Proceeding of the 3 International Conference on ...

46 downloads 414 Views 6MB Size Report
south–west while oil refinery is located in the south of the city [7]. Regarding the fact that ...... IOC and SCOR, Paris and Newark, Delaware. p.1-68. [4]. Lim, P. T. ...
3rd International Conference on Applied Life Sciences © (2014) ISALS Publishing

Proceeding of the 3rd International Conference on Applied Life Sciences (ISSN: 2304-0513)

Editor: Prof. Farhad Nejadkoorki, Yazd University, Iran

Editorial Board: Prof. Mohd Talib Latif UKM, Malaysia Dr. Bennama Rabha University of Mostaganem, Algeria Dr. Medhat Mostafa Minufiya University, Egypt Dr. Saeid Baroutian SCION, New Zealand Dr. Vrushal Ghoble University of Mumbai, India

Dr. Patel Shaikhamar Ismail University of Pune, India Prof. Sukru Dursun Selcuk University, Turkey Dr. Prudence Mutsa Mambo Rhodes University, South Africa Dr. Umair Ahsan Islamia University of Bahawalpur, Pakistan Prof. Mithat Direk Selcuk University, Turkey

ISALS Publishing International Society for Applied Life Sciences Web: http://www.isals.org E-mail: [email protected]

I

3rd International Conference on Applied Life Sciences © (2014) ISALS Publishing

International Conference on Applied Life Sciences (ICALS2014)

Edited by Farhad Nejadkoorki

ISALS PUBLISHING http://www.isals.org

II

3rd International Conference on Applied Life Sciences © (2014) ISALS Publishing

Copyright ©2014 ISALS Publishing All rights reserved. No part of the contents of this publication may be reproduced or transmitted in any form or by any means without the written permission of the publisher. International Society for Applied Life Sciences Publishing E-mail: [email protected] Web: http://www.isals.org Proceedings of International Conference on Applied Life Sciences ISSN 2304-0513

III

3rd International Conference on Applied Life Sciences © (2014) ISALS Publishing

PREFACE The organizing committee warmly welcomes our distinguished delegates and guests to the 3rd International Conference on Applied Life Sciences (ICALS 2014) held on September, 18-20, 2014 in Bangi, Malaysia. ICALS 2014 is organized by International Society for Applied Life Sciences (ISALS) and the UKM, Malaysia, and supported by ISALS Members and scholars from universities and institutes all around the world. The conference Program Committee is truly international, with membership from the Americas, Europe, Asia, Africa and Oceania. The main conference themes and tracks are Environment, Biology and Agriculture. The major goal of this event is to provide international scientific forums for exchange of new ideas in life sciences through discussions with international peers. This proceeding records the fully refereed papers presented at the conference. The conference has gathered technical research submissions related to all aspects of main conference themes. All the submitted papers in the proceeding have been peer reviewed by the reviewers drawn from the scientific committee, external reviewers and editorial board depending on the subject matter of the paper. After the careful peer-review process, the submitted papers were selected on the basis of novelty, importance, and transparency for the purpose of the conference. The selected papers and additional late-breaking contributions to be presented as lectures will make an exciting technical program. The conference will therefore be a unique event, where attendees will be able to appreciate the latest results in their field of expertise, and to attain additional knowledge in other fields. We hope that all participants and other interested readers benefit scientifically from the proceedings and also find it motivating in the process.

With the best regards, The Organizing Committee September 18-20, 2014 Bangi, Malaysia

IV

Table of Contents Title

Variation in Insecticidal Activity of Leaf Extracts of Tephrosia vogelii J.D. Hooker (Leguminosae) from West Java, Indonesia

Page 1

Djoko Prijono, Anugrah Panggraito, Gustini Syahbirin

Weekend-Weekdays Variation of Air Pollutants in Tehran

6

Farhad Nejadkoorki

Comparative pathogenicity of Beauveria bassiana, Clonostachys rosea, Metarhizium anisopliae, and Lecanicillium lecanii to adult,alfalfa weevil Hypera postica Gyllenhal (Coleoptera:Curculionidae)

11

Rebwar Ahmed Mustafa, Lazgeen Haji Assaf, Samir Khalaf Abdullah

The macrochelid mite, Macrocheles muscaedomesticae (Acarina: Macrochelidae) as a biological control agent against house fly, Musca domestica (Diptera: Muscidae) in Egypt

15

Safaa Mostafa Abo-Taka, Hany Mohamed Heikal, Ahamed Abd El-Raheem

Histopathological studies of the effects of Cyclophosphamide on liver of female albino mice

21

Intissar Numman Waheed, Khesar Hussein Khalil, Zobayda Adnan Sharafaddin

An evaluation of resistance to Fusarium disease in Korean sesame (Sesamum indicum L.) germplasm

27

Ramalingam Radhakrishnan, Sang-Mo Kang, Jae-Man Park, Soek-Min Lee, In-Youl Baek, In-Jung Lee

Studies on the bacteriological quality of water,sediment and fish samples of Poovar estuary, south west coast of India

31

Premjith Sakarapillai

Selection of new potential mutan of field corn for early maturity and yield based on line x tester analysis in Indonesia

37

Jajang Supriatna, Dedi Ruswandi

The Potential of Ornamental Plant, Sansevieria trifasciata to Inhibit the Growth of Harmful Algal Bloom Species

42

Ima Amirah Mohd Suberi, Normawaty Mohammad Noor, Deny Susanti Darnis, Yukinori Mukai, Gires Usup

Brain Regional and Hypophyseal Protein Profiles of Boars Fed Dietary Fumonisin B1

46

Francis Ayodeji Gbore

Cannibalistic behaviour of african catfish juvenile, Clarias Gariepinus under defferent light wavelengths and intensities

51

Firdaus Sallehudin, Yukinori Mukai

Diversity and distribution of scleractinian and non-scleractinian corals at selected areas in Tioman Island, Malaysia Mohd. Fikri Akmal, K., Shahbudin, S., Muhammad Hamizan Yusof V

56

Energy Efficiency through Cleaner Production Technology: an exemplary analysis of the vehicle assembly industry in Malaysia

61

Khairul Nadiah Ibrahim, Yushazaziah Mohd. Yunos, Zaida Rahayu Yet, Noraini Burok, Nurul Liyana Zaharulhisham

Investigating Economic Tourism Sustainability Indicators in Historical City (Case Study; Isfahan)

66

Mohanna Nikbin 71

Food Security: Agricultural Intra-Arab Trade Mohamed S. ElGendy, Mohamed A. ElSayed

Variability and Heritability of Green Forage and Yield Character of 26 Sorghum (Sorghum 77 bicolor (L.) Moench) Genotypes Desti Rahmaniar, Anas IR., Farida Damayanti

Seed Genetic Purity Assessment of F3 Progeny of Two Rice Lines Estimated by Simple Sequence Repeat Markers

82

Syindy Raffini Nasihin, Nono Carsono, Wieny Heriliya Rizky

Using Hydrological Model to Assess The Flash Flood Hazard in Tall Al Amarna Archeological Sites El Minya, Egypt

87

Gehan El Bayomi, El Bastawesy M.

