activity of all essential The eff ectiveness order of the above oils is in agreement with the proportion/ mentioned molecules, compared to men- toxicity rate of their individual major com- thone (saturated cetone), is the following: ponents, apart from the case of EOpip. The pulegone> piperitenone > menthone > pip- EOpip consists of menthone and menthol eritone. This ranking indicates that unsatu-
(in a total of 65%) and their LC50 values are ration might be a key factor but not nec- higher than 100 mgl-1, so it was expected to essarily the most determinant; it should be have the same toxicity pattern with the ma- taken into account in combination with the ternal essential oil. According to our experi- position factor. Our results concerning car- mental and literature data (10) EOpip did not vone (a molecule isomer to piperitenone) follow this pattern and found to be one of also support this hypothesis: carvone is less the two most drastic essential oils (LC50 val- eff ective than piperitenone while both of ue near 40mgl-1). Two suggestions could be them are showing reduced toxicity com- made: either the rest of the ingredients pos- pared to pulegone. This activity diff eren- sess independently strong larvicidal abili- tiation probably results from the diff erent ty or some kind of synergistic phenomenon location of C-C double bond (isopropenyl took place. These results are in accordance group) as well as to its endo-exocyclic di- with those reported by Amer and Mehlhorn enone character. By inference, it seems that (2) where peppermint had moderate larvi- in the case of the tested monoterpene ce- cidal activity against mosquito larvae (near tones, the presence of the isopropylidene 53% mortality after 24 h treatment). group increases the strength of larvicity, Some interesting conclusions can be comparing to isopropenyl and/or isopropyl drawn concerning the relationship be- group eff ectiveness. tween the structure of the isolated p-men- Finally, the use of the two enantiomers, thane compounds and their larvicidal ef- isomenthone and menthone, revealed that fect against Cx. pipiens. Although, pulegone the latter was more toxic, indicating that and piperitone are isomers, the fi rst is 5-fold enantioselectivity may play also an impor- more active than the later. This diff erentia- tant role for the toxicity of essential oils. This tion probably stands on account of the loca- role of enantioselectivity has already been tion of the C-C double bond that these two reported in previous projects and is a well- molecules contain. We assume that in pule- known fact: naphthoquinones (14), linalool gone the C-C double bond position on the (15) and limonene (16). chain group (isopropylidene versus isopropyl Essential oils have often proved to be group) enhances the toxicity. This hypothesis more eff ective than their ingredients, in- is strengthened by the case of piperitenone, dicating synergistic phenomena (9). Plants where toxicity ranges between pulegone usually produce essential oils as a mixture and piperitone. Piperitenone is structurally of many ingredients with strong interac- similar to both aforementioned molecules. tions among them. Results from this study
© Benaki Phytopathological Institute 42 Michaelakis et al. demonstrated that some Mentha species (M. infection in Europe. 25 Jan 2011 - 26 Jan 2011, pulegium and M. piperita) and their p-men- Thessaloniki. Organized by: ECDC, Hellenic Cen- ter for Disease Prevention and Control (Greece), thane type components (pulegone) could WHO Regional Offi ce for Europe). be potential larvicidal agents against Cx. 11. Floore, T.G. 2006. Mosquito larval control prac- pipiens. However, further investigations for tices: past and present. Journal of the American their eff ects on non-target organisms, and Mosquito Control Association, 22: 527-533. their possible toxicity against mammals 12. Kokkini, S., Hanlidou, E. and Karousou, R. 2004. should be considered to ensure safety of ap- Clinal variation of Mentha pulegium essential oils along the climatic gradient of Greece. Jour- plication. nal of Essential Oil Research, 16: 588-593. 13. Lupien, S., Karp, F., Wildung, M. and Croteua, R.B. 1999. Regiospesifi c cyrochrome P450 hy- Literature Cited drolases from mint (Mentha) species: cDNA isolation, characterization, and functional ex- 1. Adams, R.P. 2007. Identifi cation of Essential Oils pression of (-)-4S-limonene-3-hydroxylase and components by Gas Chromatography/Quadru- (-)-4S-limonene-6-hydroxylase. Archives of Bio- pole Mass Spectroscopy, 4th Edition; Allured Pub- chemistry and Biophysics, 368: 181-192. lishing Corp.: Carol Stream, Illinois. 14. Melliou, E., Michaelakis, A., Koliopoulos, G., Ska- 2. Amer, A. and Mehlhorn, H. 2006. Larvicidal ef- ltsounis, A. and Magiatis, P. 2009. High quality fects of various essential oils against Aedes, bergamot oil from Greece: chemical am=nalysis Anopheles, and Culex larvae (Diptera, Culicidae). using enantiomeric GC-MS and larvicidal activi- Parasitology Research, 99: 466-472. ty against the West Nile virus vector. Molecules, 3. Ansari, M.A., Vasudevan, P., Tandon, M. and 14: 839-849. Razdan, R.K. 2000. 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An attempt to correlate toxicity itenone via mesityl oxide and methyl vinyl ke- with chemical structure. In: A 7th Joint meeting tone. Journal of American Chemical Society, 31: of AFERP, GA, PSE & SIF. Natural products with 2026-2027. pharmaceutical, nutraceutical, cosmetic and agochemical interest, Athens. 6. CDC. Centers for Disease Control and Preven- tion, Division of Vector-Borne Infectious Dis- 17. Miyazawa, M., Watanabe, H., Umemoto, K. and eases. http://www.cdc.gov/ncidod/dvbid/west- Kameoka, H. 1998. Inhibition of acetylcho- nile/index.htm linesterase activity by essential oils of Mentha species. Journal of Agricultural and Food Chem- 7. Cheng, S.S., Huang, C.G., Chen, Y.J., Yu, J.J., istry, 46: 3431-3434. Chen, W.J. and Chang, S.T. 2009. Chemical com- positions and larvicidal activities of leaf essen- 18. Papa, A., Danis, K., Baka, A., Bakas A, Dougas tial oils from two eucalyptus species. Biore- G, Lytras T, Theocharopoulos, G., Chrysagis, D., source Technology, 100: 452-456. Vassiliadou, E, Kamaria, F., Liona A., Mellou, K., Saroglou, G. and Panagiotopoulos, T. 2010. On- 8. De Andrade, I.L., Bezerra, J.S., Lima, M.A., De Fa- going outbreak of West Nile virus infections in ria, R.P., Lima, M.S. and Andrade-Neto, M. 2004. humans in Greece, July – August 2010. Euro- Chemical composition and insecticidal activity surveillance, 15: pii=19644 (Available online: of essential oils from Vallinosmopsis pohlii baker http://www.eurosurveillance.org/ViewArticle. against Bemisia argentifolii. Journal of Agricultur- aspx?ArticleId=19644) al and Food Chemistry, 52: 5879-5881. 19. Pitarokili, D., Michaelakis, A., Koliopoulos, G., 9. Don-Pedro, K.N. 1999. Investigation of single Giatropoulos, A. and Tzakou, O. 2010. Chemi- and joint fumigant insecticidal action of citrus cal composition, larvicidal evaluation and adult peel oil components. Pest Science, 46: 79-84. repellency of endemic Greek Thymus essential 10. ECDC. European Centre for Disease Prevention oils against the mosquito vector of West Nile Vi- and Control. http://www.ecdc.europa.eu. (see rus.Parasitology Research, (DOI: 10.1007/s00436- Events: Expert consultation on West Nile Virus 011-2271-1).
© Benaki Phytopathological Institute p-menthane type compounds against mosquito 43
20. Samarasekera, R., Weerasinghe, I.S. and He- 23. Trabousli, A.F., Taoubi, K., Samih, E.H., Bessiere, malal, K.D.P. 2008. Insecticidal activity of men- J.M. and Rammal, S. 2002. Incecticidal proper- thol derivatives against mosquitoes. Pest Man- ties of essential oils against the mosquito Culex agment Science, 64: 290-295. pipiens molestus (Diptera: Culicidae) Pest Man- 21. Shaalan, E.A., Canyon, D.V., Younes, M.W.F., Ab- agment Science, 58: 491-495. del-Wahad, H. and Mansour, A.H. 2006. A review 24. WHO, Instructions for determining the susceptibil- of botanical phytochemicals with mosquitocid- ity or resistance of mosquito larvae to insecticides. al potential. Environment International, 31: 1149- Vol. WHO/VBC/81.807. 1981, Geneva: World 1166. Health Organization. p. 6. 22. Sukumar, K., Perich, M.J. and Boobar, L.R. 1991. Botanical derivatives in mosquito control: a re- view. Journal of the American Mosquito Control Association, 7: 210-237. Received: 2 May 2011, Accepted: 21 July 2011
Τοξική δράση των αιθέριων ελαίων τριών ειδών Mentha spp. και των κυριότερων συστατικών τους στο κουνούπι Culex pipiens
A. Μιχαηλάκης, Δ. Παπαχρήστος, Α. Κυμπάρης και Μ. Πολυσίου
Περίληψη Η δράση των αιθέριων ελαίων από τρία διαφορετικά είδη Mentha spp. (Lamiaceae), κα- θώς και τα σημαντικότερα συστατικά τους, αξιολογήθηκαν έναντι προνυμφών του κουνουπιού Culex pipiens (Diptera: Culicidae). Ως εκ τούτου οι ουσίες πουλεγόνη, πιπεριτενόνη, πιπεριτόνη, καρβόνη, μενθόνη και μενθόλη απομονώθηκαν με χρωματογραφία στήλης. Τα αποτελέσματα έδειξαν ισχυρή
δράση των αιθέριων ελαίων Μ. pulegium και Μ. piperita έναντι των προνυμφών (τιμές LD50 46,97 και 40,28 mgl-1 αντίστοιχα) και η πουλεγόνη είχε την πιο ισχυρή δράση ανάμεσα στα κυριότερα συστατι- κά (27,23 mgl-1). Για τα απομονωμένα μόρια, η διερεύνηση για πιθανή σχέση δομής δράσης αποκάλυ- ψε ότι η θέση του διπλού δεσμού και η παρουσία της ισοπροπυλιδένο ομάδας μπορεί να είναι σημαντι- κοί παράγοντες δραστικότητας.
Hellenic Plant Protection Journal 4: 35-43, 2011
© Benaki Phytopathological Institute Hellenic Plant Protection Journal 4: 45-52, 2011
First record and molecular identifi cation of the parasitoid Anagyrus sp. near pseudococci Girault (Hymenoptera: Encyrtidae) in Greece - Host size preference for the vine mealybug Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae)
F. Karamaouna1, G. Menounou1,2, G.J. Stathas2 and D.N. Avtzis3
Summary The parasitoid Anagyrus sp. near pseudococci (Hymenoptera: Encyrtidae) was recorded for the fi rst time in Greece and was identifi ed using a PCR-based technique. Once identifi ed, the host size preference of the parasitoid for the vine mealybug Planococcus fi cus (Signoret) (Hemiptera: Pseudococci- dae) was examined in choice experiments with four host size classes (0.5-0.9, 1-1.5, 1.6-2.3 and >2.3 mm) mostly consisting of one host stage (2nd, 3rd instar nymph, young female adult and preovipositing female adult, respectively). Anagyrus sp. near pseudococci preferably parasitized adult female mealybugs larg- er than 1.6 mm, whereas no successful parasitism was observed in smaller mealybugs (0.5-1.5 mm). The mean development time of the parasitoid at the two host size classes where parasitism occurred was 15.99 ± 0.43 days for females and 17.01 ± 0.53 days for males (28 ± 1oC, 16L:8D) and did not statisti- cally diff er between the sexes. Host size at parasitism did not aff ect the size of female and male para- sitoid off spring.
