Figures
Abstract
Background
The resistance status of malaria vectors to different classes of insecticides used for public health has raised concern for vector control programmes. Alternative compounds to supplement the existing tools are important to be searched to overcome the existing resistance and persistence of pesticides in vectors and the environment respectively. The mosquitocidal effects of Glycosmis pentaphylla using different solvents of acetone, methanol, chloroform and ethyl acetate extracts against three medically important mosquito vectors was conducted.
Methods
Glycosmis pentaphylla plant leaves were collected from Kolli Hills, India. The WHO test procedures for larval and adult bioassays were used to evaluate extracts against mosquito vectors, and the chemical composition of extracts identified using GC-MS analysis.
Results
The larvicidal and adulticidal activity of G. pentaphylla plant extracts clearly impacted the three species of major mosquitoes vectors. Acetone extracts had the highest larvicidal effect against An. stephensi, Cx. quinquefasciatus and Ae. aegypti with the LC50 and LC90 values of 0.0004, 138.54; 0.2669, 73.7413 and 0.0585, 303.746 mg/ml, respectively. The LC50 and LC90 adulticide values of G. pentaphylla leaf extracts in acetone, methanol, chloroform and ethyl acetate, solvents were as follows for Cx. quinquefasciatus, An. stephensi and Ae. Aegypti: 2.957, 5.458, 2.708, and 4.777, 3.449, 6.676 mg/ml respectively. The chemical composition of G. pentaphylla leaf extract has been found in 20 active compounds.
Conclusions
The plant leaf extracts of G. pentaphylla bioactive molecules which are effective and can be developed as an eco-friendly approach for larvicides and adulticidal mosquitoes vector control. Detailed identification and characterization of mosquitocidal effect of individual bioactive molecules ingredient may result into biodegradable effective tools for the control of mosquito vectors.
Citation: Ramkumar G, Karthi S, Muthusamy R, Suganya P, Natarajan D, Kweka EJ, et al. (2016) Mosquitocidal Effect of Glycosmis pentaphylla Leaf Extracts against Three Mosquito Species (Diptera: Culicidae). PLoS ONE 11(7): e0158088. https://doi.org/10.1371/journal.pone.0158088
Editor: Humberto Lanz-Mendoza, Instituto Nacional de Salud Pública, MEXICO
Received: March 14, 2015; Accepted: June 12, 2016; Published: July 8, 2016
Copyright: © 2016 Ramkumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Background
Control of malaria vectors has entered a new phase of challenge to public health practitioners due to increased threat of insecticides resistance among vectors [1–4]. Large proportions of the human population has been annually affected by vector-borne diseases. Effective vector control includes both larval and adult mosquito control measures, which have great impact in disease control. Synthetic insecticides are currently the most widely used tool for fighting against malaria vectors. However, these synthetic chemicals pose threat against non-target organisms and increase in resistance against targeted vector population [5]. Biopesticides have shown promise of being a source for the environmental eco-friendly and an alternative to synthetic pesticides [6], and for several decades plants have been a source of traditional medicine with least toxicity to mankind and domesticated animals [7,8]. Plant based extracts are presumed to have potential high toxicity and eco-friendly compared to synthetic insecticides for vector of veterinary and medical significance [9]. The current methods used for the control of mosquito-borne diseases -are, the use of insecticides for Indoor residual spray, and long lasting insecticidal nets. It has also provoked undesirable effects, including toxicity to non-target organisms, and fostered environmental and human health concerns [10]. Plant material extracts have been found to be useful and effective as larvicides, repellent or deterrent agents, oviposition attractants or repellent and insect growth hormone regulators [8,11,12]. Plant based materials, whole plants or their extracts have been potentially used for killing and repelling mosquitoes and other nuisance vectors [13]. Previous studies revealed that, different solvents such as acetone, petroleum ether, ethyl acetate and methanol used for extracting active ingredients of C. dentata had also attributed to effect differences on larvicidal activity against mosquitoes [14,15].