Ecosystems Health Status Through Metal Analysis and Profiling Ahmad Zaharin

VI

91

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Variation in Insecticidal Activity of Leaf Extracts of Tephrosia vogelii J.D. Hooker (Leguminosae) from West Java, Indonesia Djoko Prijono 1 , Anugrah Panggraito 2, and Gustini Syahbirin 2 1

Department of Crop Protection, Bogor Agricultural University, Bogor, Indonesia 2 Department of Chemistry, Bogor Agricultural University, Bogor, Indonesia

Abstract. This study was conducted to determine the variation in insecticidal activity of acetone extracts of Tephrosia vogelii leaves from West Java, Indonesia, against the cabbage head caterpillar, Crocidolomia pavonana. A total of ten leaf samples were collected from seven different locations in the study area between February 1 and May 19, 2010. Leaf extract of T. vogelii with purple flower from Cipanas-Cianjur was the most toxic to C. pavonana larvae (LC50 at 96 hours post-treatment was 0.137%), followed by that with purple flower from Pangalengan-Bandung (LC50 0.147%), and those with white flower from Lembang-Bandung and Cisarua-Bogor (LC50 0.168% and 0.186%, respectively). LC50 of T. vogelii extracts of the other six samples, i.e. those with purple flower from Cisarua-Bogor, Megamendung-Bogor, Cikalong Wetan-Bandung, Lembang-Bandung, and Tanjungsari-Sumedang, and that with white flower from Pangalengan-Bandung, ranged from 0.208% to 0.371%. The order of toxicity of the test T. vogelii extracts did not correlate with rotenone content of the extracts (r = 0.015) suggesting that some other active compounds were also responsible for the extract toxicity. The treatment with T. vogelii extracts also delayed the development of the surviving C. pavonana larvae from the second to fourth instar by 1.72-2.61 days compared with control larvae.

Keywords: Botanical insecticides, comparative toxicity, cabbage pest, Tephrosia vogelii, rotenone.

1. Introduction Insecticidal activity of Tephrosia vogelii J.D. Hooker (Leguminosae) has long been known. T. vogelii leaf powder has been commonly used by farmers in East Africa to protect stored seeds from insect infestation [1]. Leaves and some other parts of T. vogelii contain insecticidal rotenoid compounds such as rotenone, deguelin, and tephrosin [2, 3]. Rotenone is active as a stomach as well as contact poison against various insect pests with biting and sucking mouthparts [4, 5]. Rotenone-containing preparations from Derris spp. roots and some other Leguminosae plants were among botanical insecticides commonly used in pest control until the early 1950s before being superseded by the more effective synthetic insecticides [6]. In recent years, however, there is a revived interest in the use of botanical insecticides as an attempt to alleviate various negative impacts of injudicious use of broadspectrum synthetic insecticides. Moreover, the increasing practice of organic farming worldwide, which precludes the use of synthetic insecticides, has brought back the increased demand for botanical insecticides [7]. Delfel et al. [2] reported that rotenone content in T. vogelii leaves was higher than that in the other plant parts such as leaf stalks, stems, and roots. Thus, the use of T. vogelii leaves as a rotenone source for botanical insecticide production will be more advantageous than Derris roots since leaves are more amenable to 

Corresponding author. Tel.: +62-251-8629364; fax: +62-251-8629362. E-mail address: [email protected]. 1

handle than roots. Rotenoid content in T. vogelii, and accordingly the insecticidal activity of this plant, may vary with geographical locations [8]. Thus, it is necessary to evaluate the insecticidal activity of T. vogelii materials from different locations in order to determine the source plant material with the highest insecticidal activity. This study was conducted to determine the variation in insecticidal activity of acetone extracts of ten leaf samples of T. vogelii from seven different locations in West Java, Indonesia, against the cabbage head caterpillar, Crocidolomia pavonana.

2. Materials and Methods 2.1.

Preparation of Test Materials

The test insect, C. pavonana, was reared in the laboratory following the procedures described by Prijono and Hassan [9]. Briefly, the larvae were fed pesticide-free broccoli leaves and the adults were provided 10% honey solution in cotton swab. Leaf samples of T. vogelii as the insecticidal plant materials were collected between February 1 dan May 19, 2010 from seven different locations in West Java as follows: (i) organic farm, Cisarua-Bogor, 6°41’18.5’’ S, 106°56’53” E, 946 m asl; (ii) forest margin, Megamendung-Bogor, 6°42’43’’ S, 106°55’17” E, 1034 m asl; (iii) forest margin, Cipanas-Cianjur, 6°43’23’’ S, 107°0’26” E, 1283 m asl; (iv) tea plantation, Cikalong Wetan-Bandung, 6°43’33’’ S, 107°26’43” E, 689 m asl; (v) experimental farm of Spices and Aromatic Plants Research Institute, Lembang-Bandung, 6°48’30’’ S, 107°36’51” E, 1200 m asl; (vi) forest margin, Pangalengan-Bandung, 7°10’38’’ S, 107°36’44” E, >1500 m asl; and (vii) Winayamukti University garden, Jatinangor-Sumedang, 6°53’55’’ S, 107°49’9” E, ca. 850 m asl. In Cisarua, Lembang, and Pangalengan, leaf samples were collected from T. vogelli plants with purple-colored flowers and those with white flowers, while in the other four locations, leaf samples were collected only from T. vogelli plants with purple flowers. In the laboratory, T. vogelii leaf samples were cut into small pieces and then dried in a fume hood for one week. Air-dried T. vogelii leaf cuts were ground with a blender and then sieved with 0.5 mm wire screen mesh. Three-hundred grams of the ground leaves of each sample were extracted with 6 x 1500 ml of acetone by a maceration (immersion) method. The extract liquid was filtered with a No. 41 Whatman filter paper and then evaporated to dryness using a rotary evaporator at 50 C. The solvent-free extract obtained was kept in a refrigerator (ca. 4 C) until used for bioassays.

2.2.