Additional keywords: development, host selection, parasitism, vine mealybug
Introduction Grapevine Leafroll Associated Virus 3 (GLRa V-3) (13) so it is considered economically im- The vine mealybug P. fi cus is a pest of grape- portant even at low densities (15). vine in the Medditerranean region, North Chemical control of P. fi cus is diffi cult and South Africa, Middle East, Mexico, Cali- and ineff ective as the mealybug feeds on fornia and Argentina. It is the most common all parts of the grapevine (roots, trunk, cor- mealybug species infesting grapevine in dons, canes, leaves, fruit) and a portion of its Greece (Heraklio); it has been a key pest on population often resides in protected loca- grapevine in South Africa for more than sev- tions i.e. under the bark of the trunk or cor- enty years and it is a pest of economic im- don (8, 11, 15, 34). Biological control through portance damaging table and wine grapes introducing and/or fostering populations of as well as raisins in California (6, 10, 28, 30, natural enemies has provided an alternative 31, 33). Furthermore, P. fi cus is a vector of the method to suppress vine mealybug popu- lations (7, 12, 34). The parasitoid Αnagyrus 1 Laboratory of Biological Control of Pesticides, De- pseudococci (Girault) (Hymentopeta: En- partment of Pesticides Control and Phytopharmacy, cyrtidae) is the most commonly reared spe- Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia (Athens), Greece cies among the natural enemies of the vine 2 Technological Educational Institute of Kalamata, mealybug P. fi cus (23) as usual with many School of Agricultural Technology, Department of Planococcus spp. and Pseudococcus spp. (31). Crop Production, Antikalamos, GR-241 00 Kalamata, Greece Similarity in favourable climatic conditions 3 NAGREF, Forest Research Institute, Laboratory of For- and geographic distribution between A. est Entomology, GR-570 06 Vasilika (Thessaloniki), Greece pseudococci and the vine melybug renders it Corresponding author: [email protected] one of the most important biological agents
© Benaki Phytopathological Institute 46 Karamaouna et al. of the pest (15, 16). spring (idiobionts) or can only produce small Anagyrus pseudococci is reported as a parasitoids in size which consequently reduc- native endoparasitoid in many countries in- es components of fi tness such as longevity, cluding, Argentina, Israel, Italy, South Afri- fecundity and searching effi ciency (idiobionts ca and Turkey bu Gülek et al. (15) but mor- and koinobionts) (1). Small host size is often phological and molecular studies as well as associated with lengthened development cross-breeding experiments in cultures dif- and/or reduced survival (14). ferent origin which were originally consid- In the present work, the incidenece of ered as P. pseudococci revealed two distinct the parasitoid A. sp. near pseudococci was taxonomic forms: Anagyrus sp. near pseudo- proved from genetically identifi ed samples cocci and A. pseudococci (31). in Greece. Moreover, the host size prefer- Anagyrus sp. near pseudococci is a main- ence for the vine mealybug P. fi cus and the ly Palearctic species, common in the Med- eff ect of host size on development time and itterranean region, which has also been es- size of the parasitoid off spring were investi- tablished after introduction in California (31). gated as indices of the parasitoid fi tness. Anagyrus pseudococci is considered aslo of Palearctic origin, believed to have been in- intentioanally introduced in Argentina with Materials and methods seedlings from Italy (31). Both species coexist in Sicily. Anagyrus sp. near pseudococci resem- Anagyrus sp. near pseudococci identifi - bles with A. pseudococci and the only mor- cation phological feature that discriminates them The parasitoid samples were collected is found in the antennae: the fi rst antennal as parasitized mealybugs on Mentha spp. funicle segment of female is partially black (Lamiaceae) at the region of Κifi ssia- Ath- (basal half or so) and white (distal half or so) ens, Greece. The parasitoid species was fi rst in A. pseudococci whereas it is entirely black identifi ed as Anagyrus sp., presumably A. sp. in A. sp. near pseudococci (31). However, it is near pseudococci versus Anagyrus pseudococ- generally admitted that the subtle morpho- ci (Girault) (Karamaouna, personal commu- logical diff erences between A. pseudococci nication) based on morphological features and A. sp. near pseudococci, can sometimes (24, 31). Samples of both sexes of the para- be misleading (Noyes, personal communi- sitoid were sent to Dr John Noyes (Natural cation), and thus only an expert could distin- History Museum in the U.K.) for the system- guish between them. DNA barcoding, based atic taxonomy of the species and they were on a sequence from the Cytochrome Oxidase identifi ed as Anagyrus sp. of the pseudococ- One (COXI) mitochondrial gene, has assisted ci complex, which includes both A. dacty- in the resolution of various biological queries, lopii and A. pseudococci, sensu stricto and ranging from detection of intraspecifi c varia- the so-called “Israeli” strain (Noyes, person- tion to cryptic species recognition (4, 5, 26, 27, al communication). 28) and therefore could apply for the genetic Molecular techniques were used for the identifi cation of A. sp. near pseudococci. verifi cation of the sampled species. Insect Host preference has been defi ned as DNA was extracted using the GenEluteTM the relative frequency of host types chosen 100 Kit (Sigma) following the protocol of the for parasitism compared with the frequen- manufacturer. An amplicon of about 900 bp cy of host types available (18). Host stage or from the mitochondrial COI gene was po- host size selection behaviour within a patch lymerised in 25μl reactions containing 1.5 may be related to several factors such as dif- mM MgCl2, 50 M dNTPs, 0.2 _M of the prim- ferences in the profi tability of each develop- ers MTDNA04 (forward) (AGGAAC(AT)GG(AG) mental stage e.g. small hosts may not provide TGAACNGTTTA(TC)CC(AT)CC) (Avtzis, Nav- adequate amounts of resource to support rozidis and Tsoktourides, unpublished data) the successful development of parasitoid off - and UEA10 (reverse) (22), and 1U of Taq poly-
© Benaki Phytopathological Institute Anagyrus sp. near pseudococci on P. fi cus in Greece 47
merase. PCR was performed with a thermo- The boxes were kept in a Gallenkamp CΟ2 cycler (Primus 25, PeqLab) with the following growth chamber at 26oC and constant dark. amplifi cation conditions: initial denaturation All biological stages of the mealybug were phase at 97°C for 4 min, 35 cycles of denatur- present in the culture. ation at 96°C for 25 s, annealing at 48°C for The parasitoid colony was reared on 25 s and extension at 68°C for 90 s were fol- sprouted potatoes from the P. fi cus mass lowed by a fi nal extension step at 68°C for culture in plexiglass cages [50x40x40 cm 10 min. PCR products were purifi ed using the with two net covered ventilation openings QIAquickTM 108 Kit (QiaGen) and directly se- (30x20 cm)] in the insectary at 28 ± 1oC and quenced with both primers described above L16:D8 h. In order to schedule parasitoid on an ABI 3770 capillary sequencer (Applied emergence for the experiment, parasitoids Biosystems), in order to exclude cases of of both sexes from the colony were released base misincorporation due to PCR error (20) in sandwich boxes (5-10 individuals/box) and visualised using CHROMAS LITE (37). and were let to parasitise mealybugs on the The 818 bp long sequences were aligned infested potatoes every week. by eye and were put into the GenBank (36). Individuals of the pest were transferred There, the similar sequences were deter- from the mass culture on leaf sections (cen- mined using the Basic Local Allignment tral part) of Nerium oleander (Apocynaceae), Search Tool (BLAST) by nucleotide. In ad- which is a host plant of the mealybug (Sca- dition, twelve nucleotide sequences of lenet database), a few hours before the exper- three Anagyrus species already submit- iment. The leaf sections were kept with their ted to GenBank were used in the construc- lower surface upwards on top of a layer of 8 g/l tion of a phylogenetic tree. Specifi cally, An- Agar, which had previously been autoclaved, agyrus pseudococci (DQ667743, DQ667745, in Petri-dishes of 9 cm diameter. Size classes of DQ667744, DQ667746, DQ667747) Anagyrus the host were used rather than host stages but sp. near pseudococci (DQ667737, DQ667738, they were selected so that each size class com- DQ667739, DQ667740, DQ667741, DQ667742) prised mostly one stage according to sampled and Anagyrus dactylopii (DQ667736) se- sizes of mealybugs measured after moults. The quences were used. A Neighbor-Joining (NJ) size classes were 0.5-0.9 mm (2nd instar nymph), approach (25) was applied to construct a 1-1.5 mm (3rd instar nymph), 1.6-2.3 mm (young tree from the pairwise distances that were female adult), >2.3 mm (preovipositing female estimated using the substitution model adult) [Classifi cation by Karamaouna and Cop- of Tamura and Nei (29) as implemented in land (21)]. Only female mealybugs were used MEGA version 3.1 (21). in the experiments apart from the two small size classes where males and females could Bioassay not be distinguished. Five mealybugs of each The primary culture of the mealybug P. size class (20 in total) were placed randomly fi c u s was established in the insectary of the in each Petri dish. Biological Control of Pesticides Laboratory Every 1-3 days, females of A. sp. near at Benaki Phytopathological Institute from pseudococci were collected from the sandwich individuals which were collected in an in- boxes of the mass parasitoid culture, in which fested vineyard at the region of Helia-Pelo- both female and male wasps were present. ponnese. Taxonomic identifi cation of the These 1-3 days old females were assumed species was done according to Cox and Ben- to have mated and were used in the experi- Dov (6) key (P. Milonas and F. Karamaouna, ments. During the experiments, wasps were personal communication). The culture was released individually in the Petri-dishes for 24 maintained on sprouted potatoes of the va- hours. At the end of the experiment, the ovi- riety ‘Marfona’ in sandwich boxes (17x11x5 positing female wasps were measured (head- cm: Length x Width x Height) with net cov- width) and dissected to confi rm that they had ered openings (d= 1.5 cm) for ventilation. been inseminated (17). The Petri-dishes were
© Benaki Phytopathological Institute 48 Karamaouna et al. kept in a growth room at 28 ± 1oC and L16:D8 BLAST indicated a high similarity of the 818 h until the mealybugs became mummifi ed. bp long sequence with Anagyrus sp. near The mummies were collected, measured (tip pseudococci sequences, whereas it was less of the head to the end of the abdomen) and similar with the sequences of A. pseudococ- kept individually in 0.5 ml Eppendorf tubes, ci and A. dactylopii. Furthermore, the dis- under the aforementioned conditions of tem- tance-based calculation of the phylogenet- perature and light until parasitoid emergence. ic tree clustered the sequences in the clade A binocular stereo-microscope (Χ 5 – Χ 50) of the A. sp.near pseudococci sequences ob- with a linear scale graticule fi tted in one of tained from the NCBI GeneBank, supporting its lenses was used for all the necessary meas- this clade with high bootstrap value (Boot- urements of the host and the parasitoid. strap Value: 100%) whereas sequences from Proportion of mealybugs mummifi ed A. pseudococci individuals were separately out of the total number in each size class of clustered into another distinct monophylet- the host (index of host size preference) as ic clade (Figure 1). well as the sex, development time and size (headwidth) of the parasitoid off spring were Host size preference of the parasitoid A. recorded (body size is usually a good predic- sp.near pseudococci tor of fecundity in parasitoids). The evaluation of host size preference was based on the rate of parasitized mealy- Data analysis bugs (mummies) in each size class. No para- The four host size classes, off ered simul- sitism was observed in the smaller host size taneously to the parasitoid in each Petri dish, classes (0.5-0.9 mm and 1-1.5 mm). The rate were the treatments. Each Petri-dish was of parasitism in the larger size classes was considered as one experimental unit – repli- 0.18-0.22 and did not statistically diff er be- cation of the experiment. Fifty experimental tween them. (ANOVA, F1, 48 = 0,37, P= 0,544) units were set but parasitism was observed (Table 1). only in 34 Petri dishes in the two larger class- es of the host. Because observations of host Eff ect of host size of P. fi cus on develop- size classes within the same experimental ment time and size of the parasitoid off - unit were not independent, the 34 observa- spring A. sp. near pseudococci tions were divided into two groups and data The mean development time of female on development time and headwidth of one and male off spring of A. sp. near pseudococ- parasitized host size class from one group ci in female adults did not signifi cantly dif- were compared with the respective data of fer between the size classes ‘1.6-2.3 mm’ and the other size class from the second group ‘>2.3 mm’ (females: ANOVA, F1,15 = 0.02, P= using ANOVA (α = 0.05) in MINITAB (Release 0.877; males: ANOVA, F1,20 = 0.94, P= 0.344) 10.51 Xtra). Data on parasitism rate were sub- (Table 2). No signifi cant diff erence was jected to angular transformation to fulfi ll the found in development time of female and assumptions of normality and homogeneity male parasitoids, when the pooled data of of variance before ANOVA. the two size classes were analysed (ANOVA,
F1, 37 = 2.06, P= 0.160) (Table 2). The mean size (headwidth) of female Results and male off spring of the parasitoid A. sp. near pseudococci in female adults (1.6-2.3 Molecular identifi cation of A. sp.near mm and >2.3 mm) of P. fi cus did not signif- pseudococci icantly diff er signifi cantly between the size
The employment of a PCR-based meth- classes (females: ANOVA, F1,10 = 0.92, P= od in the identifi cation of the Anagyrus spe- 0.360; males: F1,30 = 0.01, P= 0.932) (Table 2). cies, through either BLAST or phylogenetic Female parasitoids were larger (headwidth) tree contruction yielded a similar outcome. than male parasitoids based on the pooled
© Benaki Phytopathological Institute Anagyrus sp. near pseudococci on P. fi cus in Greece 49
Figure 1. Neighbor-Joining phylogenetic tree of the samples and the sequences retrieved from NCBI databank based on the Tamura-Nei nucleotide substitution model (bootstrap repeats = 500). The bar indicates a TN genetic distance of 0.01.