Glycosmis pentaphylla (Family: Rutaceae) is an evergreen shrub commonly called an orange berry grown in eastern Asia in Bangladesh, India, and Thailand. G. Pentaphylla (Rutaceae) is used for treatments of boils, chest pain, hookworm infestation and urinary tract infections in human [16]. Juice extracted from G. pentaphylla leaves is used for treatment of fever and liver problems. A decoction of roots is used for the facial inflammations treatment [17]. The roots pounded and mixed with sugar are used as an antidote and fever treatment regime [17]. G. Pentaphylla is reported to contain arborinine among active ingredients [18]. Leaf extract and crude alkaloid possesses antibacterial and antifungal properties [19].
The current study assessed the mosquitocidal activity of different extracts of Glycosmis pentaphylla against larvae and adults of three mosquito species.
Materials and Methods
Collection of plants
The plant leaves of Glycosmis pentaphylla (Fig 1) were sampled from Kolli Hills of Eastern Ghats in a region that is rich in biodiversity and indigenous populations. Kolli Hills are located in the Namakkal district which lies between 10°12'–11°7'N, and 76°–77°56'E with an altitude 1300 meters above sea level and the taxonomic identification of plant was made by Dr. D. Natarajan, Assistant Professor, from Department of Biotechnology, Periyar University, Tamil Nadu, India, did the taxonomic identification of the plant and the voucher specimen was deposited in the herbarium. Periyar university approved ethical clearance for this research. In Kolli Hills, the management was informed about the study objectives and written consent was obtained with permission to collect plant material. This study did not involve any endangered or highly protected species.
Plant extracts preparations
Plant leaves were dried under shade for a period of 7 to 10 days in the shade at environmental temperatures (27–37°C daytime). A blander was used for powdering the dried leaves of which three hundred grams were extracted separately using the following chemicals for eight hours, chloroform, ethyl acetate, acetone and methanol in a Soxhlet apparatus with boiling point ranging between 50–80°C. The leaf extracts were concentrated at a pressure of 22–26mm Hg at 45°C. The obtained active ingredients were stored in a room temperature (in a dark room).
Mosquitoes culture
The National Centre for Disease Control (NCDC), Mettupalayam, Tamil Nadu, India provided larvae of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus for this study. The larvae were kept in plastic trays containing tap water was used for rearing under laboratory conditions at 27±2°C and relative humidity of 75–85%. The photophase was maintained at 14:10 light and dark. Ground dog biscuit and yeast powder was used as larvae food in the ratio of 3:1.
Larvicidal bioassay
WHO standard protocol for evaluating the larvicides was used to evaluate the plant extracts toxicity against mosquito larvae[20], with some minor modifications done by previous studies, [21]. Twenty five (25) fourth instar larvae were transferred to a small disposable paper cups with a mixture of 249 ml of water and 1.0 ml of proposed plant extract. The control used as DMSO. Dosage evaluation started with the lowest dosage of 0.01mg/ml to 2 mg/ml. Larvae mortality was monitored after 24 hours of exposure in all replicates of each dosage. The percentage mortality was reported from three replicates. The LC50 (lethal concentration that kills 50% of the exposed larvae) and LC90 (lethal concentration that kills 90% of the exposed larvae) values were calculated after 24 h using probit analysis method [22].
Adulticidal bioassay
Adulticidal bioassay was performed by using a clean glass test tube and different concentrations of five solvent extracts were made ranging from 0.1, 0.3, 0.5, 1 and 2mg/ml was coated with test tube and allow to dry the solvent extract [22–25]. There were three replicates per concentration and twenty unfed female mosquitoes were released in each replicates. Tubes were tightly covered with netting materials and coated with solvent alone served as control. Mosquito mortality was determined 24 hours post exposure. LD50, LD90 with their 95% confidence limits were determined using Log Probit analysis test [22].
GC–MS analysis
The plant extracts of G. pentaphylla were analysed using gas chromatography (Polaris Q Ion Trap GC/FID) and mass spectrometry (Perkin Elmer Q-700 equipment). We followed the method used by previous published research findings [26,27], which used one type of column (preferably Polaris Q Ion Trap GC/FID). The column temperature programme was 35°C for 2 min, increased to 180°C at 4°C/min, then increased to 280°C at 20°C/min. Helium was used as a carrier gas at a 0.9 ml/min. The best mass spectrum was obtained at 70 eV ionization voltage in the machine. Individual compounds were identified using Wiley/ NBS Registry of mass spectral database, the NIST machine (version 3.0) database. The Retention Time (RT) and Kovats Index (KI) values of several authentic reference compounds were compared with isolated compounds for identification.