Bioassay of T. vogelii Extracts

The toxicity testing of T. vogelii extracts against C. pavonana larvae was conducted with a leaf-residue feeding method [10]. Each extract was tested at six concentration levels that were expected to give 15%-95% mortality in the test insects as determined in the preliminary (range finding) tests. An appropriate amount of each extract was mixed with methanol and an emulsifier Tween 80 (5:1 v/v, final concentration 1.2%), then was diluted with distilled water to the predetermined volume. Pesticide-free broccoli leaves were cut into 4 cm x 4 cm portions and then dipped in appropriate extract dilutions to complete wetness and then air-dried. Control leaves were dipped in distilled water containing 1% methanol and 0.2% Tween 80. Treated and control leaf portions were placed separately in upside-down petri dishes (diameter 9 cm) lined with towel paper, then 15 second-instar larvae C. pavonana (ca. 15-30 minutes after moulting) were placed in the petri dishes. The test larvae were fed treated or control leaves for 2 x 24 hours, and then they were provided untreated leaves for the subsequent 48 hours. The number of dead larvae was recorded daily until day-4 (96 hours after treatment [HAT]) and larval mortality data at 96 HAT were analyzed with the probit method using POLO-PC [11].

2.3.

Analysis of Rotenone Content

Rotenone content in the test T. vogelii extracts was determined by HPLC using the method described by Cabizza et al. [12]. The content of other rotenoid compounds was not determined due to the unavailability of standard compounds. 2

3. Results and Discussion The yield of leaf extracts of T. vogelii with purple flower ranged from 8.2% to 11.1%, while that with white flower ranged from 10.6% to 13.7% (Table 1). Insecticidal activity of those extracts varied with location and flower color of T. vogelii plants. Among the leaf extracts of T. vogelii with purple flower, that from Cipanas-Cianjur was the most toxic to C. pavonana larvae (LC50 at 96 hours post-treatment was 0.137%), followed by that from Pangalengan-Bandung, Lembang-Bandung, Megamendung-Bogor, Cikalong Wetan-Bandung, Tanjungsari-Sumedang, and Cisarua-Bogor (LC50 ranged from 0.147% to 0.358%). Among the three extracts of T. vogelii with white flower, that from Lembang-Bandung was the most toxic (LC50 0.168%), followed by that from Cisarua-Bogor (LC50 0.186%) and Pangalengan-Bandung (LC50 0.371). Extract of T. vogelii with purple flower from Pangalengan-Bandung (LC50 0.147%) was 2.52 times more toxic than that with white flower (LC50 0.371%), while those with white flower from Cisarua-Bogor and Lembang-Bandung (LC50 0.186% and 0.168%, respectively) were 1.92 and 1.22 times, respectively, more toxic than those with purple flower (LC50 0.358% and 0.206%, respectively). Table 1. Rotenone content and toxicity of Tephrosia vogelii extracts on Crocidolomia pavonana larvae at 96 hours post-treatment

Location of leaf sample collection 1 Cisarua-Bogor (p) Megamendung-Bogor (p) Cipanas-Cianjur (p) Cikalong Wetan-Bandung (p) Lembang-Bandung (p) Pangalengan-Bandung (p) Tanjungsari-Sumedang (p) Cisarua-Bogor (w) Lembang-Bandung (w) Pangalengan-Bandung (w) 1

Extract yield (%) 2 11.1 9.9 8.2 10.1 9.2 9.6 9.9 11.0 13.7 10.6

Rotenone content (%) 0.194 0.180 0.191 0.386 0.240 0.233 0.283 0.156 0.075 0.072

2

b ± SE 3

LC50 (%)

4.09 ± 0.48 4.67 ± 0.36 5.54 ± 0.39 5.17 ± 0.41 4.86 ± 0.37 5.29 ± 0.37 3.57 ± 0.42 2.92 ± 0.24 4.43 ± 0.35 2.64 ± 0.30

0.358 0.208 0.137 0.215 0.206 0.147 0.290 0.186 0.168 0.371

Relative toxicity 4 1.04 1.78 2.71 1.73 1.80 2.52 1.28 1.99 2.21 1.00

3

p = purple flower, w = white flower. On a dry-weight basis. b = slope of probit regression line, SE = standard error. 4 Relative toxicity = LC50 of extract from Pangalengan-Bandung (w) divided by LC50 of extract from a particular location.

LC50 of extract of T. vogelii with white flower from Pangalengan-Bandung, which was the highest among the extracts tested, was used to calculate the relative toxicity of the test extracts (Table 1). The order of toxicity of the test T. vogelii extracts did not correlate with their rotenone content (r = 0.015). This suggests that some other active compounds were also responsible for the extract toxicity. In addition to rotenone, T. vogelii leaves have been known to contain some other rotenoid compounds such as tephrosin, deguelin, and elliptone [2, 3]. The difference in insecticidal activity of the test T. vogelii extracts may be due to the difference in total rotenoid content of the extracts, and this in turn may be affected by the difference in environmental condition of location where plant materials were collected, genetic traits and age of the source plants, and the season during which the source plants were collected [13]. In addition to mortality, the treatment with T. vogelii extracts also delayed the development of the test insects. As an illustration, the effect of five most active T. vogelii extracts on development time of the surviving C. pavonana larvae is shown in Table 2. The treatment with those extracts at three lowest concentrations, which caused less than 50% mortality, delayed development of the surviving C. pavonana larvae from the second to fourth instar by 1.72-2.61 days compared with their respective control (Table 2). The delay in larval development was longer at the higher extract concentrations. This delay in larval development could be indirectly due to reduced feeding or directly due to impairment of primary metabolism that supports insect growth and metabolism, or combination of both effects. Visually, treated leaves were 3

less consumed by the test larvae than control leaves and feeding reduction was more noticeable at the higher extract concentrations. Rotenone as one of the insecticidal rotenoids in T. vogelii leaves is known as a poison of cellular respiration, which acts by blocking electron transfer at Complex I of the electron transport chain in the mitochondria [14]. This kind of inhibition of mitochondrial respiration can cause the depletion of ATP as a cellular energy source which then can result in impairment of the functioning of bodily organs and eventually can lead to death or delay in the development of survivors. The delay in larval development can increase risk of being found by natural enemies and in the field this can result in further suppression of pest population after most individuals of the target pest are killed by T. vogelii extract that is applied. Table 2. Development time from the second to fourth instar of the surviving Crocidolomia pavonana larvae as affected by the treatment with extracts of five leaf samples of Tephrosia vogelii collected from West Java, Indonesia

Location of leaf sample collection 1 Cipanas-Cianjur (p)

Lembang-Bandung (p)

Pangalengan-Bandung (p)

Cisarua-Bogor (w)

Lembang-Bandung (w)

1

Extract concentration (%, w/w) 0 0.05 0.075

Development time from 2nd to 4th instar (days) 2 4.02 ± 0.15 (89) 6.09 ± 0.57 (86) 6.17 ± 0.47 (81)

0.11 0 0.05 0.075 0.11 0 0.05 0.075 0.11 0 0.05 0.075 0.11 0 0.05 0.075 0.11

6.63 ± 0.76 (59) 4.02 ± 0.15 (90) 5.74 ± 0.47 (87) 6.02 ± 0.34 (89) 6.18 ± 0.42 (82) 4.01 ± 0.11 (88) 5.82 ± 0.39 (88) 5.92 ± 0.41 (87) 6.09 ± 0.36 (86) 4.00 ± 0.00 (90) 5.98 ± 0.26 (86) 6.23 ± 0.45 (83) 6.34 ± 0.51 (65) 4.06 ± 0.23 (87) 5.91 ± 0.45 (88) 6.19 ± 0.43 (79) 6.53 ± 0.53 (72)

2

p = purple flower. w = white flower. Figures in parentheses represent the number of larvae that survived to fourth instar.