Table 1. Rate of P. fi cus mealybugs parasitized by A. sp. near pseudococci when diff erent host size classes were simultaleously off ered to the parasitoid for 24 hours.
Rate of parasitism Host size classes (mm) n x ± s.e. 1.6–2.3 25 0.22 ± 0.05 >2.3 25 0.18 ± 0.05 n: number of replications
Table 2. Development time and size (headwidth) of female and male off spring of the para- sitoid A. sp. near pseudococci, which emerged from diff erent size classes of female adults of the mealybug P. fi cus.
Parasitoid development time (days) Headwidth (mm)
Host size classes (mm) Females Males Females Males n n n n x ± s.e. x ± s.e. x ± s.e. x ± s.e. 1.6–2.3 8 15.92 ± 0.66 10 17.58 ± 0.99 5 0.51 ± 0.03 19 0.45 ± 0.01 > 2.3 9 16.06 ± 0.59 12 16.54 ± 0.52 7 0.54 ± 0.02 13 0.46 ± 0.01 >1.6 (both size classes) 17 15.99 ± 0.43 22 17.01 ± 0.53 12 0.53 ± 0.02 21 0.46 ± 0.009 n: number of observations
© Benaki Phytopathological Institute 50 Karamaouna et al. data of both parasitized size classes (ANO- ied then. Gülec et al. (15) reported successful rd VA, F1,31 = 15.04, P= 0.001) (Table 2). parasitism of 3 instar nymphs and young Size of male wasps was related positive- female adults of P. fi cus by A. pseudococci in ly to a straight line with the host size (Linear no-choice experiments, indicating that even rd Regression ANOVA, F1,30 = 11.79, P= 0.002) 3 instar nymphs provide adequate resourc- but the size of female wasps was not (ANO- es for successful parasitoid development.
VA, F1,10 = 3.56, P= 0.088); however the fe- Parasitoid fi tness, in particular short- male sample was low. In addition the re- er development time and larger size, of gression line coeffi cient (r2) of the estimated Anagyrus sp. near pseudococci was not di- regression line for males (headwidth = 0.276 rectly related with the host size at parasit- + 0.016 x mumy size) was low (r2 = 0.282). ism. The development time of Anagyrus sp. near pseudococci in young and pre-ovipos- iting female adults of P. fi cus was 16-17 days Discussion (for female and male parasitoids, respective- ly), which is comparable to the development The parasitoid Anagyrus sp. near pseudo- time of 15.2-16.6 days (for male and female cocci was recorded for the fi rst time in parasitoids, respectively) in A. pseudococ- Greece and identifi ed with accuracy using ci found by Gülec et al. (15) at the same a PCR-based technique. Varikou et al. (33) mealybug species and similar temperature have reported the presence of A. pseudo- and photoperiod conditions (28 ± 1oC and cocci in Crete and attributed eff ective bio- 16L:8D). The host size at parasitism did not logical control of P. fi custo the parasitoid in aff ect the size of off spring of Anagyrus sp. organic vineyards. In addition there is only near pseudococci in either sex, which prob- one record of Anagyrus sp. on Planococcus ably means that this is a koinobiont parasi- citri (Risso) (Hemiptera: Pseudococcidae) in toid like A. pseudococci (15). Greece (2). Males and females of Anagyrus sp. near The phylogenetic comparison of the se- pseudococci took similar time to develop in quences of Anagyrus sp. near pseudococ- our study whereas Gülec et al. (15) report- ci with those of A. dactylopii and A. pseudo- ed that males of A. pseudococci developed cocci revealed that A. sp.near pseudococci faster than females in young female adult is genetically closer to A. dactylopii than to mealybugs. However, female parasitoids A. pseudococci, an outcome fully congruent emerging from hosts larger than 1.6 mm with the conclusion of Triapitsyn et al. (31). were larger than male parasitoids emerg- Anagyrus sp. near pseudococci seems ing from the same host size class as it usu- to prefer parasitizing female adults of the ally occurs in solitary Hymenoperan para- vine mealybug P. fi cus, larger than 1.6 mm, sitoids of other mealybug species or other whereas no successful parasitism is ob- insect pests (3, 19). served in smaller mealybugs (0.5-1.5 mm). Should A. pseudococci and Anagyrus sp. Female adults (young and pre-oviposit- near pseudococci be biologically separate ing) appear to be the most profi table host species or not, the present preliminary host stage of P. fi cus for the development of A. size preference bioassay supports that both near pseudococci based on host preference of them have a preference for larger hosts of and successful parasitism. Laboratory ex- the vine mealybug P. fi cus. periments by Daane and Bendley (10) with the parasitoid A. pseudococci indicated also a host size preference for larger hosts of the We would like to thank Dr John Noyes (Natural mealybug P. fi cus based on the parasitoid’s History Museum, U.K.) for the identifi cation of attempts for oviposition in 2nd and 3rd instar the parasitoid samples and the valuable com- nymphs and female adults but successful ments on the systematic taxonomy of the spe- parasitism by A. pseudococci was not stud- cies.
© Benaki Phytopathological Institute Anagyrus sp. near pseudococci on P. fi cus in Greece 51
Literature Cited 15. Gülec, G., Kilincer, A.N., Kaydan, M.B. and Ulgen- turk, S. 2007. Some biological interactions between 1. Alphen, J.J.M. van and Jervis, M.A. 1996. Foraging the parasitoid Anagyrus pseudococci (Girault) (Hy- Behaviour. pp. 1-62. In: M. Jervis and N. Kidd (eds.) menoptera: Encyrtidae) and its host Planococcus Insect Natural Enemies: Practical Approaches to their fi c u s (Signoret) (Hemiptera: Coccoidea: Pseudo- Studies and Evolution, Chapman and Hall, London. coccidae). Journal of Pest Science, 80: 43-49. 2. Argyriou, L.C., Stavraki, H.G. and P.A. Mourikis 16. Gutierez, A.P., Daane, K.M., Ponti, L., Walton V.M. 1976. A list of recorded entomophagous insects and Ken Ellis C. 2008. Prospective evaluaton of of Greece. Benaki Phytopahtological Institute, the biological control of the vine mealybug: re- Kifi ssia-Athens, Greece. fure eff ects and climate. Journal of Applied Ecol- 3. Asante, S.K. and Danthanarayana, W. 1993. Sex ogy, 45: 524-536. ratios in natural populations of Aphelinus mali 17. Jong de, P.W., and van Alphen, J.J.M. 1989. Host (Hym: Aphelinidae) in relation to host size and size selection and sex allocation in Leptomastix host density. Entomophaga, 38: 391-403. dactylopii, a parasitoid of Planococcus citri. Ento- 4. Avise J.C. 2000. Phylogeography - The history and mologia Experimentalis et Applicata, 50: 161-169. formation of species. Harvard University Press. 18. Hopper, K.R. and King, E.G. 1984. Preference of Mi- 5. Avtzis, D.N., Arthofer, W. and Stauff er, C. 2008. croplitis croceipes (Hymenoptera: Braconidae) for Sympatric occurrence of diverged mtDNA lin- instars and species of Heliothis (Lepidoptera: Noc- eages of Pityogenes chalcographus (Coleoptera, tuidae). Environmental Entomology, 31: 1145-1150. Scolytinae) in Europe. Biological Journal of the 19. Karamaouna, F. and Copland, M.J.W. 2000 Host Linnean Society, 94: 331–340. suitability, quality and host size preference of 6. Ben-Dov, Y., Miller, D.R. and Gibson, G.A.P. (2011) Leptomastix epona and Pseudaphycus fl avidulus, Scalenet: Α datadase of the scale insects of the two endoparasitoids of the mealybug Pseudo- world
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27. Smith, M.A., Wood, D.M., Janzen, D.H., Hallwachs, tylopii. Biological Control, 41: 14-24. W. and Hebert, P.D. 2007. DNA barcodes affi rm that 32. Τzanakakis, Μ.Ε. and Katsoyannos B.I. 2003. In- 16 species of apparently generalist tropical para- sects of fruit trees and grapevine. Agrotypos, sitoid fl ies (Diptera: Tachinidae) are not all gener- Marousi-Greece, p. 360 (in greek). alists. Proc. Natl. Acad. Sci. U.S.A., 104: 4967–4972. 33. Varikou, K., Birouraki, A., Bagis, N. and Kontodi- 28. Stauff er, C., Riegler, M. and Lakatos, F. 2001. Ips mas, D.C. 2010. Eff ect of temperature on the de- typographus f. japonicus Niijima (Coleotpera, velopment and longevity of Planococcus fi cus Scolytidae): A genetic analysis by allozymes (Hemiptera: Pseudococcidae). Annals of Ento- and mitochondrial sequence data. In: Jandl R, mological Society of America, 103: 943-948. Devall M, Khorchidi M, Schimpf E, Wolfrum G & Krishnapillay B (eds.). Forests and Society: The 34. Walton, V.M. 2003. Development of an integrat- Role of Research. XXI IUFRO World Congress 7-12 ed pest management system for vine mealy- August 2000. Kuala Lumpur, Malaysia: Pramaju bug, Planococcus fi cus (Signoret), in vineyards Sdn. Bhd, Vol. 3, 397. in the western CapeProvince, South Africa. PhD Thesis. University of Stellenbosch, Stellenbosch, 29. Tamura, K. and Nei, M. 1993. Estimation of the South Africa. number of nucleotide substitutions in the con- trol region of the mitochondrial DNA in humans 35. Walton, V.M and Pringle K.L. 2005. Develop- and chimpanzees. Molecular Biology and Evolu- mental biology of vine mealybug, Planococcus tion, 10: 512-526. fi c u s (Signoret) (Homoptera: Pseudococcidae), and its parasitoid Coccidoxenoides perminutus 30. Tranfaglia, A. 1981. Les pseudococcines de la vi- (Timberlake) (Hymenoptera: Encyrtidae). Afri- gne. Atti Terzo Incontro “La Difesa Integrata del- can Entomology, 13: 143-147. la Vite” Latina, 3-4 Dec. 1981, pp. 211-215. 31. Triapitsyn, S.V., Gonzalez, D., Vickerman, D.B., Web sites Noyes, J.S. and White, E.B. 2007. Morphological, 36. http://www.ncbi.nlm.nih.gov/ Biological and molecular comparisons among the doiff erent geographical populations of An- 37. http://www.technelysium.com.au agyrus pseudococci (Hymenoptera: Encytridae), parasitoids of Planococcus spp. (Hemiptera: Pseudococcidae), with notes on Anagyrus dac- Received: 10 May 2011, Accepted: 14 July 2011
Πρώτη καταγραφή και μοριακή ταυτοποίηση του παρασιτοειδούς Anagyrus sp. near pseudococci Girault (Hymenoptera: Encyrtidae) στην Ελλάδα – Προτίμηση ως προς το μέγεθος του ψευδόκοκκου-ξενιστή Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae)
Φ. Καραμαούνα, Γ. Μενούνου, Γ.Ι. Σταθάς και Δ.Ν. Αβτζής
Περίληψη Το παρασιτοειδές Anagyrus sp. near pseudococci (Hymenoptera: Encyrtidae) καταγράφη- κε για πρώτη φορά στην Ελλάδα και ταυτοποιήθηκε με την βοήθεια τεχνικών PCR. Επίσης εξετάστη- κε η προτίμηση του παρασιτοειδούς ως προς το μέγεθος του ψευδόκοκκου-ξενιστή Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae) σε εργαστηριακά πειράματα όπου ήταν διαθέσιμες τέσσερις κλάσεις μεγέθους (0.5-0.9, 1-1.5, 1.6-2.3 and >2.3 mm) οι οποίες αντιστοιχούν κυρίως σε ένα βιολογικό στάδιο του εντόμου (νύμφες 2ης και 3ης ηλικίας, νεαρά ακμαία θηλυκά και ακμαία θηλυκά πριν την ωο- τοκία, αντίστοιχα). Το Anagyrus sp. near pseudococci παρασίτησε κατά προτίμηση ακμαία θηλυκά άτο- μα του ψευδόκοκκου μεγαλύτερα από 1.6 mm, ενώ δεν παρατηρήθηκε επιτυχής παρασιτισμός σε μι- κρότερα άτομα (0.5-1.5 mm). Η μέση διάρκεια ανάπτυξης του παρασιτοειδούς στις δύο κλάσεις μεγέ- θους του P. fi cus που παρατηρήθηκε παρασιτισμός ήταν 15.99 ± 0.43 ημέρες για τα θηλυκά άτομα και 17.01 ± 0.53 ημέρες για τα αρσενικά (28 ± 1oC, 16L:8D) χωρίς στατιστικά σημαντική διαφορά μεταξύ των δύο φύλων. Το μέγεθος του ξενιστή κατά τον παρασιτισμό δεν είχε επίδραση στο μέγεθος των απογό- νων του παρασιτοειδούς.