Statistical analysis
The mean mortality data were analysed using probit analysis for calculating LC50, LC90 and other statistics at 95% confidence interval limits of upper and lower confidence limits. The Chi-square values were calculated using the SPSS Statistical software package version 16.0 for windows.
Results
Larval and adult bioassay
The larvicidal activity results were obtained from bioassays of the crude extracts by acetone, methanol, chloroform and ethyl acetate solvent leaf extracts of Glycosmis pentaphylla against the larvae of three important vector mosquitoes in different concentration varied among species; Cx. Quinquefasciatus (Table 1), An. stephensi (Table 2) and Ae. aegypti (Table 3). Among the plant extracts tested, the highest larvicidal activity was observed in acetone extracted compounds against Cx. quinquefasciatus, An. stephensi and Ae. aegypti with the LC50 and LC90 values of 0.0005, 138.54; 0.267, 73.741 and 0.058, 303.746 mg/ml, respectively. The 95% confidence limits LC50 (95%CI) and LC90 (95%CI), chi-square and degree of freedom (df) values were also calculated. In control assays we did not find any significant mortality.
The adulticidal activity of acetone, methanol, chloroform and ethyl acetate solvent leaf extracts of G pentaphylla against the adults of three important vector mosquitoes are reported for Cx. quinquefasciatus, An. stephensi, and Ae. Aegypti (Table 2). The plant crude extracts mortality increased proportionally with dosage. Among the tested plant extract, the highest adulticidal effect was found to be in chloroform extract against Cx. quinquefasciatus, An. stephensi and Ae. aegypti. The control was tested without solvent showing no mortality. The LC50 and LC90 values of G pentaphylla leaf extracts of acetone, methanol, chloroform and ethyl acetate against adulticidal activity of Cx. quinquefasciatus, An. stephensi and Ae. aegypti were as follows: 2.957and 5.458;2.708 and 4.777; 3.449 and 6.676 mg/ml, respectively.
GC-MS analysis and identification of compounds
GC-MS analysis of acetone and chloroform solvent extracts of G. pentaphylla extracts showed 40 peaks which indicating the presence of 20 phytochemical compounds (Figs 2 and 3). On the comparison of the mass spectra of the constituents with the NIST library, the 20 compounds were characterized and identified (Tables 3 and 4). The major chemical compounds which were identified in acetone solvent extracts based on the 5 highest peaks were 4h-1-Benzopyran-4-one 2-(3,4-Dimethoxyphenyl)-3,5,6,7-Tetramethoxy (36.061%), Cyclopropane carboxylic Acid, 2-Methyl-, 2,6-Di-T-Butyl-4-Methylphenyl Ester (17.035%), 2h-1-Benzopyran-2-One, 6-(1-Hydroxy-3-Methylbutyl)-7-Methoxy(6.314%),4,4'-ethylenebis(2,6-di-tert-butylphenol) (4.775%), Hexane,3-(1,5-dimethyl-4-heanyl)-6-metheline-,(s-(R,S) (4.650%). For the chloroform solvent extracts based on the 5 highest peaks, the identified compounds were 4h-1-Benzopyran-4-One, 2-(3,4-Dimethoxyphenyl)-3,5,6,7-Tetramethoxy (20.326%), Glaucyl Alcohol (15.876%), Cyclopropanecarboxylic Acid, 2-Methyl-, 2,6-Di-T-Butyl-4-Methylphenyl Ester (11.695%), 3-methyl-2-(2-oxopropyl)furan (9.511%), 2r-acetoxymethyl-1,3,3-trimethyl-4t-(3-methyl-2-buten-1-yl)-1t-cyclohexanol (5.923%).