4. Summary and Conclusion Insecticidal activity of extract of ten leaf samples of T. vogelii collected from West Java, Indonesia varied with location and flower color of the source plants. All test extracts had strong insecticidal activity against the cabbage head caterpillar, C. pavonana (LC50 < 0.4%). LC50 of the test extracts ranged from 0.137% to 0.371% in which leaf extract of T. vogelii with purple flower from Cipanas-Cianjur was the most toxic and that with white flower from Pangalengan-Bandung was the least toxic. The order of toxicity of the test extracts did not correlate with their rotenone content (r = 0.015) suggesting that some other active compounds were also responsible for the extract toxicity. In addition to mortality, the treatment with T. vogelii extracts also delayed the development of the surviving C. pavonana larvae from the second to fourth 4

instar by 1.72-2.61 days compared with control larvae. Good insecticidal properties of T. vogelii as reported in this study warrant further studies to develop this plant material as a source of effective botanical insecticides.

5. Acknowledgements The authors thank the Incentive Research Program Management, State Ministry of Research and Technology for funding this study under Contract No. 021/RT/D.PSIPTN/Insentif/PPK/I/2010. The authors also thank Mr. Saodik, Ms. Astri Febrianni, Ms. Catur Hertika, Ms. Petronella Sy. Nenotek, Mr. Ahmad Sifa, Mr. Ridho Putrotomo, and Ms. Nelly Nailufar for their technical assistance.

6. References [1]

Koona, P. and S, Dorn, 2005. Extracts from Tephrosia vogelii for the protection of stored legume seeds against damage by three bruchid species. Annals of Applied Biology, 147(1): p. 43-48.

[2]

Delfel, N.E., W.H. Tallent, D.G. Carlson, and I.A. Wolff, 1970. Distribution of rotenone and deguelin in Tephrosia vogelii and separation of rotenoid-rich fractions. Journal of Agricultural and Food Chemistry, 18(3): p. 385-390..

[3]

Lambert, N., M.F. Trouslot, C. Nef-Campa, and H. Crestin, 1993. Production of rotenoids by heterotrophic and photomixotrophic cell cultures of Tephrosia vogelii. Phytochemistry, 34: p. 1515-1520.

[4]

Prakash, A. and J. Rao, 1997. Botanical Pesticides in Agriculture. CRC Press, Boca Raton, USA..

[5]

Tomlin, C.D.S., ed., 2005. The e-Pesticide Manual: A World Compendium [CD-ROM]. 13th ed. Version 3.1. British Crop Protection Council, Farnham, UK.

[6]

Klocke, J.A., 1987. Natural plant compounds useful in insect control. in Allelochemicals: Role in Agriculture and Forestry (Waller, G.R., ed.). American Chemical Society, Washington D.C., USA. p. 396-415.

[7]

Isman, M.B., 2006. Botanical insecticides. deterrents. and repellents in modern agriculture and an increasingly regulated world. Annual Review of Entomology, 51: p. 45-66.

[8]

Gaskins, M.H., G.A. White, F.W. Martin, N.E. Delfel, E.G. Ruppel, and D.K. Barnes, 1972. Tephrosia vogelii: A Source of Rotenoids for Insecticidal and Piscicidal Use. United States Department of Agriculture, Washington D.C., USA.

[9]

Prijono, D. and E. Hassan, 1992. Life cycle and demography of Crocidolomia binotalis Zeller (Lepidoptera: Pyralidae) on broccoli in the laboratory. Indonesian Journal of Tropical Agriculture, 4(1): p. 18-24.

[10]

Prijono, D., J.I. Sudiar, and Irmayetri, 2006. Insecticidal activity of Indonesian plant extracts against the cabbage head caterpillar. Crocidolomia pavonana (F.) (Lepidoptera: Pyralidae). Journal of the International Society for Southeast Asian Agricultural Sciences, 12(1): p. 25-34.

[11]

LeOra Software, 1987. POLO-PC User’s Guide. LeOra Software, Petaluma, USA.

[12]

Cabizza, M., A. Angioni, M. Melis, M. Cabras, C.V. Tuberoso, and P. Cabras, 2004. Rotenone and rotenoids in cubè resins. formulations. and residues on olives. Journal of Agricultural and Food Chemistry, 52(2): p. 288293.

[13]

Kaufman, P.B., A. Kirakosyan, M. McKenzie, P. Dayanandan, J.E. Hoyt, and C. Li, 2006. The uses of plant natural products by humans and risks associated with their use. in Natural Products from Plants (Cseke L.J., A. Kirakosyan, P.B. Kaufman, S.L. Warber, J.A. Duke, and H.L. Breilmann, eds.). CRC Press, Boca Raton, USA. p. 441-473.

[14]

Hollingworth, R.M., 2001. Inhibitor and uncouplers of mitochondrial oxidative phosphorylation. in Handbook of Pesticide Toxicology (Krieger R., J. Doull, D. Ecobichon, D. Gammon, E. Hodgson, L. Reiter, and J. Ross, eds.). Vol 2: Agents. Academic Press, San Diego, USA. p. 1169-1227.

5

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Weekend-Weekdays Variation of Air Pollutants in Tehran Farhad Nejadkoorki1 1

Department of Environmental Engineering, Yazd University, Yazd, Iran

Abstract. Tehran, capital city of Iran suffers from severe air pollution due to a rapid increase in urbanization and industrialization over the past few decades. In this study, three years (2000-2003) of sulfur dioxide, nitric oxide, nitrogen dioxide, nitrogen oxide, carbon monoxide, ozone, MSCON and particulate matter concentrations are reported for North of Tehran. The temporal variations in the concentrations of pollutants were investigated. The concentrations namely sulphur dioxide, nitric oxide, nitrogen dioxide, nitrogen oxide were not found to differ significantly between days of a weeks. However, carbon monoxide, ozone, MSCON and PM10 were found to differ meaningfully among days of a week.