Hellenic Plant Protection Journal 4: 45-52, 2011
© Benaki Phytopathological Institute Hellenic Plant Protection Journal 4: 53-56, 2011
SHORT COMMUNICATION
First record of Lixus umbellatarum Fabricius (Coleoptera: Curculionidae) in Greece, on the medicinal herb Opopanax chironium
C.Th. Buchelos1 and E. Evergetis2
Summary In summer 2004, adults of a Coleopteran species were observed piercing with their ros- trum the stems of the plant Opopanax chironium at an altitude of 1850 m on Parnassus Mountain. Upon insect’s feeding, the eff used sap of the plant turned into droplets of a resinous fl uid which is known to have pharmaceutical and other useful properties. The insect was identifi ed as Lixus umbellatarum Fab- ricius of the Curculionidae family and it is recorded for the fi rst time in Greece.
Additional keywords: Apiaceae, Coleoptera, Greece, Panacea, Panax, Umbelliferae
In summer 2004, Opopanax sp. plants were ple was found to be totally devoured by the observed on the northwest (NW) side of recorded weevil which had been acciden- Mount Parnassus, Greece to bare light-or- tally introduced into the sample and sur- ange coloured droplets of a resinous liq- vived in -22o C for about a two month peri- uid on their stems lengthwise, in a rather od. In mid April of 2008, when the Opopanax straight line and regular distance between them. Because of the late season, the plants carried only mature umbels; therefore prop- er identifi cation of the taxon was not possi- ble. In May 2006, the droplets observed on the Opopanax sp. plants were associated with an adult weevil, which was found at the same site to feed on the stem, causing eff u- sion of the sap (Fig.1). At that time, a plant specimen was collected in order to form a proper herbarium for its defi nite identifi ca- tion. After the ordinary procedure, which included drying and disinfestations at low temperatures, the plant sample was stored among the other Apiaceae (Umbelliferae) collections of the Agricultural University of Athens (AUA). One year later, the plant sam-
1 Emeritus Professor at Agricultural University of Ath- ens, 52 Agias Varvaras Str., GR-152 31 Halandri (Ath- ens), Greece 2 Laboratory of Chemistry, Department of Science, Ag- ricultural University of Athens, 75 Iera Odos Str., GR- 118 55 Athens, Greece Figure 1. Lixus umbellatarum feeding on Opopanax chironi- Corresponding author: [email protected] um stem.
© Benaki Phytopathological Institute 54 Buchelos & Evergetis sp. plants possess only their lower leaf ro- cialization of the related keys, the fi rst one settes and the basal part of the previous referring to the mountain fl ora of Greece year’s stem, living weevil adults were found and the second to Turkey and East Aegean. in the openings of exit holes at the base of Another inquiring character, observed in the dead stems, indicating that the insect the specimen and was never described be- had survived the severe winter conditions fore, relates to the presence of reticulate, at 1850 m. altitude. In summer of 2008, sev- ribbed and brightly yellow coloured veins eral individuals of the weevil were collected both in basal and cauline leaves. This sec- and identifi ed in the Laboratory of Entomol- ondary character, absent in the population ogy of AUA. of O. chironium from Peloponnesus and the All biological material was collected in island of Rhodes, which was not infected the NW side of the Mountain Parnassos – by the insect, could be considered as a re- Greece at the location “Kokinorachi” (lati- sponse to the insect’s presence. However, tude 38 34´ N., longitude 22 28´ E., altitude this hypothesis needs further experimental 1850 m), in the clearings of an Abies cephal- confi rmation. lonica forest with limestone substrate, in The identifi cation of the insect to the consequent years from 2004 to 2008. species was made by the fi rst author (spe- Plant material presented a distinct and cialist on Curculionidae) based on classi- unique combination of characters attrib- cal monographs (9, 14, 15). The genus Lix- uted to genus Opopanax W.D.J. Koch. (3, 7, us is characterized by a cylindrical rostrum 16, 17). These are the excessive presence and elytra rather narrow and oblong end- of stellate hairs in the stem and the leaves, ing in two separate points. The body surface the large 2-pinnate basal leaves with ovate seems to be covered with yellow or reddish leaf-lobes, the yellow petals and the strong- ‘dust’ (effl orescence) while its basic colour ly compressed obovate fruit (17). Opopa- is black with dense and thin punctuation nax W.D.J. Koch consists of stout perenni- and the abdominal pubescence is yellowish al herbs, including 3 species worldwide (13). with bi- or trifi de setae. Lixus (Eulixus) umbel- The species which occur in Greece include latarum belongs to Rhynchophora Coleop- O. hispidus (Friv.) Griseb. and O. chironium (L.) tera, the subfamily Cleoninae and the tribe Koch (17) but the presence of the later above Lixini, with the following taxonomic history: 1.500 m is dubious (16). Lixus (Eulixus) umbellatarum Fabricius, 1787 The herbarium identifi cation was based primary genus: Curculio, subgenus Eulixus on the four most prominent identifi cation Reitter, 1916 = Promecaspis Hoff mann, 1958. keys of these species (3, 7, 16, 17). The orig- species: pardalis Boheman, 1835 –submacu- inal key character is related to the size and latus Boheman, 1842 –brevicaudatus Lucas, the border of the fruit, which in the exam- 1848 –brevicaudis Küster, 1849. Species’ char- ined herbarium was 6-7 mm with a narrow acteristics include: Body length 8-14 mm. Ef- and thickened border, concluding to O. chi- fl orescence of pronotum, greenish-yellow. ronium (7, 17). Later key characters advocat- Rostrum of female slender, cylindrical, con- ing this identifi cation are the rays’ number, vex, shinny, longer than the prothorax. Ros- which in the herbarium was counted up to trum of male less convex, opaque, densely 23 (3) and the leaf lobes length which was punctuated. Antennae short, fairly slender measured up to 14 cm (16, 17). A deviation with the fi rst two segments of the fl agellum from the described key characters was ob- equal, the 3rd to 6th being of the same diame- served in relation with the thickened bor- ter and the 7th larger. Antennal club ellipsoid der’s width, which was measured from 1 to and sharp- pointed. Antennae (except the 2 mm, when in O. chironium is described less club) and tarsi, ferruginous. Eyes oval and than 1 mm, and in O. hispidus between 2-3 convex. Prothorax conical, more slender in (3, 16). This deviation can be explained by the male. Elytra, at their base, slightly larger the geographical (16) or territorial (3) spe- than prothorax. Tarsi short (Hoff mann, Por-
© Benaki Phytopathological Institute Lixus umbellatarum on the Opopanax chironium 55 ta, Portevin). stitutive antifungal agents or phytoalexins The presence of L. umbellatarum has (10). Experimental investigation is required been confi rmed in France (Corsica) (14, 15) in order to ascertain the recorded insect- and in Italy (Latsio, Campania, Pulia, Sardin- plant interaction and determine the role of ia) (14). According to Hoff man (9), the insect is the weevil in the induction of Panacea. rare in the French mainland but abundant in Corsica, feeding on miscellaneous Apiaceae such as Ferula communis L., Pastinaca lutea Literature cited L. and O. chironium. It is also found in Spain, 1. Appendino, G., Bianchi, F., Bader, A., Campag- Portugal, Algeria and Morocco. There are ‘no nuolo, C., Fattorusso, E., Taglialatela-Scafati, data’ for its presence in the Greek mainland O., Blanco-Molina, M., Macho, A., Fiebich, B.L., and the islands (19). Bremner, P., Heinrich, M., Ballero, M. and Munoz, E. 2004. Coumarins from Opopanax chironium. The host plants of L. umbellatarum be- New Dihydrofuranocoumarins and Diff erential long to the families Apiaceae, Chenopo- Induction of Apoptosis by Imperatorin and Her- diaceae, Polygonaceae, Caryophylaceae, aclenin. Journal of Natural Products, 67: 532-536. Brassicaceae (Cruciferae), Lamiaceae (Labi- 2. Bohlmann, F. 1971, Acetylenic compounds in atae), Papilionaceae, Capparidae, Malvace- the Umbelliferae, Botanical Journal of the Linne- an Society, 64: 279-291. ae and Geraniaceae. The larvae of the wee- 3. Davis, P.H. 1984. Flora of the Turkey and the East vil feed on the internodes of the stem; they Aegean islands, Edinburg University Press, vol. 4, bore straight descending galleries through pp 471-473. petioles and stems attaining the plant’s 4. Dioscorides, 1st-2nd cent. A.D., De Materia Medi- ‘neck’ or the upper part of the roots. (12). ca, Codex Neapolitanus Graecus 1 of the Nation- Hibernation takes place in the adult stage, al Library of Naples, Photographic reprint by Ge- nus Publications, Athens, Greece, 2001. usually in the soil, appearing during March 5. French D.H., 1971, Ethnobotany of the Umbel- and April and feeding up to September. Pu- liferae, Botanical Journal of the Linnean Society, pation takes place at the end of the galleries 64: 385-412. and life cycle usually lasts one year. 6. Gijbels, M.J.M., Bos, R., Scheff er, J.J.C. and Sv- The present report associates feeding endsen, A.B. 1983. Phthalides in roots Opopanax of the weevil L. umbellatarum on O. chiro- chironium, Planta Medica, 47: 3-6. nium with the produce of resin by the plant. 7. Halacsy, E. 1900. Conspectus Florae Graecae, Lip- Opopanax chironium is richly celebrated siae Sumptibus Guilelmi Engelmann, vol. 1, p 637. through history for the medicinal properties 8. Hegnauer R., 1971, Chemical patterns and rela- of its resin, Panacea, applied mainly as anal- tionships of Umbelliferae, Botanical Journal of gesic and venom’s antidote (4). Among the the Linnean Society, 64: 267-277. natural products that have been identifi ed 9. Hoff mann, A. 1950. Faune de France, 52 (2ème par- in O. chironium are C-17 acetylenes (2), vari- tie): Curculionidae, Librairie de la Faculté des ous phthalides in the roots (6), coumarin de- Sciences, Paris. rivatives (1, 8) and irregular diterpenoids (11), 10. Harborne, J.B. and Williams, C.A. 2000. Advan- ces in Flavonoid research since 1992. Phyto- while crude extracts of the plant present sig- chemistry, 55: 481-504. nifi cant apoptotic activity (1). Opopanax sp. 11. Muckenstrum, B., Foechterlen, D., Reduron, J.- resin and oil are also used as a direct food P., Danton, P. and Hildenbrand, M. 1997. Phyto- additive (18), expectorant and antispasmod- chemical and chemotaxonomic studies of Foe- ic (5, 11) and in perfumery (5). Despite this niculum vulgare. Biochemical Systematics and Ecology, 25: 353-358. extensive use of Opopanax sp. resin, the 12. Perrier, R. 1932. La Faune de France, Fasc.6: Colé- presence of L. umbellatarum has never been optères, Librairie Delagrave, Paris. reported before as a stimulating agent that 13. Pimenov, M.G., Leonov, M.V. 1993. The genera of induces resin production. This suggestion the Umbelliferae. Royal Botanic Gardens Kew & is advocated by the fact that several of its Botanical Garden of Moscow State University, compounds, such as coumarins, furanocou- Kent U.K., pp 156. marins and fl avonoids, are identifi ed as con- 14. Porta, A. 1932. Fauna Coleopterorum Italica, Vol.