Discussion
The findings of this study have shown that phytochemical compounds extracted from G. pentaphylla might be innovative, alternative to synthetic insecticides in the future as these are safe for non-target organisms. Chemical compounds extracted from different parts of plant have different active ingredients with different activity against larvae and adult mosquitoes. Plant crude extracts might be more effective than individual active compound due to active ingredients synergisms which can be effective in managing resistant population of mosquitoes. Our results show that the crude acetone, methanol, chloroform and ethyl acetate solvent leaf extracts of G pentaphylla were effective against the larvae of three important vector mosquitoes, Cx. quinquefasciatus, An. stephensi and Ae. aegypti. These results are similar to previous reported study findings [14,15] who observed the larvicidal activity of C. dentata plant extract against vector mosquitoes, namely, Ae. aegypti (LC50 = 0.169 mg/ml) Cx. quinquefasciatus (LC50 = 0.150 mg/ml) and An. stephensi (LC50 = 0.046 mg/ml). Previous studies have shown that, petroleum ether of R. nasutus have stronger larvicidal effect with LC50 values ranging from 3.9 to11.5 mg/l, while Derris elliptica had LC50 values varying from 11.2 to18.84 mg/l against Ae. aegypti, Cx. quinquefasciatus, An. dirus, and Mansonia uniformis [28]. Khanna and others found that, crude leaf extracts of Gymnema sylvestre had the highest mortalities at the concentration of 1000 ppm against the larvae of An. subpictus (LC50 = 166.28 ppm) and Cx. quinquefasciatus (LC50 = 186.55 ppm). Among its active ingredients, gymnemagenol compound isolated from petroleum ether had LC50 of 22.99 ppm for larvae of An. subpictus and 15.92 ppm for Cx. quinquefasciatus [29].
In other studies, the limonoids extracted from neem seeds evaluated for larvicidal, pupicidal, adulticidal, and anti-ovipositional activity against An. stephensi using different solvent had larval mortality which was dose dependent[30]. Ethanol extract induced mortality to C. sinensis at a concentration of 272.19 ppm (LC50) and 457.14 ppm (LC90) while for An. stephensi LC50 was 289.62 and LC90 was 494.88 ppm. In Ae. aegypti LC50 was 320.38ppm and LC90 was 524.57 ppm [30,31]. In the present study, the adulticidal results had the LC50 and LC90 values were 205.78 and 459.51 ppm for An. stephensi, 242.52 ppm and 523.73 ppm for Ae. aegypti respectively which was similar to study by Murugan and others with the extracts from Citrus peals [31].
The adulticidal activity results of acetone, methanol, chloroform and ethyl acetate solvent leaf extracts of G. pentaphylla against adult vector mosquitoes, Cx. quinquefasciatus, An. stephensi and Ae. aegypti showed to have dose-dependent mortality. At higher concentrations, the adult showed restless movement for some times with abnormal wagging and then died. Among the extracts tested, the highest adulticidal activity was observed in chloroform extract against Cx. quinquefasciatus, An. stephensi and Ae. aegypti. The lethal dose to kill 50% and 90% (LC50 and LC90) values of G. pentaphylla leaf extracts against adulticidal activity of (acetone, methanol, chloroform and ethyl acetate), Cx. quinquefasciatus, An. stephensi and Ae. aegypti were 2.957and 5.458; 2.708 and 4.777;3.449 and 6.676 mg/ml. Rajkumar and others found that, the leaf extract of C. asiatica has larvicidal and an inhibitory properties for the emergence adults against Cx. quinquefasciatus [32]. The reported results in this study in adult emergence inhibitory and mosquitocidal effects are by far greater that those reported by other studies using different plant extracts. The above findings support the results observed in this study that, plant G. pentaphylla act as potent larvicide and adulticide as well as disrupting the growth of larvae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti.
The isolation of compounds from leaf extracts of G pentaphylla, as described here, could lead to the development of natural mosquitocidal products to replace the synthetic insecticides. The use of natural plant based products by individuals and communities can enhance their better diseases vector control, plant species protection and propagations hence become source of income to community to eradicate poverty[33]. For example, our previous study provides the active ingredients of C. dentata plant extracts of acetone solvent against mosquito vectors [15]. Our study shows that besides mosquito larval and adult control efficacy, the compounds in the extracts which may be useful as further studies are needed to identify structure of the bioactive molecules.