Keywords: Air pollution, daily variation, Tehran

1. Introduction The direct relation of particulate matter and gaseous pollutants and health effects has been well recognized [1]. These effects include premature death, as well as increases in the incidence of chronic heart and lung disease [2]. Several attempts have been performed to analyse pollution data in Tehran. Spatial patterns of carbon monoxide dispersion and its temporal trends were studied in Tehran using proper orthogonal decomposition (POD) basis functions [3]. Carbon monoxide concentration data of 15 monitoring stations were interpolated to generate grid based data and a time series of data. In another study a time series analysis, frequency distribution and prediction of sulphur dioxide level were performed in five different locations in Tehran[4]. The results confirmed higher pollution in autumn and winter. In an another attempt a study [5] was conducted an analysis on the concentration of sulphur dioxide in the atmosphere of Tehran. The measured data from seven different monitoring stations were used to find the effect of the meteorological parameters on the concentration of pollutant. Several studies focusing on air quality and dust over Iran have already been carried out. Therefore, prediction or detecting air pollutant concentration is of crucial interest. The current paper addresses the daily variations of key air pollutants to investigate possible contribution of different sources.

2. Material and methods 2.1. Study area and data Tehran, the capital city of Iran with a current population of more than 12 million and area of 2000 km2 suffers from severe air pollution. Among the Tehran pollution sources, the mobile sources play the major role in this city. Pollutants such as SO2, NO, PM10 and CO are the major air pollutants in Tehran. It is proven that most of Tehran’s air pollution comes from the motor-vehicles [1]. Approximately 1.1 million (often old) motor vehicles produce 70 % of the city's air pollution [6]. About 40% of country-wide industries are concentrated in Tehran area. In Tehran industrial factories are predominantly located in the west and south–west while oil refinery is located in the south of the city [7]. Regarding the fact that geographical 

6 Corresponding author. Tel.: +98-351-8122798; Fax: +98-351-8210312 E-mail address: [email protected]

considerations have been disregarded for industrial site surviving, industries are responsible for 15-20 percentage of air pollution in Tehran. Corrected, edited, and adjusted electronic data was obtained from Aghdasyeh Air Quality Monitoring Centre, during August, 2000 to October, 2003. The hourly data collected from Aghdasyeh Centre includes the concentration sulphur dioxide, nitric oxide, nitrogen dioxide, nitrogen oxide, ozone and carbon monoxide, in PPB, pollutant MSCON and PM10 measured in µg/m3.

Figure 1. Location of the air quality monitoring station in Tehran, Iran

2.2. Data Analysis In this work, to analyse the data, statistical software package SPSS (version 15.0) was used. Means, standard deviations, and simple box plots of the data were constructed in order to show simple differences between categories, while t-tests and ANOVAs and Tukey Honestly Significantly Different (HSD) procedure were used to show the statistical significance of the differences.

3. Results and discussion Descriptive statistics of pollutant concentration in Aghdasyeh air monitoring stations during the study period are depicted in Table 1. The table provides a set of descriptive statistics characterizing the distribution of the pollutant concentrations monitored at Aghdasyeh. Table 1. Descriptive Statistics of pollutant in Aghdasyeh, Tehran during study period

N Mean SD Minimum Maximum Percentiles 10 Percentiles 90

Sulfur Nitric Nitrogen Nitrogen Carbon Ozone MSCON dioxide oxide dioxide oxide Monoxide 11042 11042 11042 11042 11042 11042 11042 35.543 57.790 103.890 162.140 4.176 16.484 89.312 59.169 42.119 76.057 108.325 2.315 27.099 85.994 0.300 3.000 1.300 5.000 0.100 0.100 2.500 405.000 452.500 499.000 891.000 20.700 198.100 895.000 0.500 12.860 33.000 54.500 2.000 1.300 20.000 73.300 95.800 185.300 282.500 7.100 38.800 182.500 7

PM10 11042 82.224 72.812 2.500 830.000 17.500 162.500

Analysis of the day of week of pollutant distributions were conducted to determine if a typical weekend effect was being observed in Tehran as was currently observed in other similarly sized metropolitan areas. The day of week analysis reflects the daily changes in emissions patterns due to variations in weekly activity patterns. The analysis was conducted to examine whether the daily emissions correction factor can be used in conjunction with persistence as a predictor variable. As shown in Figure 2, it has been observed that particulate matter, MSCON, carbon monoxide, nitric oxide, were lower in Fridays. Likewise sulphur dioxide was lower in Thursdays. Similarly nitrogen dioxide and nitrogen oxide and ozone were lower on Wednesdays. In Tehran, sulphur dioxide, nitrogen dioxide, nitrogen oxide, carbon monoxide attained their maximum concentrations in Mondays. Similarly, nitric oxide was peaked on Thursdays and ozone was peaked on Fridays. The weekend effect on O3, a phenomenon with a higher O3 concentration during weekends compared to weekdays in spite of the low concentration of O3 precursors (NOx and volatile organic compounds ) at weekends, has been reported since the 1970s [8-10]. The weekend effect on O3 has also been reported in various locations [11-14].

Figure 2. Daily variation of pollutant concentrations

Regarding to particulate matter concentration PM10 and MSCON, were peaked on Sundays and Wednesdays, respectively. In Teheran pollutant concentrations exhibit a weekend effect whereby concentrations drop on Thursday/Friday peak through Sunday/Monday. Mondays had the highest concentration of any day of the week. These observations suggest a strong degree of pollutant concentration carry over from day to day indicating that in Tehran, during the week days average pollutant concentrations were higher in comparison with weekends. For a better comparison, an independent sample t-test was carried out for pollutants during week days and weekends. As can be seen in Table 2, sulphur dioxide, carbon monoxide, MSCON and PM10 concentrations were significantly higher during working days whereas ozone was significantly lower in working day. As can be seen in the ANOVA table for the daily analysis (Table 2), the concentrations namely sulphur dioxide, nitric oxide, nitrogen dioxide, nitrogen oxide were not found to differ significantly between days of a weeks as indicated by p-value (0.0000). However, carbon monoxide, ozone, MSCON and PM10 were found to differ significantly among days of a week.

8

Table 2. ANOVA table for daily pollution data

Pollutant Sulfur dioxide

Between groups Within groups Total

Sum of Squares 23489.47 38630569 38654058

Nitric oxide

Between groups Within groups Total

7506.165 19579416 19586922

6 11035 11041

1251.027 1774.301

0.705

0.646

Nitrogen dioxide

Between groups Within groups Total

54718.66 63814479 63869197

6 11035 11041

9119.776 5782.916

1.577

0.149

Nitrogen oxide

Between groups Within groups Total

79042.75 1.29E+08 1.3E+08

6 11035 11041

13173.79 11733.61

1.123

0.346

Carbon monoxide

Between groups Within groups Total

416.487 58755.4 59171.89

6 11035 11041

69.414 5.324

13.037

0.000*

Ozone

Between groups Within groups Total

38752.18 8069018 8107770

6 11035 11041

6458.696 731.22

8.833

0.000*

Between groups Within Groups Total

216634 81431888 81648522

6 11035 11041

36105.67 7379.419

4.893

0.000*

Between Groups Within Groups Total

240478.6 58293696 58534175

6 11035 11041

40079.77 5282.619

7.587

0.000*

MSCON

PM10

df 6 11035 11041

Mean Square 3914.912 3500.731

F

Sig.