© Benaki Phytopathological Institute 56 Buchelos & Evergetis
V: Rhynchophora – Lamellicornia, Stab. Tipogr. 18. United States of America. 1966. Legislation Re- Piacentino, Piacenca. gulations governing the use of colour additives, 15. Portevin, G. 1935. Histoire Naturelle des Coléoptè- pesticides and food additives, Fd Cosmet. Toxi- res de France, Tome IV: Polyphaga – Rhynchopho- coL Vol. 4, pp. 63-72. Pergamon Press 1966. Prin- ra, Paul Lechevalier, Paris. ted in Great Britain. 16. Strid, A. 1986. The mountain fl ora of Greece, Cam- 19. www.faunaeur.org/distribution.php, 2011 bridge University Press, vol. 1, p 714. 17. Tutin, G.T., Heywood, H.V., Burges, A.N., Moore, M.D., Valentine, H.D., Walters, M.S. and Webb, A.D. 1972. Flora Europaea, Cambridge Universi- ty Press, vol. 3, p 360. Received: 15 May 2011, Accepted: 14 July 2011
ΣΥΝΤΟΜΗ ΑΝΑΚΟΙΝΩΣΗ
Πρώτη καταγραφή του Lixus umbellatarum Fabricius (Coleoptera: Curculionidae) στην Ελλάδα. H παρουσία του στο φαρμακευτικό φυτό Opopanax chironium
Κ.Θ. Μπουχέλος και Ε. Ευεργέτης
Περίληψη Το καλοκαίρι του 2004, παρατηρήθηκαν τέλεια άτομα κολεοπτέρου εντόμου της οικογέ- νειας Curculionidae να διατρέφονται σε στελέχη φυτών Opopanax chironium στο Όρος Παρνασσός, σε υψόμετρο 1850 μ. Το ρητινώδες κόμμι που εκκρίνεται από τις οπές των νυγμάτων του εντόμου, είναι ο γνωστός από την αρχαιότητα, πλούσιος σε φαρμακευτικές και άλλες χρήσιμες ιδιότητες «οποπάναξ» ή «πανάκεια», ο οποίος έδωσε το όνομα του γένους στο εν λόγω φυτό. Το έντομο προσδιορίστηκε ως Lixus umbellatarum Fabr. (Col.: Curculionidae) και αποτελεί πρώτη αναφορά για την Ελλάδα.
Hellenic Plant Protection Journal 4: 53-56, 2011
© Benaki Phytopathological Institute Hellenic Plant Protection Journal 4: 57-69, 2011
Bioaccumulation of thiram in Mytilus galloprovincialis and its eff ect on diff erent tissues
Ch. Emmanouil1, K.M. Kasiotis1 and K. Machera1
Summary The accumulation of thiram, a characteristic dithiocarbamate fungicide, in Mytilus gallopro- vincialis soft tissues and its eff ect on bivalvian DNA integrity has been examined through a series of in vivo exposures of mussels to 0.1, 1.0 and 10.0 mg thiram/L in saltwater for 48 h. Regarding bioaccumu- lation of the fungicide, a dose-dependent increase of the sodium adduct ion of thiram was observed in mussel soft tissues after the end of the exposures. This identifi cation/quantifi cation of thiram and one of its metabolites was achieved by Liquid Chromatography Mass Spectrometry (LC-MS) analysis. Regarding DNA integrity (DNA Single Strand Breaks) these were strongly dose-dependent. They were also tissue-dependent in concordance with the diff erent susceptibility of certain tissues to pollutants and the levels of metabolism that takes place in them. Further investigation in oxidative and apoptotic DNA damage revealed that a) oxidative stress was evident in all the tissues examined b) apoptotic cell morphology was detected in gill and digestive gland. Imbalance of the antioxidant/prooxidant status in favour of the latter and initiation of apoptosis may be a causative mechanism of DNA damage in M. galloprovincialis, as it has already been shown for DNA damage caused by thiram in mammals.
Additional Keywords: apoptosis, dithiocarbamates, LC-MS, mussel, oxidative stress, Single Strand Breaks
Introduction also an intermediate metabolite of two oth- er dithiocarbamates-ferbam and ziram (41). The dithiocarbamate class (DTCs) of fungi- Several analytical methods are available for cides comprises a group of plant protection the detection and quantifi cation of thiram products (PPP) which are widely used world- and its metabolites. Gas chromatographic wide. They are the main group of fungicides techniques have been applied with determi- used to control approximately 400 patho- nation of carbon disulfi de (CS2), the product gens of more than 70 crops and are regis- of the rapid degradation of DTCs. However, tered in all the EU member states and many these methods are laborious and this has di- other countries (18). They are of relatively rected scientists to the exploitation of LC- low acute toxicity for humans with an aver- MS methods which off er the advantage of age LD50 of more than 2523 mg/kg bw (41). the direct determination of DTCs and thiram Thiram (tetramethylthiuram disulfi de) is one in particular. of the most characteristic dithiocarbamates. Thiram, like the majority of dithiocar- It was synthesized in 1931 and since then has bamates, exerts its toxic action via creation been extensively used as a fungicide (41), as of metabolites of carbon disulfi de (43) and it a seed treatment in maize, cotton and cere- is of low mammalian toxicity (17). However, als (25, 35), as an accelerator and vulcanis- in certain animal models it has caused he- ing agent during rubber processes as well as patotoxicity (24, 25) and adverse develop- for treatment of human scabies and in anti- mental and reproductive eff ects (24, 27, 40). septic soaps and preparations (21, 22). It is It has also caused eczema, contact dermati- tis and skin lesions to exposed workers (21, 1 Laboratory of Toxicological Control of Pesticides, De- 22, 40). partment of Pesticides Control and Phytopharmacy, Since thiram is so extensively used, it is Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia, (Athens), Greece commonly found in aquatic environments Corresponding author: [email protected] (31). According to U.S. Environmental Pro-
© Benaki Phytopathological Institute 58 Emmanouil et al. tection Agency “thiram is expected to be prepared fresh in distilled water from com- suffi ciently mobile and persistent in some mercially available thiram 80% w/w and was cases to reach surface waters in concentra- diluted to nominal concentrations of 0.1, 1.0 tions high enough to impact aquatic life” and 10.0 mg thiram/L in saltwater. Thiram an- (20). Moreover, it may not be readily catab- alytical standard was purchased from Fluka olised and it may persist in soil for several (Buchs, Switzerland) and a stock solution of weeks. 100 μg mL-1 was prepared in methanol. The A series of in vivo experiments have working solutions were prepared from this shown that thiram may aff ect certain bio- stock solution in methanol. Methanol and ac- logical or biochemical aspects in aquatic etonitrile were purchased from Merck (Darm- organisms namely cladocerans (31), fi sh (6, stadt, Germany) and were LC-MS grade. Eth- 42) and mussels (15). The aim of the present yl acetate (pro analysi) was also purchased project was to test for bioaccumulation and from Merck (Darmstadt, Germany). eff ects of thiram, which is in vivo non-clas- togenic for mammals (19) in the model ma- Experimental set up rine mussel of the Mediterranean Sea Myti- Healthy mussels from the aquaria were lus galloprovincialis, in relation to tissue and exposed in 2 L glass beakers (Simax, Czech dose. Following positive results, mechanistic Republic) at a density of 4 mussels/beaker aspects of this interaction have been sought to fi nal concentrations of the agrochemi- (oxidative DNA damage and early apoptotic cal stated in 2.2. The exposures lasted for DNA damage). These pathways are implicat- 48 h after which the animals were sacrifi ced. ed in thiram toxicity in mammals but they The water was changed every 12 h and was have not yet been examined in bivalve spe- spiked with thiram after each renewal. Dur- cies. ing the exposure the animals were not fed. The general condition of the animals and the mortalities were recorded daily. Materials and Methods Chemical analysis Test animals Commercially available Mytilus gallopro- Soxhlet Extraction vincialis from a mussel farm in Evia, Greece Lyophilized mussels (7 g) were placed were purchased. Mussels of similar size (8-9 with equal amount of sodium sulfate at the cm) were kept in continuously aerated glass bottom of the Soxhlet apparatus covered aquaria of 15 L saltwater (salinity 33‰) at with glass wool. The solvent of choice was a ambient temperature of 25°C, under natu- mixture of ethyl acetate/hexane (1:1 respec- ral light. The mussels were fed with approxi- tively, 200 mL) and the extraction time was mately 0.05 g powdered Spirulina (M.Rohrer, 4 h. After the extraction the solvent was re- Netherlands) every day. Nitrate and nitrite moved in vacuum and the mixture was re- levels were periodically checked (API phar- constituted with ethyl acetate (2 mL). maceuticals, USA) and they did not exceed 0.5 and 0.25 μg mL-1 respectively. The mus- Removal of sulfur compounds and Solid sels were acclimatized in laboratory condi- Phase Extraction (SPE) tions for at least 5 days before the beginning The removal procedure was the follow- of the experiments. These species absorb ing: 1 g of activated copper was added to quickly contaminants from their environ- the reconstituted mixture (2 mL) and stirred ment but they depurate in an equally rapid vigorously for 2 min. Centrifugation (Her- manner when found in clean water (2). aeus Labofuge 400R Thermo Electron Cor- poration) of the mixture was followed by Fungicide and Solvents the separation of the organic phase. Then A stock solution of 10 mg thiram/L was the organic phase was passed from Flori-
© Benaki Phytopathological Institute Thiram Bioaccumulation in Mytilus galloprovincialis 59 sil SPE cartridge (Waters, SEP-PAK® Cartridg- it of Detection (LOD) and Limit of Quantifi - es), fi ltered (Whatman, PuradiscTM 25 TF fi l- cation (LOQ), recoveries and matrix depen- ters, 0.45 μm), evaporated under a gentle dent variations as it is established by the EU stream of nitrogen, reconstituted with ac- guidelines. Linearity and matrix eff ect were etonitrile (0.5 mL) and 10 μL were injected assessed by analyzing standard solutions to Liquid Chromatograph Mass Spectrome- and matrix matched standards at six points ter (LC/MS). in the range of 0.20-7.88 μg mL-1 to cov- er the expected range of concentrations in Experimental Procedure for Extraction of samples. Recovery values derived from for- Thiram from Water Samples tifi ed experiments at two levels of concen- An aliquot of 2 mL of water sample was tration and were considered as the measure mixed with 2 mL of ethyl acetate (vortex, a of the trueness of the analytical method. For MS1 Minishaker, IKA) for 2 minutes. Then the repeatability and reproducibility studies of mixture was placed in an appropriate falcon the LC-MS methodology, fi ve replicate de- tube (15 mL) and centrifuged at 4000 rpm terminations on the same day and on fi ve for 5 min, at 4°C. The organic layer was col- diff erent days of a standard solution (0.78 lected, fi ltered (Whatman, PuradiscTM 25 TF μg mL-1 of thiram) were performed. Repeat- fi lters, 0.45 μm), evaporated under a gentle ability and reproducibility is considered ac- stream of nitrogen, reconstituted with ace- ceptable when relative standard deviation tonitrile (0.5 mL) and then an amount of 10 values (RSD%) are < 20%. Based on the sta-