Conclusion
In conclusion, the findings of this study have shown a great effectiveness of Glycosmis pentaphylla extracts by different solvents against the three major mosquito species in aquatic stages and adults. Our findings showed that leaf extract of G pentaphylla bioactive molecules to be an effective and can be developed as an eco-friendly larvicides and adulticides for mosquito vector control. This study suggests that future research work on the use of individual active ingredient to evaluate its mosquitocidal effect on the three species used in this study in semifield and small scale field trials for invention of environmentally safe botanical insecticide for the control of mosquito vectors.
Acknowledgments
We thank the Department of Biotechnology, Periyar University, Salem for providing the infrastructural facility for carrying out this research work.
Author Contributions
Conceived and designed the experiments: GR MSS. Performed the experiments: SK RM PS. Analyzed the data: SK RM PS. Contributed reagents/materials/analysis tools: EJK. Wrote the paper: SK EJK GR DN MSS. Revised the manuscript critically: EJK and MSS.
References
- 1. Kumar D, Andimuthu R, Rajan R, Venkatesan M (2014) Spatial trend, environmental and socioeconomic factors associated with malaria prevalence in Chennai. Malaria Journal 13: 14. pmid:24400592
- 2. Bharati K, Ganguly NK (2013) Tackling the malaria problem in the South-East Asia Region: need for a change in policy? Indian J Med Res 137: 36–47. pmid:23481050
- 3. Sharma SK, Upadhyay AK, Haque MA, Tyagi PK, Kindo BK (2012) Impact of changing over of insecticide from synthetic pyrethroids to DDT for indoor residual spray in a malaria endemic area of Orissa, India. Indian J Med Res 135: 382–388. pmid:22561626
- 4. Das A, Anvikar AR, Cator LJ, Dhiman RC, Eapen A, Mishra N, et al. (2012) Malaria in India: The Center for the study of complex malaria in India. Acta Tropica 121: 267–273. pmid:22142788
- 5. Amer A, Mehlhorn H (2006) Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitology Research 99: 466–472. pmid:16642386
- 6.
Liu SQ, Shi Cao JJ, Jia H, Liu FB, Shi G. Survey of pesticidal component in plant. In: Press SaT, editor; 2000; China. 1098–1104.
- 7. Chogo JBA, Crank G (1981) Chemical composition and biological activity of the Tanzania plant Ocimum suave. J Nat Prod 44: 308–309. pmid:6114991
- 8. Das N, Baruah I, Talukdar PK, Das SC (2003) Evaluation of botanicals as repellents against mosquitoes. J Vector Borne Dis 40: 49–53. pmid:15119071
- 9. Sarwar M, Ahmad N, Toufiq M (2009) Host plant resistance relationships in chickpea (Cicer arietinum L.) against gram pod borer (Helicoverpa armigera Hubner). Pak J Bot 41: 3047–3052.
- 10. Lee SE, Kim JE, L HS Insecticide resistance in increasing interest. Agric Chem Biotechnol 44: 105–112.
- 11. Okumu F, Knols B, Fillinger U (2007) Larvicidal effects of a neem (Azadirachta indica) oil formulation on the malaria vector Anopheles gambiae. Malaria Journal 6: 63. pmid:17519000
- 12. Kilonzo BS, Ngomuo AJ, Sabuni CA, Mgode GF (2001) Effects of Azadirachta indica (Neem) extract on livestock fleas in Morogoro district, Tanzania. International Journal of Tropical Insect Science 21: 89–92.
- 13. Seyoum A, Killeen GF, Kabiru EW, Knols BGJ, Hassanali A (2003) Field efficacy of thermally expelled or live potted repellent plants against African malaria vectors in western Kenya. Tropical Medicine & International Health 8: 1005–1011.
- 14. Govindarajan M (2010) Chemical composition and larvicidal activity of leaf essential oil from Clausena anisata (Willd.) Hook. f. ex Benth (Rutaceae) against three mosquito species. Asian Pacific Journal of Tropical Medicine 3: 874–877.
- 15. Manjari M, Karthi S, Ramkumar G, Muthusamy R, Natarajan D, Shivakumar M (2014) Chemical composition and larvicidal activity of plant extracts from Clausena dentata (Willd) (Rutaceae) against dengue, malaria, and filariasis vectors. Parasitology Research 113: 2475–2481. pmid:24802866
- 16.