1.118

0.349

4. Summary and conclusion This paper has reported results of daily variation of a set of different air pollutants in Tehran. Three years of main air pollutants for a monitoring site at the north of Tehran were considered to discover the possible variations. Of the studied pollutants some appears to be significantly different. The concentrations namely sulphur dioxide, nitric oxide, nitrogen dioxide, nitrogen oxide were not found to differ significantly between days of a weeks. However, carbon monoxide, ozone, MSCON and PM10 were found to differ meaningfully among days of a week.

9

5. References [1] Halek, F., A. Kavouci, and H. Montehaie, Role of motor-vehicles and trend of air borne particulate in the Great Tehran area, Iran. International Journal of Environmental Health Research, 2004. 14(4): p. 307-313. [2] Hooyberghs, J., et al., A neural network forecast for daily average PM10 concentrations in Belgium. Atmospheric Environment, 2005. 39(18): p. 3279-3289. [3] Ashrafi, K., Determining of spatial distribution patterns and temporal trends of an air pollutant using proper orthogonal decomposition basis functions. Atmospheric Environment, 2012. 47(0): p. 468-476. [4] Hassanzadeh, S., F. Hosseinibalam, and R. Alizadeh, Temporal Variations of Major Air Pollutants and Pollution Standard Index in the Great Tehran Area. Environmental Forensics, 2012. 13(1): p. 55-67. [5] Asrari, H., V.S. Ghole, and P.N. Sen, Study on the Status of SO2 in the Tehran- Iran. Journal of Applied Science and Environmenal Management, 2006. 10(2): p. 75-82. [6] Madanipour, A., City profile: Tehran. Cities, 1999. 16(1): p. 57-65. [7] Estelaji, A. and M.A.G. Nejad. A Study on Effective Factors in Environmental Alterations in Mega Cities. Case Study: Tehran Mega City. in Proceedings of the 5th WSEAS International Conference on Waste Management, Water Pollution , Air Pollution , Indoor climate. 2011. Iasi, Romania. [8] Cleveland, W.S., et al., Sunday and Workday Variations in Photochemical Air Pollutants in New Jersey and New York. 10.1126/science.186.4168.1037. Science, 1974. 186(4168): p. 1037-1038. [9] Lebron, F., A comparison of weekend-weekday ozone and hydrocarbon concentrations in the BaltimoreWashington metropolitan area. Atmospheric Environment (1967), 1975. 9(9): p. 861-863. [10] Elkus, B. and K.R. Wilson, Photochemical air pollution: Weekend-weekday differences. Atmospheric Environment (1967), 1977. 11(6): p. 509-515. [11] Pun, B.K., C. Seigneur, and W. White, Day-of-week behavior of atmospheric Ozono in three US cities. Proceedings of the 5th WSEAS International Conference on Waste Management, Water Pollution , Air Pollution , Indoor climate, 2003. 53: p. 789-801. [12] Qin, Y., G.S. Tonnesen, and Z. Wang, One-hour and eight-hour average ozone in the California South Coast air quality management district: trends in peak values and sensitivity to precursors. Atmospheric Environment, 2004. 38(14): p. 2197-2207. [13] Jimenez, P., et al., Modeling the ozone weekend effect in very complex terrains: a case study in the Northeastern Iberian Peninsula. Atmospheric Environment, 2005. 39(3): p. 429-444. [14] Pudasainee, D., et al., Influence of weekdays, weekends and bandhas on surface ozone in Kathmandu valley. Atmospheric Research, 2010. 95(2-3): p. 150-156.

10

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Comparative pathogenicity of Beauveria bassiana, Clonostachys rosea, Metarhizium anisopliae, and Lecanicillium lecanii to adult,alfalfa weevil Hypera postica Gyllenhal (Coleoptera:Curculionidae) Rebwar Ahmed Mustafa 1, Lazgeen Haji Assaf 2, and Samir Khalaf Abdullah 3+ 1

2

Agriculture Technical College in Halabja, Sulaimani Technical University, Iraq Department of Plant Protection, Faculty of Agriculture and Forestry, Duhok University, Iraq 3 Department of Biology, Faculty of Science, University of Zakho, Iraq

Abstract. Alfalfa weevil Hypera postica (Gyllenhal) (Coleoptera:Curculionidae) is a destructive pest on alfalfa Medicago sativa L. Pathogenicity test with conidial suspension of entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Clonostachys rosea isolates had a significant effect on H.postica adults and the mortality percentage caused by them significantly differed from the control treatment. Isolates of B.bassiana ,M.anisopliae, and L.lecanii were the most effective species on adults, incontrast with the isolates of C.rosea which they had a slow effect on adult and recorded the lowest mortality percentage.

Keywords: Entomopathogenic fungi, alfafa weevil,pathogenicity.

1. Introduction Alfalfa (Medicago sativa L.) is one of the most prized forages and is grown worldwide, and provided benefits to farmers as well as to agro ecosystems, due to its wide adaptation, biological nitrogen fixation, soil improvement and benefits to subsequent crops for being often grown in rotation with wheat, corn and cotton. Alfalfa is attacked by a diversity of insect's pest; however, the alfalfa weevil H. postica Gyllenhal is the most destructive pest of alfalfa in the world [1] and in Iraq [2]. H. postica is a typical oligophagous insect that feeds almost exclusively on leguminous plant of the genus Medicago, although it may occasionally feed on a few species of related genera including Melilotus, Trifolium and Trigionella in most regions of the world. Both larvae and adults of alfalfa weevil are voracious feeders damaging terminal leaves and new crown shoots[1]. Biological control of insect pests is an important feature to their management programs by it can avoid using insecticides and reduce risks to people, water resource and wild life. The objective of this study was to determine the pathogenicity of native isolates of four fungal pathogens against adults of alfalfa weevil.

2. Materials and methods 2.1. Sampling alfalfa weevil adults population Alfalfa weevil becomes active in northern region of Iraq in late-fall. Oviposition occurs in late-October or November and continued through the winter and spring [2]. Alfalfa adults were sampled in the fields in October, November, December, January, March and April during the 2012-2013. Twenty samples of soil and litter were weekly collected from the field using a sampling quadrate measured (0.5 × 0.5) m as developed by Hilburn [3]. Corresponding author: Tel: +9647704574841 E-mail address: [email protected]

11

2.2. Preparation of Alfalfa weevil Hypera postica (Gyllenhal) for laboratory experiments The adults of H. postica were collected in October in the fields of Faculty of Agriculture and Forestry/ University of Duhok and transferred to laboratory, kept in wooden cages measured (80×40×40 cm) covered with muslin clothes perforated from their base. The cage was also supplied each three days with fresh branches of alfalfa fixed inside conical flasks and filled with water daily to prevent branches drying.