μL was injected for analysis to the Liquid tistical defi nition 3.3 (SY/x)/α and 10(SY/x)/α, of Chromatograph Mass Spectrometer. the LOD and LOQ respectively, they were de- termined at the concentration levels rang- -1 Liquid Chromatography Mass Spectrometry ing from 0.20 to 7.88 μg mL . SY/x represents A Shimadzu (Kyoto, Japan) LCMS-2010 EV the residual standard deviation and α is the Liquid Chromatograph Mass Spectrometer slope of the respective calibration plot. instrument was used with the LCMS solution version 3.0 software consisting of a SIL-20A Tissue preparation for biochemical as- prominence autosampler and an SPD-M20A says diode array detector. The latter were cou- The three main tissues of mussels (gill, pled in series with a mass selective detec- digestive gland and haemolymph) were tor equipped with an atmospheric pressure extracted from sacrifi ced animals after the ionization. The LC separation was achieved end of the experiment. Briefl y, for gills, the working in positive Electron Spray Ioniza- valves of the mussel were fully opened with tion (ESI) mode, on a Shim-Pack XR-ODS 2.2 a metallic scalpel and wet gill tissue (< 0.2 μm, 100×4.6 mm i.d. chromatographic col- g) was extracted. The gill was gently disag- umn using a gradient system consisting of gregated in HEPES buff ered saline (2 mL). methanol and water. The fl ow rate was set at After centrifugation a fraction of the pel- 0.8 mL min-1 and the column gradient pro- let was rediluted in saline (100 μL) and kept gram consisted of 40% methanol and 60% on ice until further processing. For haemo- water, ramped linearly over the course of 7.5 lymph the valves of the mussel were slight- min at 70 % methanol. Then methanol re- ly opened and haemolymph was abstracted turned in the course of 2 min at 60% con- from the posterior adductor muscle accord- centration and the mobile phase was held at ing to Rank and Jensen (36) with a 23G nee- that composition from 9.5 min to 15 min. dle and a 1 mL syringe (PiC Insumed, Italy). Approximately 100 μL were abstracted from Validation procedure each animal and used without further treat- For the validation procedure the follow- ment. Digestive gland suspension prepa- ing parameters were determined: linearity, ration was performed according to Birmel- repeatability, reproducibility, analytical Lim- in et al. (8) with minor modifi cations. Briefl y,
© Benaki Phytopathological Institute 60 Emmanouil et al. digestive glands (< 0.2 g) were abstracted were rinsed 3 times in 1mL of Fpg buff er (KCl from the animal and cut in small pieces with 0.1M, HEPES 40 mM, Na2EDTA 0.5mM, bovine surgical scissors. The pieces were gently dis- serum albumin 0.2 mg/mL, pH 8.0) each. aggregated in HEPES-NaOH buff ered saline Four slides per animal were prepared. One (10 mL) and agitated on a rocking platform slide per pair was incubated with one unit of (Innova2000, Brunswick Scientifi c) on ice for Fpg enzyme (AMS Biotechnology, UK) in Fpg 2 h. Every 30 min aliquots of cell suspension buff er (50 μL) for 1 h as described by Collins (2 mL) were taken out and replaced with et al. (14). The remaining slide of each pair new HEPES-NaOH buff ered saline. All the was incubated with 50 μL Fpg buff er only. 80 aliquots from the same animal were fi nal- randomly selected nucleoids were analyzed ly pooled (8 mL). After centrifugation a frac- from the non-Fpg incubated slides and 80 tion of the pellet was rediluted in saline (100 randomly selected nucleoids were analyzed μL) and kept on ice until further processing. from the Fpg incubated slides. A total of 160 The preparations provided whole and single cells were scored. The net diff erence (Fpg-in- cells with negligible additional damage. cubated minus non Fpg-incubated) is pro- portional to oxidative DNA damage. Single Cell Gel Electrophoresis on mus- sel tissue (Comet assay) Modifi ed Single Cell Gel Electrophoresis A fraction of the cell suspension (15 μl) (Halo assay) was mixed with 150 μL low melting point The procedure was run as described in agarose. The mixture was spread on an aga- 2.6 with the complete omission of the elec- rose-precoated slide and lowered in lysis trophoresis step. A positive apoptosis con- buff er (NaCl 2.5M, Na2EDTA 0.1M, Tris base 10 trol was co-evaluated: staurosporine (Sig- mM, Triton-X 1% v/v and DMSO 10% v/v, pH ma-Aldrich) was diluted in DMSO to a fi nal 10.0) for 1 h at 4º C in the dark. Two slides per concentration of 1 μΜ (26) and was injected animal were prepared. The slides were then (100 μL) in the adductor muscle directly be- rinsed in distilled water (1mL/slide) and left low the mantle of two individuals. The ani- in electrophoresis buff er (pH 13.0) in a hor- mals were returned to a plastic aquarium of izontal electrophoresis tank (Cleaver Scien- 1 L and sacrifi ced 4 h post-injection. Select- tifi c, Ltd) for 30 min. The slides were subse- ed tissues were collected and processed to- quently subjected to electrophoresis at 25 V gether with the thiram samples. 50 random- for 20 min, neutralised with Tris buff er (Tris ly selected nucleoids were checked per slide 0.4M, pH 7.5) and stained with propidium io- in two slides so that a total of 100 cells per dide (2.5 μg/mL) (Research Organics, Cleve- animal were scored. The percentage of char- land, USA). Each slide was analyzed using a acteristic halo images of apoptotic cells (39) fl uorescent microscope (Zeiss AxioCam MRC, was calculated. Carl Zeiss Inc., Germany) at 200 x magnifi ca- tion, with an excitation fi lter of 515-560 nm Statistical Analysis and a barrier fi lter of 590 nm and scored us- Diff erences between groups for SSB ing an image analysis package (TriTekCom- were assessed using the parameter %DNA in etScoreTM). 40 randomly selected nucleoids tail. Normality of data was tested by the Sha- were analyzed per slide in two slides so that piro-Wilk W-test. Since data did not follow a a total of 80 cells (per animal) were scored. Gaussian distribution median values were used for each set of cells (16). Data were an- Modifi ed Single Cell Gel Electrophoresis alyzed by 2-way ANOVA. Means were sep- (Comet coupled with formamidopyrimi- arated by Tukey’s HSD test (α=0.05). Diff er- dine glycosylase) ences between groups for % Fpg sensitive The procedure was as described in 2.6 sites and for % apoptotic cells were assessed with the exception that, after lysis and be- by Student’s t-test. Analyses were conduct- fore unwinding in high pH buff er, the slides ed using the statistical package JMP.
© Benaki Phytopathological Institute Thiram Bioaccumulation in Mytilus galloprovincialis 61
Results Figure 3. For the sample of 10 μg mL-1, thiram was determined as the sum of areas of peaks Validation results which correspond to thiram and its metab- The analytical method applied produced olite as equivalent of thiram. The sum con- good response of linearity in the range of centration measured was 8.19 μg mL-1. 0.20-7.88 μg mL-1 with correlation coeffi cient value r2 = 0.998 (equation, y=241932x-4695). Mussel mortality LOD and LOQ were determined from the The general condition of the animals (i.e. calibration plot and were 0.32 μg mL-1 and reaction to stimuli, excretion of mucus, at- 0.96 μg mL-1 respectively. Satisfactory re- tachment to glass-walls) was recorded daily sults were obtained for all levels with recov- and dead animals were discarded as soon as eries for low and high concentrations, well possible. Profuse mucus secretion was de- above the cut off value of 70%. RSDs % for tected only in the high dose groups. Mortal- both low and high concentrations were < ities were low and did not exceed 12.5 % in 5% therefore acceptable. RSDs % for repeat- all exposed groups. ability and reproducibility were 3.69% and 4.49% respectively. Finally no matrix eff ect SSB in relation to dose group and tissue was observed. type SSB values were aff ected by both tissue Identifi cation of thiram, accumulation and dose, while there was signifi cant inter- in mussel soft tissues and concentration action between dose and tissue (Table 2). levels in aqueous solutions Since signifi cant interaction was observed, The identifi cation of thiram (6.61 min, re- multiple comparisons were conducted at tention time) was achieved by the LC-ESI-MS each tissue for all doses and at each dose for system functioning on the positive mode. all tissues (Figure 4). Selected Ion Monitoring mode (SIM mode) For gill, exposure to 0.1 mg/L thiram was applied and the characteristic sodi- caused a statistically signifi cant increase in um adduct [M+Na]+ ion of thiram was ob- SSB (see also Figure 5A,B). Higher doses (1.0 served (Figure 1). Accumulation of thiram in and 10.0 mg/L) also caused an increase in mussel soft tissues for exposure of 0.1 mg/L relation to control but these were indistin- (100 μg/kg) was below the LOD and thus guishable from each other. For haemolymph, not quantitated. Accumulation for the 1.0 exposure to the high dose only (10.00 mg/L) and 10.0 mg/L (1000 and 10000 μg/kg cor- caused an increase in relation to control respondingly) exposure group was 375 μg/ whereas the low and medium dose did not kg d.w. and 3115 μg/kg d.w respectively (Ta- produce a statistically signifi cant increase ble 1). Next step was the determination of in SSB. For digestive gland there was also a the actual concentrations of thiram in aque- dose response with the low dose group be- ous solutions. Thus three diff erent concen- ing indistinguishable from the control group tration levels were investigated same with and the medium and high dose groups be- those presented in Table 1. Surprisingly for ing diff erent from the control group but not 0.1 and 1 μg mL-1 the only peak which ap- diff erent from each other. peared (3.55 min, retention time) and was identifi ed was the peak of another sodium Oxidative DNA damage in relation to + adduct [C3H7NS2+Na] (47). It seems that in dose group and tissue type more dilute aqueous solutions of thiram For gill, exposure to the medium dose of + the peak of [C3H7NS2+Na] predominates. 1.0 mg/L caused a very signifi cant increase One possible fragmentation pathway is de- (P<0.001) in Fpg-sensitive sites which corre- picted in Figure 2. The aqueous solution of spond to oxidative DNA damage (see also 10 μg mL-1 contained both sodium adducts Figure 5C). For haemolymph and digestive + + [M+Na] and [C3H7NS2+Na] as it is shown in gland, exposure to the medium dose of 1.0
© Benaki Phytopathological Institute 62 Emmanouil et al.
Figure 1. Total Ion Chromatogram (TIC) of Thiram in mussels sample at 0.788 μg mL-1 and the chromatogram of its Sodi- um Adduct (m/z 263).
Table 1. Accumulation of thiram in mussel soft tissues.
Treatment Thiram (μg/kg d.b.w.) Control n.d. 100 μg/kg n.d. 1000 μg/kg 375 10000 μg/kg 3115 n.d.: non detected Figure 2. ESI-MS fragmentation of Thiram.
Figure 3. Total Ion Chromatogram (TIC) of Thiram in aqueous solution at 10 μg mL-1. The SIM chromatograms of its Sodium Adduct (m/z 263) and the other identifi ed adduct at m/z 143.
Table 2. F- values of the eff ect of tissue and dose and their interaction on SSB values for Myti- lus galloprovincialis (2-way ANOVA).
df MS F p Tissue 2, 24 234.02 12.32 0.0002 Dose 3, 24 993.18 52.31 <0.0001 tissue/dose 6, 24 128.82 6.78 0.0003
© Benaki Phytopathological Institute Thiram Bioaccumulation in Mytilus galloprovincialis 63
Figure 4. Percentiles of the values of SSB for each tissue of the 3 mussels in each group. Values followed by diff erent letter diff er signifi cantly (Tukey’s HSD test at α=0.05). Capital letters (A, B, C) indicate diff erences within tissue among concentra- tions while small letters (a, b, c) indicate diff erences among tissues within a concentration.
© Benaki Phytopathological Institute 64 Emmanouil et al.
A B
C D
Figure 5. PI stained nuclei (A): control, gill (B): high dose, gill (C): Fpg-treated, medium dose, gill (D): apoptotic cell, me- dium dose, gill.
Table 3. Percentage of apoptotic cells and of Fpg-sensitive sites in mussels exposed to 1.0 mg/L thiram (*: P<0.05 in relation to control, ** P<0.01, ***P<0.001).