Uddin S (2006) Traditional uses of Ethnomedicinal plants of the Chittagong Hill Tracts. Dhaka: Bangladesh National Herbarium.
- 17.
Chopra N, Chopra P (1969) Glossary of Indian Medicinal Plants. New Delhi.
- 18. Quader MA, Nutan MTH, Rashid MA (1999) Antitumor alkaloid from Glycosmis pentaphylla. Fitoterapia 70: 305–307.
- 19. Athikomkulchai S, Prawat H, Thasana N, Ruangrungsi N, Ruchirawat S (2006) COX-1, COX-2 Inhibitors and Antifungal Agents from Croton hutchinsonianus. Chemical and Pharmaceutical Bulletin 54: 262–264. pmid:16462080
- 20.
WHO (2005) Guidelines for laboratory and field testing of mosquito larvicides. Communicable disease control, prevention and eradication, WHO pesticide evaluation scheme. WHO, Geneva,.
- 21. Suganya G, Karthi S, Shivakumar M (2014) Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti. Parasitology Research 113: 1673–1679. pmid:24553980
- 22.
Finney DJ (1971) Probit analysis. London: Cambridge University Press.
- 23.
WHO (1981) Instructions for determining the susceptibility or resistance of adult mosquitoes to organochlorine, organophosphate and carbamate insecticides: diagnostic test. Geneva,: WHO.
- 24. Brogdon WG, M JC. (1998) Simplification of adult mosquito bioassays through use of time-mortality determinations inglass bottles. J Am Mosq Control Assoc 14: 159–164. pmid:9673916
- 25.
CDC (2014) Evaluating mosquitoes for insecticide resistance: a web based instruction. Education and Training. Available: http://www.cdc.gov/ncidod/wbt/resistance/assay/bottle/index.htm. Accessed 25 July 2014: Centre for Diseases Control.
- 26. Cheng S-S, Chua M-T, Chang E-H, Huang C-G, Chen W-J, Chang S-T (2009) Variations in insecticidal activity and chemical compositions of leaf essential oils from Cryptomeria japonica at different ages. Bioresource Technology 100: 465–470. pmid:18178080
- 27. Mdoe FP, Cheng S-S, Lyaruu L, Nkwengulila G, Chang S-T, Kweka EJ (2014) Larvicidal efficacy of Cryptomeria japonica leaf essential oils against Anopheles gambiae. Parasites & Vectors 7: 1–6.
- 28. Komalamisra N, Trongtokit Y, Rongsriyam Y, Apiwathnasorn C (2005) Screening for larvicidal activity in some Thai plants against four mosquito vector species. Southeast Asian J TropMed Public Health 36: 1412–1422.
- 29. Gopiesh Khanna V, Kannabiran K, Rajakumar G, Rahuman A, Santhoshkumar T (2011) Biolarvicidal compound gymnemagenol isolated from leaf extract of miracle fruit plant, Gymnema sylvestre (Retz) Schult against malaria and filariasis vectors. Parasitology Research 109: 1373–1386. pmid:21537987
- 30. Nathan SS, Kalaivani K, Murugan K (2005) Effects of neem limonoids on the malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Acta Tropica 96: 47–55. pmid:16112073
- 31. Murugan K, Mahesh Kumar P, Kovendan K, Amerasan D, Subrmaniam J, Hwang J-S (2012) Larvicidal, pupicidal, repellent and adulticidal activity of Citrus sinensis orange peel extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitology Research 111: 1757–1769. pmid:22797605
- 32. Rajkumar S, Jebanesan A (2005) Larvicidal and adult emergence inhibition effect of Centella asiatica Brahmi (Umbelliferae) against mosquito Culex quinquefasciatus Say (Diptera: Culicidae). African J Biomed Res 8: 31–33.
- 33. Bowers WS, Sener B, Evans PH, Bingol F, Erdogan I (1995) Activity of Turkish medicinal plants against mosquitoes Aedes aegypti and Anopheles gambiae. Inst Sci Appl 16: 339–342.