2.3. Pathogenicity to adults Groups of 10 adults/ replicate were sprayed with (3ml) of conidial suspension (10 7 concentration) of each selected fungal isolate inside a Petri- dish of (9 cm) diameter lined with moisten filter paper, and supplied with fresh alfalfa leaves daily while the control treatment sprayed with water by a new perfume sprayer (35 ml capacity). The mortality percentage was recorded after 2, 4 and 6 days of treatment. Cumulative mortality counts obtained from experiments was corrected for natural mortality using Abbott 's formula [4]. Pr = (Po – Pc)/ (100 – Pc) × 100 Pr = Corrected mortality percentage Po = treatment mortality percentage Pc = Control mortality percentage The data were statistically analyzed by SAS program using Randomized Complete Block Design (RCBD) with four replicates the means were compared using Duncan's multiple range tests at P ≤ 0.05.

4. Results and discussion The results in Table (1) showed that there were significant differences among the fungi effect on adults mortality percentage. M. anisopliae Am2 and B. bassiana Ab1 recorded the highest mortality percentage after two days of treatment which reached 66.667% and 66.667% respectively, followed by B. bassiana Fb1and M. anisopliae Pm1 which recorded 53.333% and 46.667% respectively and did not differ significantly from mortality percentage that caused by C. rosea Bc1 as 43.333 % after the same period. Also the results showed that M. anisopliae Am2, M. anisopliae Pm1, and B. bassiana Ab1 recorded the highest mortality percentage after 4 days of treatment which reached 100% for the three fungi mentioned, in spite of that it did not differ significantly from the mortality caused by B. bassiana Fb1as 90.00 %. The lowest mortality percentage was recorded when the adult treated with C. rosea Bc2 and C. rosea Fc2 suspension after the same period (4 days) as 66.67 % and 40.00% respectively and significantly differed from the mortality caused by control treatment 0.00 %. The mortality percentage of adults treated with L. lecanii Pl2 conidia suspension after 6 days of treatment, reached 100%, in contrast with the C. rosea Fc2 and C. rosea Bc1 which they had a slow effect on adults and recorded the lowest mortality percentage as 55.172% and 89.655 % and 58.620% and 96.55% after 6, 8 days respectively. These results indicated that the conidial suspension of fungi used have a significant effect on H. postica adults (Fig. 1) and the mortality percentage caused by them significantly differed from that of control treatment(Table 1). Also we showed that M. anisopliae Am2 and M. anisopliae Pm1 and B. bassiana Ab1 were the most effective species on adults. Burdeos and Villacarlos [5] stated that M. anisopliae was more pathogenic to sweet potato weevil Cylas fornicarius F. than B. bassiana and Paecilomyces lilacinus under laboratory conditions. Moino et al. [6] mentioned that the infection process varies among the fungi and added that penetration, colonization and sporulation occurs faster with M. anisopliae (Metsch.) than with B. bassiana, resulting in the earlier death of hosts infected with the former fungus. The entomopathogenic fungi M. anisopliae process a chitinolytic syste, which is believed to have a role in the digestion of insect's cuticle during the development of diseases [7]. The results also agreed with Shams et 12

al., [8] who mentioned that the mortality percentage for adult C. maculatus after (3-5-7 and 9 days) of treatment with B. bassiana was 50%. Kram [9] mentioned that nut-leaf weevil Strophosoma melangrammum (Forster) infected with entomopathogenic fungi began to die (2-11 days) after inoculation with Metarhizium spp and B. bassiana isolates. The two species found to be highly virulent with mortalities of 93-91% respectively.

A

B

C

D

E

F

Figure (1): Infection of adult of alfalfa weevil by entomopathogenic fungi:. A, B- B. bassiana E- L. lecanii F- C. rosea .

13

C-, D- M. anisopliae

Table (1): Pathogenicity of different fungi to adult of alfalfa weevil Hypera postica Fungi 107 conidia/ ml

2 days after treatment

4 days after treatment

Beauveria bassiana Ab1

66.667 ± 6.667 a

100.000 ± 0.000 a

100.000 ± 0.000 a

Beauveria bassiana Fb1

53.333 ± 3.333 ab

90.000 ± 10 .000 ab

100.000 ± 0.000 a

Metarhizium anisopliae Am2

66.667 ± 3.333 a

100.000 ± 0.000 a

100.000 ± 0.000 a

Metarhizium anisopliae Pm1

46.667 ± 8.819 bc

100.000 ± 0.000 a

100.000 ± 0.000 a

Clonostachys rosea Fc2

16.667 ± 3.333 d

40.000 ± 5.774 d

55.172 ± 3.333 c

Clonostachys rosea Bc1

43.333 ± 6.667 bc

66.667 ± 3.333 c

89.655 ± 5.774 b

Lecanicillium lecanii Pl2

33.333 ± 3.333 c

86.667 ± 3.333 b

100.000 ± 0.000 a

Control

0.000 ± 0.000 e

0.000 ± 0.000 e

3.333 ± 3.333 d

6 days after treatment

Means followed by a common letter within the same column are not significantly different the 5% level by DMRT

References [1] [2]

Summers,C.G., L.D. Godfrey and T.E.Natwick, 2007. Managing insects in alfalfa. University of California, Division of Agriculture and Natural Resources, Publication 8295, 12/2007. USA. Khaphy, A. A., 1981. Ecological, biological and chemical control studies on alfalfa weevil Hypera fascocinera (Marsh.) (Coleoptera: Curculionidae) in Ninevah. M.Sc. Tthesis. College of Agriculture and Forestry, Mosul University, Iraq. pp.178. (In Arabic)

[3]

Hilburn, D.J., 1985. Population dynamics of overwintering life stages of the alfalfa weevil, Hypera postica (Gyllenhal). Ph.D. dissertation, Virginia polytechnic Institute and State University, Blacksburg.

[4]

Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18: p. 265-267.

[5]

Burdeos, A. T. and L. T. Villacarlos, 1989. Comparative pathogenicity of Metarhizium anisopliae, Beauveria bassiana and Paecilomyces lilacinus to adult sweet potato weevil, Cylas formicarius (F.) (Coleoptera: Curculionidae). Philippine Entomologist, 7: p. 561-571.