Treatment Apoptotic cells (%) Haemolymph Gill Digestive gland Control 0 0.25 ± 0.5 0.5 ± 0.6 1.0 mg/L 0 2 ± 0.41* 1.5 ± 0.6* % Fpg sensitive sites Haemolymph Gill Digestive gland Control 0.37 ± 0.05 3.96 ± 1.91 12.46 ± 3.57 1.0 mg/L 3.80m ± 0.76** 28.04 ± 6.93*** 27.79 ± 6.09** mg/L caused a signifi cant increase in Fpg- phology to the cells observed in the stau- sensitive sites (Table 3). rosporine-injected mussels. These cells are characteristic of apoptotic DNA dam- Apoptotic DNA damage in relation to age (see also Figure 5D). For haemolymph, dose group and tissue type exposure to the medium dose of 1.0 mg/L For gill and digestive gland, exposure to did not cause a signifi cant increase in halo the medium dose of 1.0 mg/L caused a sig- cells. Staurosporine injection caused a sig- nifi cant increase in halo cells of similar mor- nifi cant increase in apoptotic cells (P<0.001)
© Benaki Phytopathological Institute Thiram Bioaccumulation in Mytilus galloprovincialis 65 for all cell types in relation to control. Injec- vivo, despite being Ames positive (19). In vit- tion and/or DMSO does not cause additional ro alkaline fi lter elution has also produced DNA damage as proved by a series of exper- positive results (increase in SSB) in rat and iments in tissues of Mytilus edulis and Mytilus human cells (9). The discrepancy between galloprovincialis (data not shown) in vivo and in vitro results for thiram is fur- ther mirrored in the experiments conducted by Villani et al. (45), where exposure of mice Discussion to the maximum tolerated dose caused a borderline increase in SSB in lymphocytes Even though thiram is toxic to fi sh (19), bi- and no increase in splenocytes. In contrast, valves (Unio tumidus) were able to withstand concomitant exposure of human lympho- 0.1 mg/L thiram for 3 days without mortal- cytes to thiram in vitro caused a signifi cant ities (15). Taking into account the apparent increase in SSB. Our results for Mytilus gal- lack of thiram LC50 for bivalves in bibliogra- loprovincialis can therefore mostly relate to phy, the aforementioned concentration was the in vitro outcomes of Bjørge et al. (9) rath- used as the lowest one which can elicit bio- er than to the in vivo ones of Villani et al. (45). chemical responses. This concentration elic- Bivalves possess unique physiology and bio- ited responses in some of M. galloprovincial- chemistry quite diff erent from higher organ- is tissues as mentioned later in this section. isms. As a result, innocuous substances for Usually, the determination of thiram is higher organisms may be biologically active associated with its decomposition to car- for bivalves and vice versa. bon disulfi de (CS2) in acidic medium, fol- The eff ect of thiram (SSB increase) was lowed mainly by spectrometry (5, 12, 38) diff erent in each tissue as shown in Figures and head space gas chromatography (1, 34). 4 and 5 and there was a signifi cant interac- The drawback of these methods is that they tion between dose and tissue (Table 2). The are time consuming and they also lack of se- route of exposure (water-spiking) may be an lectivity or sensitivity Another way to de- important factor for gill susceptibility since tect DTCs is to employ Liquid Chromatog- gill cells would be the fi rst ones to come into raphy (LC) and capillary electrophoresis (CE) contact with thiram. The direct contact of with UV and/or electrochemical detection. the large surface area of gills with the dilut- Recently Blasco et al. (10) applied a quan- ed pollutants may also contribute to this (3). titative matrix solid-phase dispersion and This diff erence may also stem from the cell liquid chromatography-atmospheric pres- subpopulations: whereas the main gill cell sure chemical ionization mass spectrome- type and the agranular haemocytes of Myti- try (LC-APCI-MS) method for the simultane- lus galloprovincialis were sensitive to the ous analysis of DTCs and their degradation genotoxic eff ects of benzo[a]pyrene (BaP), products in plants. Among the compounds the agranular ones which are present at var- analyzed was thiram which was detected by ied concentrations were relatively refractory both APCI and ESI methods with diff erent to these eff ects (44). Regarding the digestive corresponding ions each time. This method gland, it is considered to be the main organ was also applied here and was able to de- of metabolism of organic xenobiotics and tect concentration-related diff erences in the the main site of biotransformation enzyme way mussels bioaccumulate thiram. activities (8). As a result a number of reactive Even though the concentrations test- intermediates produced may directly attack ed were high, only certain signs of morbidi- vicinal digestive gland DNA. Furthermore, ty (profuse mucus secretion and delayed re- the highly condensed chromatin of diges- action to stimuli) were observed. Similarly, tive gland nuclei which creates additional al- high concentrations of agrochemicals have kali labile sites also contributes to % tail DNA not produced mortalities in bivalves (37). (33). This higher baseline damage may have Thiram is not considered genotoxic in masked here the eff ect of thiram, at least in
© Benaki Phytopathological Institute 66 Emmanouil et al. the low dose group. for thiram reduction in the cell, minimiz- Thiram, in the present experiments, has ing in this way the regeneration of reduced caused a signifi cant increase in oxidative GSH. In more detail, it is postulated that di- DNA damage as measured through Fpg in- thiocarbamates undergo oxidation by Cu2+ cubation, which quantifi es mainly 8-oxo-dG ions within the cell to their corresponding but also other damaged purines according thiuram disulfi des. These intermediates are to Collins et al. (14). This increase was most then reduced by GSH, regenerating the par- prominent in gill cells rather than in haemo- ent compound and oxidized glutathione cytes and in digestive gland cells. The meta- (11). GSH aberrant metabolism due to thi- bolic system of bivalves, even though mark- ram was further corroborated in studies in edly diff erent from the mammalian ones is V79 CHC which showed a decrease in total capable of producing oxidizing intermedi- GSH/oxidised GSH ratio (22). Therefore, the ates (28) which in their turn aff ect the cell’s pro-oxidant eff ects of thiram are considered metabolism and components (46). For ex- to be indirect and mainly due to the lower- ample, in a similar way to the present re- ing of antioxidant defenses. sults, the common aquatic pollutant BaP Finally, a series of important experiments which necessitates metabolic activation in aquatic organisms showed that thiram ox- to redox quinones to exert its toxic action, idative eff ects are not peculiar to terrestrial caused formation of 8-oxo-dG in Mytilus gal- organisms. Namely, incubation of Oncho- loprovincialis digestive gland (2, 29). rhynchus mykiss liver with thiram led to loss An oxidative mode of action is not un- or decrease of activity of SOD and GSH-Px usual for thiram as it has been shown in oth- respectively (6). Exposure of the mussel Unio er animal models. Any imbalance between tumidus to thiram has also caused decrease prooxidant substances and antioxidant de- of activity of selenium-dependent glutathi- fenses in favor of the prooxidants causes ox- one peroxidase and glutathione reductase idative stress associated with damage to cel- as well as decrease in reduced and oxidised lular macromolecules (40). Thiram has acted GSH in both gills and digestive gland (15). as a potent oxidative agent in the liver of Regarding testing for apoptosis, apop- broilers fed with this fungicide. The activi- totic cells give a characteristic image of large ties of the antioxidant enzymes superoxide fan-like tail and small head (ghost cells) in dismutase (SOD) and glutathione peroxi- the conventional comet assay. However dase (GSH-Px) were decreased and this low- due to their extensive fragmentation, they ering of antioxidant defenses has led to lip- may become lost during the electropho- id peroxidation in the liver of broilers (27). resis step (32). In contrast, omission of the Thiram has also caused deregulation in ac- electrophoresis step but retention of alka- tivities of key antioxidant enzymes (cata- line unwinding in the alkaline halo assay de- lase, GSH-Px, SOD, glutathione reductase) picts successfully the unique morphology of in an in vitro model of V79 Chinese hamster apoptotic cells which present a difuse, spot- ovary cells (22). These results may be part- ted halo and a pin-like head clearly delin- ly explained by glutathione (GSH) depletion eated from the halo (39). The assay has here caused by thiram (13, 23). GSH off ers one of revealed a signifi cant increase in apoptotic the most effi cient non-enzymatic protective cells in gills and digestive glands but no in- mechanisms by its conjugation with elec- crease in haemolymph when mussels were trophilic and/or oxidised components. Thi- exposed to 1.00 mg/L thiram (approximate- ram however possesses a reactive disulfi de ly 4 μM) for 48 h. In related bibliography, tri- bond which may react with thiols of critical closan (3 nM) caused more extensive dam- cellular proteins such as GSH forming mixed age (approximately 16%) after 48 h exposure disulfi des and other products (23), thus ren- to Dreissena polymorpha haemolymph (45). dering them inactive. Furthermore, the GSH This diff erence cannot be readily explained, redox cycle off ers the reducing equivalents however it may be linked to the apoptotic
© Benaki Phytopathological Institute Thiram Bioaccumulation in Mytilus galloprovincialis 67 potency of the tested chemical, the mussel 2008. Evaluation of thiram induced oxidative species used and possibly the lower sensitiv- stress, immunosuppression and apoptosis in avian lymphocytes culture system. Journal of ity in our experiments (staurosporine posi- Biotechnology, 136: S620-S632. tive control also caused less than 16% apop- 5. Association of Offi cial Analytical Chemists, 2000. totic cells). An apoptotic mode of action, as Offi cial Methods of Analysis, 16th ed., AOAC, Ar- this recorded here for Mytilus galloprovincia- lington, USA lis gill and digestive glands, has been shown 6. Binelli, A., Cogni, D., Parolini, M., Riva, C. and in a variety of animal models for thiram. Dis- Provini, A. 2009. In vivo experiments for the evaluation of genotoxic and cytotoxic eff ects of arrangement of redox related mechanisms Triclosan in Zebra mussels hemocytes. Aquatic and disintegration of important thiol-con- Toxicology. 91: 238-244. taining proteins eventually led to apoptot- 7. Babo, S. and Vasseur, P. 1992. In vitro eff ects of ic cell death in the V79 CHC cells (22). Thiram Thiram on liver antioxidant enzyme activities has also caused apoptosis in avian lympho- of rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology, 22 : 61-68. cytes (4), in bovine capillary endothelial cells 8. Birmelin, C., Mitchelmore, C.L., Goldfarb, P.S. (30) and in PC12 cells, the latter due to mas- and Livingstone, D.R. 1998. Characterisation of 2+ sive Ca intracellular infl ux (24). biotransformation enzyme activities and DNA Overall, a simple and convenient ana- integrity in isolated cells of the digestive gland lytical method for the determination of the of the common mussel, Mytilus edulis L. Compar- ative Biochemistry and Physiology Part A, 120: 51- concentration of thiram in mussel samples 56. revealed that M. galloprovincialis may ac- 9. Bjørge, C., Brunborg, G., Wiger, R., Holme, J.A., cumulate this fungicide in a dose-depen- Scholz, T., Dybing, E. and Søderlung, E.J. 1996. A dent way. This accumulation elicited dose- comparative study of chemically induced DNA and tissue-related SSB and it also caused damage in isolated human and rat testicular cells. Reproductive Toxicology, 10: 509-519. increased 8-oxo-dG levels (in all tissues) 10. Blasco, C., Font, G. and Picó, Y. 2004. Determi- and % apoptotic cells (in gill and digestive nation of dithiocarbamates and metabolites in gland). The two last parameters have also in- plants by liquid chromatography-mass spec- creased in a number of in vivo mammalian trometry. Journal of Chromatography A, 1028: models. It is possible that a common mech- 267-276. anism (reduction, depletion or disintegra- 11. Burkitt M.J., Bishop, H.S., Milne, L., Tsang S.Y., Provan G.J., Nobel C.S.I., Orrenius S. and Slater, tion of important thiol-containing proteins) A.F.G, 1998. Dithiocarbamate toxicity towards is implicated in both mammalian and non- thymocytes involves their copper-catalyzed mammalian thiram toxicity. conversion to thiuram disulfi des, which oxidize glutathione in a redox cycle without the release of reactive oxygen species. Archives of Biochem- istry and Biophysics, 353: 73-84. Literature cited 12. Caldas, E.D., Conceicao, M.H., Miranda, M.C.C., 1. Ahmad, N., Guo, L., Mandrakas, P. and Apple- de Souza, L.C.K.R. and Lima, J.F. 2001. Determi- by, S. 1995. Determination of dithiocarbamate nation of Dithiocarbamate Fungicide Residues and its breakdown product ethylenethiourea in in Food by a Spectrophotometric Method Using fruits and vegetables J. AOAC Int., 78: 1238-1243. a Vertical Disulfi de Reaction System. Journal of 2. Akcha, F., Izuel., C., Venier, P., Budzinski, H., Bur- Agricultural and Food Chemistry, 49: 4521-4525. geot, T. and Narbonne, J.-F. 2000. Enzymatic 13. Careser, C., Boget, S., Parvaz, P. and Revol, A. biomarker measurement and study of DNA ad- 2001. An evaluation of thiram toxicity on cul- duct formation in benzo[a]pyrene-contaminat- tured human skin fi broblasts. Toxicology, 162: ed mussels, Mytilus galloprovincialis. Aquatic 89-101. Toxicology., 49: 269-287. 14. Collins, A.R., Dobson, V.L., Dusinska, M., Ken- 3. Almeida, E.A, Bainy, A.C.D., Dafre, A.L., Gomes, nedy, G. and Stetina, R. 1997. The comet assay: O.F., Medeiros, M.H.G. and Di Mascio, P. 2005. what can it really tell us? Mutation Research, 375: Oxidative stress in digestive gland and gill of 183-193. the brown mussel (Perna perna) exposed to air 15. Doyotte, A., Cossu, C., Jacquin, M.C., Babut, M. and re-submersed. Journal of Experimental Ma- and Vasseur, P. 1997. Antioxidant enzymes, glu- rine Biology and Ecology, 318: 21– 30. tathione and lipid peroxidation as relevant bio- 4. Ambwani, S., Ambwani, T.J. and Chauhan, R.S. markers of experimental or fi eld exposure in the
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© Benaki Phytopathological Institute Thiram Bioaccumulation in Mytilus galloprovincialis 69
42. Teraoka, H., Urakawa, S., Nanba, S., Nagai, Y., iuram disulfi de (thiram). Food and Chemical Toxi- Dong, W., Imagawa, T., Tanguay, R.L., Svoboda, cology, 36: 155-164. K., Handley-Goldstone H.M., Stegeman, J.J. and 46. Vlahogianni, T., Dassenakis, M., Scoullos, M.J., Hiraga, T. 2006. Muscular contractions in the ze- and Valavanidis, A. 2007. Integrated use of bio- brafi sh embryo are necessary to reveal thiuram- markers (superoxide dismutase, catalase and li- induced notochord distortions. Toxicology and pid peroxidation) in mussels Mytilus gallopro- Applied Pharmacology, 212: 24-34. vincialis for assessing heavy metals’ pollution in 43. Thiruchelvam, M. 2005. Thiram. In: Wexler P. coastal areas from the Saronikos Gulf of Greece. (ed.), Encyclopedia of Toxicology, Elsevier, Marine Pollution Bulletin, 54 :1361-1371. pp.179-181. 47. Walia, S., Kumar Sharma, R. and Parmar, B.S. 44. Venier, P., Maron, S. and Canova, S. 1997. Detec- 2009. Isolation and Simultaneous LC Analysis of tion of micronuclei in gill cells and haemocytes Thiram and Its Less Toxic Transformation Pro- of mussels exposed to benzo[a]pyrene. Muta- duct in DS Formulation. Bulletin of Environmen- tion Research, 390, 33–44. tal Contamination and Toxicology, 83: 363-368. 45. Villani, P., Andreoli, C., Crebelli, R., Pacchierot- ti, F., Zijno, A. and Carere, A. 1998. Analysis of micronuclei and DNA single-strand breaks in mouse splenocytes and peripheral lympho- cytes after oral administration of tetramethylth- Received: 21 May 2011 ; Accepted: 27 June 2011
Μελέτη βιοσυσσώρευσης του thiram στους μαλακούς ιστούς του μυδιού Mytilus galloprovincialis
Χ. Εμμανουήλ, Κ.Μ. Κασιώτης και Κ. Μαχαίρα
Περίληψη Η βιοσυσσώρευση του thiram, ενός χαρακτηριστικού μυκητοκτόνου της κατηγορίας των διθειοκαρβαμιδικών, εξετάσθηκε όσον αφορά στους μαλακούς ιστούς του μυδιού Mytilus galloprovin- cialis. Επίσης εξετάσθηκε η επίδραση του thiram στην ακεραιότητα του DNA του Mytilus galloprovin- cialis, μέσω μια σειράς in vivo εκθέσεων σε 0,1, 1,0 and 10,0 mg thiram/L σε θαλάσσιο νερό για 48 h. Παρατηρήθηκε μια δοσοεξαρτώμενη βιοσυσσώρευση συμπλόκων ιόντων του νατρίου με το thiram μετά το πέρας των εκθέσεων. Αυτή η ταυτοποίηση/ποσοτικοποίηση του thiram και των μεταβολιτών του πραγματοποιήθηκε μέσω Υγρής Χρωματογραφίας Φασματομετρίας Μάζας (LC-MS). Όσον αφορά στην ακεραιότητα του DNA του M. galloprovincialis (παρατηρούμενες μονές αλυσίδες DNA) η εμφάνι- σή τους ήταν ισχυρά δοσοεξαρτώμενη. Αυτό το αποτέλεσμα επηρεάστηκε εξίσου σημαντικά από τον ιστό υπό εξέταση , όπως ήταν αναμενόμενο, δεδομένης της διαφορετικής ευαισθησίας του κάθε ιστού στο thiram και του μεταβολισμού που λαμβάνει χώρα σε αυτόν. Περαιτέρω αναλύσεις DNA βλαβών λόγω οξειδωτικού στρες και απόπτωσης κατέδειξαν πως α) οξειδωτικό στρες ήταν εμφανές σε όλους τους εξεταζόμενους ιστούς και β) κυτταρικά μορφολογικά σχήματα χαρακτηριστικά αποπτωτικών δι- εργασιών παρουσιάστηκαν τόσο στα βράγχια όσο και στον πεπτικό αδένα του οργανισμού. Από τα παραπάνω αποτελέσματα μπορούμε να υποθέσουμε πως ανισορροπία μεταξύ των αντιοξειδωτικών/ οξειδωτικών μηχανισμών των κυττάρων του M. galloprovincialis, προς όφελος των δεύτερων, καθώς και έναρξη αποπτωτικών κυτταρικών διεργασιών είναι δυνατόν να αποτελέσουν τον κύριο λόγο βλα- βών DNA στον M. galloprovincialis λόγω του thiram. Αντίστοιχοι μηχανισμοί έχει αποδειχθεί ότι ισχύ- ουν για τα θηλαστικά.
Hellenic Plant Protection Journal 4: 57-69, 2011
© Benaki Phytopathological Institute Hellenic Plant Protection Journal Volume 4, 2011 Contents
D. Makowski Uncertainty and sensitivity analysis for models used in pest risk analysis 1-11
Y.E. Troyanos and E. Roukounaki Response of young olive trees to nitrogen fertilization 13-19
I. Anastasiadis, A.C. Kimbaris, M. Kormpi, M.G. Polissiou and E. Karanastasi The eff ect of a garlic essential oil component and entomopathogenic nematodes on the suppression of Meloidogyne javanica on tomato 21-24
E.A. Tzortzakakis, I.L.P.M. da Conceição, M.C.V. dos Santos and I.M. de O. Abrantes Root-knot nematodes (Meloidogyne spp.) in Greece 25-30
A.F. Martinou, D. Papachristos and P.G. Milonas Report of the Geranium Bronze Butterfl y, Cacyreus marshalli for mainland Greece 31-34
A. Michaelakis, D. Papachristos, A. Kimbaris and M. Polissiou Larvicidal evaluation of three Mentha species essential oils and their isolated major components against the West Nile virus mosquito 35-43
F. Karamaouna, G. Menounou, G.J. Stathas and D.N. Avtzis First record and molecular identifi cation of the parasitoid Anagyrus sp. near pseudococci Girault (Hymenoptera: Encyrtidae) in Greece - Host size preference for the vine mealybug Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae) 45-52 C.Th. Buchelos and E. Evergetis First record of Lixus umbellatarum Fabricius (Coleoptera: Curculionidae) in Greece, on the medicinal herb Opopanax chironium 53-56
Ch. Emmanouil, K.M. Kasiotis and K. Machera Bioaccumulation of thiram in Mytilus galloprovincialis and its eff ect on diff erent tissues 57-69
© Benaki Phytopathological Institute www.bpi.gr Hellenic Plant Protection Journal Τόμος 4, 2011 Περιεχόμενα
D. Makowski Ανάλυση αβεβαιότητας και ευαισθησίας των μοντέλων που χρησιμοποιούνται στις αναλύσεις επικινδυνότητας επιβλαβών οργανισμών 1-11
Γ.Ε. Τρωγιάνος και Ε. Ρουκουνάκη Αντίδραση νεαρών δενδρυλλίων ελιάς στην αζωτούχο λίπανση 13-19
I. Αναστασιάδης, A.K. Κυμπάρης, M. Κορμπή, M.Γ. Πολυσίου και E. Καραναστάση Επίδραση ενός αιθερίου ελαίου του σκόρδου και εντομοπαθογόνων νηματωδών στην καταστολή ου Meloidogyne javanica στην τομάτα 21-24
E.A. Tζωρτζακάκης, I.L.P.M. da Conceição, M.C.V. dos Santos and I.M. de O. Abrantes Νηματώδεις του γένους Meloidogyne στην Ελλάδα 25-30
Α.Φ. Μαρτίνου, Δ. Παπαχρήστος και Π.Γ. Μυλωνάς Αναφορά του Cacyreus marshalli στην Ηπειρωτική Ελλάδα 31-34
A. Μιχαηλάκης, Δ. Παπαχρήστος, Α. Κυμπάρης και Μ. Πολυσίου Τοξική δράση των αιθέριων ελαίων τριών ειδών Mentha spp. και των κυριότερων συστατικών τους στο κουνούπι Culex pipiens 35-43
Φ. Καραμαούνα, Γ. Μενούνου, Γ.Ι. Σταθάς και Δ.Ν. Αβτζής Πρώτη καταγραφή και μοριακή ταυτοποίηση του παρασιτοειδούς Anagyrus sp. near pseudococci Girault (Hymenoptera: Encyrtidae) στην Ελλάδα – Προτίμηση ως προς το μέγεθος του ψευδόκοκκου-ξενιστή Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae) 45-52 Κ.Θ. Μπουχέλος και Ε. Ευεργέτης Πρώτη καταγραφή του Lixus umbellatarum Fabricius (Coleoptera: Curculionidae) στην Ελλάδα. H παρουσία του στο φαρμακευτικό φυτό Opopanax chironium 53-56
Χ. Εμμανουήλ, Κ.Μ. Κασιώτης και Κ. Μαχαίρα Μελέτη βιοσυσσώρευσης του thiram στους μαλακούς ιστούς του μυδιού Mytilus galloprovincialis 57-69
© Benaki Phytopathological Institute www.bpi.gr Τόμος 4, Τεύχος 2, Ιούλιος 2011 ISSN 1791-3691
Περιεχόμενα
Α.Φ. Μαρτίνου, Δ. Παπαχρήστος και Π.Γ. Μυλωνάς Αναφορά του Cacyreus marshalli στην Ηπειρωτική Ελλάδα 31-34
A. Μιχαηλάκης, Δ. Παπαχρήστος, Α. Κυμπάρης και Μ. Πολυσίου Τοξική δράση των αιθέριων ελαίων τριών ειδών Mentha spp. και των κυριότερων συστατικών τους στο κουνούπι Culex pipiens 35-43
Φ. Καραμαούνα, Γ. Μενούνου, Γ.Ι. Σταθάς και Δ.Ν. Αβτζής Πρώτη καταγραφή και μοριακή ταυτοποίηση του παρασιτοειδούς Anagyrus sp. near pseudococci Girault (Hymenoptera: Encyrtidae) στην Ελλάδα – Προτίμηση ως προς το μέγεθος του ψευδόκοκκου-ξενιστή Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae) 45-52
Κ.Θ. Μπουχέλος και Ε. Ευεργέτης Πρώτη καταγραφή του Lixus umbellatarum Fabricius (Coleoptera: Curculionidae) στην Ελλάδα. H παρουσία του στο φαρμακευτικό φυτό Opopanax chironium 53-56
Χ. Εμμανουήλ, Κ.Μ. Κασιώτης και Κ. Μαχαίρα Μελέτη βιοσυσσώρευσης του thiram στους μαλακούς ιστούς του μυδιού Mytilus galloprovincialis 57-69
Περιεχόμενα Τόμου 4 (2011)
Hellenic Plant Protection Journal www.bpi.gr © Benaki Phytopathological Institute Volume 4, Issue 2, July 2011 ISSN 1791-3691
Contents H Hellenic Plant Protection Journal e l
A.F. Martinou, D. Papachristos and P.G. Milonas l Report of the Geranium Bronze Butterfl y, Cacyreus marshalli for e mainland Greece 31-34 n i c A. Michaelakis, D. Papachristos, A. Kimbaris and M. Polissiou
Larvicidal evaluation of three Mentha species essential oils and P
their isolated major components against the West Nile virus l mosquito 35-43 a n
F. Karamaouna, G. Menounou, G.J. Stathas and D.N. Avtzis t
First record and molecular identifi cation of the parasitoid P Anagyrus sp. near pseudococci Girault (Hymenoptera: Encyrtidae) in Greece - Host size preference for the vine mealybug r o Planococcus fi cus (Signoret) (Hemiptera: Pseudococcidae) 45-52 t e C.Th. Buchelos and E. Evergetis First record of Lixus umbellatarum Fabricius (Coleoptera: c Curculionidae) in Greece, on the medicinal herb Opopanax t i chironium 53-56 o n Ch. Emmanouil, K.M. Kasiotis and K. Machera
Bioaccumulation of thiram in Mytilus galloprovincialis and its J eff ect on diff erent tissues 57-69 o u
Contents of Volume 4 (2011) r n a l
© Benaki Phytopathological Institute www.bpi.gr