[6]

Moino,A., S.B. Alves, R. B. Lopez, P.M.O.J. Neves, R.M. Pereira, and S, A. Vieira, 2002. External development of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in the subterranean termite Heterotermes tenuis. Scientific Agriculture, 59: p. 267 – 273.

[7]

Valadares-Inglis M.C. and J.F. Peberdy, 1997. Location of chitinolytic enzymes in protoplasts and whole cells of the entomopathogenic fungus Metarhizium anisopliae. Mycological. Research, 101: p. 1393–1396.

[8]

Shams,G.,M. H.Safaralizadeh,.S. Ismail, M. shajai, and S. Aramodeh, 2011. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin)to control Callosobruchus maculates(F.) (Coleoptera:Bruchidae) and Sitophilus granaries (L.) (Coleoptera:Curculiondae). African Journal of Microbiology Research, 5: p. 1192-1196.

[9]

Kram, A. A., 2010. Mortality of the nut-leaf Strophosoma melanogrammum (Forster) and damage rate of needles after treatment with entomopathogenic fungi. Journal of Plant Protection Research, 50 (4): p. 545-550.

14

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Macrochelid Mite, Macrocheles Muscaedomesticae (Acarina: Macrochelidae) As A Biological Control Agent Against House Fly, Musca Domestica (Diptera: Muscidae) In Egypt Safaa Mostafa Abo-Taka 1, Hany Mohamed Heikal 2, Ahamed Abd El-Raheem 3 1,2,3

Economic Entomology and Agricultural Zoology Dept. Faculty of Agric., Menoufia University, Egypt

Abstract. Numerous species of macrochelids (Acarina; Mesostigmata) have shown capability to attack housefly larvae and eggs, but it is presumed that only a few of them play a significant role in the control of flies in nature. Macrocheles muscaedomesticae (Scopoli) is one of several mites that feeds on eggs, newly hatched and small larvae of house fly Musca domestica L. This study provide evidence that macrochelid mite is attacking not only housefly larvae and eggs but also housefly adults. M. muscaedomesticae mite was reared in the laboratory on house fly frozen eggs and first instar of larvae at constant conditions of 28 °C±1 and 90 % relative humidity using sterilized artificial diet. The mean incubation period of eggs, total immatures, female longevity were 0.7, 4.0 and 25.2 days when fed on frozen eggs, meanwhile means were 0.8, 7.0 and 22.6 days respectively, when fed on first instar larvae of M. domestica. The total consumption of female was 131.1 eggs/female or 82.7 larvae. Results of the present study showed that, the mean mortality percentages of eggs due to predation of three levels of predator 2, 5 and 10 individuals were 57.2, 74.9 and 96.5 after 5, 4, 2 days, respectively. Also, the larval stage of M. domestica was introduced with three levels of 10, 20 and 25 individuals for each level of predatory mite 2, 5 and 10 adults, respectively . Results revealed that the best results were recorded at the level of 5 mites, where the mean mortality percentage of larvae was 100 % after one day when introduced with 10 housefly’s larvae, but it was 96.0 % after two days when introduced with 20 housefly’s larvae, and 76.2 % after three days when introduced with 25 houseflies larvae at level 5. In addition the present study provide evidence that mites can consume the adult stage. Our findings indicated that the best results were recorded at the level of 10 mites, where the mean mortality percentages of adults were 83.55 % the fly died after two days of one prey treatment, 62.5 % after four days of two preys treatment, while it was only 55.57 % after three days of three preys treatment.

Keywords: Macrocheles muscaedomestiace, house fly, predation, bio-control.

1. Introduction A number of mite’s species encountered in dung are predators on nematodes, Oligochaetes or arthropods. Family Machrochelidae is one of the six families of the suborder Gamasida and some species of this family prey on different stages of house fly [20]. Several species of this family are predacious on the eggs and firstinstar larvae of the house fly and cause substantial reductions in housefly production from manure [4, 5, 6]. The pioneering research of Filipponi and coworkers in Italy on the biology of Macrochelidae associated with synanthropic flies verified that macrochelid mites hold considerable promise as a means of controlling fly populations [11, 12]. The macrochelid mite, Macrocheles muscaedomesticae, is the most common mite in poultry manure. The reddish-brown mite, slightly less than 1/16 inch in size, feeds on house fly eggs and first-instar larvae and it can consume up to 20 house fly eggs per day. Mites are found on the outermost layer of the manure, particularly its peak. Macrochelids can cause substantial reductions in house fly numbers, but large mite populations are required for appreciable impact. Rates of predation by M. muscaedomesticae and Fuscuropoda vegetans [21, 25, 8] were studied extensively under a variety of experimental conditions and results were variable. The mite M. muscaedomesticae (Scopoli) is one of the most ______________________________ Corresponding author. Hany M. Heikal 2 Tel.: +2 (01004990202); fax: +2 (0482228187). E-mail address: ([email protected]).

15

abundant predators of fly immatures in poultry production systems [23, 70, 15, 17]. There are several advantages that make M. muscaedomesticae an attractive bio-control agent such as short development time, high attacking rate, ability to reproduce on alternative prey, and proclivity for dispersal into new fly breeding areas via phoresy [14, 8, 13, 19, 10]. [16] Found that 54 fly immatures were destroyed per predator per day, at 15 and 35°C predation rates of M. muscaedomesticae were 5.0 and 36.3 per day respectively. [3] Revealed that Macrocheles glaber (Müller) is one of several mites that feeds on eggs, newly hatched & small larvae of house fly Musca domestica L. This mite was reared in the laboratory on house fly frozen eggs at constant conditions of 28° C ± 1 and 90 % relative humidity using sterilized horse dung substrate. The predation rate of adult female and male on frozen eggs was 18.3 eggs/mite/day. The aim of this study is to investigate developmental stages of Macrocheles muscadomestica and its potential to control different stages of Musca domestica using three levels of this predator.

2. Materials and Methods House fly culture reared on artificial diet consisted of 9 g powder milk and 5 g yeast dissolved in 100 ml water in addition to 100 g fine bran (Wilkins and Khalequzzaman, 1993). The mixture was stirred and put into the cups 3 cm from the top. The cups were transferred to an entomological glass cages (60×35×40 cm) which used for rearing house fly larvae under laboratory conditions (25 ±5 C˚, 60±5% RH) , and a 12:12 light: dark cycle (Palacios et al., 2009). These cages were covered with mesh screen with cloth sleeve opening at top. When adult house fly emerged in cages, granulated sugar and milk soaked cotton wool balls were provided in Petri dishes to house fly adults. The emerged flies were also fed with full fat fresh milk in Petri dishes. After two days of fly emergency, the beakers containing larval food was placed for egg laying process. Beakers were removed from cages after 2 - 3 days when eggs were visible and attached to food along the sides of beakers. The food was changed after 2 - 4 days depending upon the numbers of larvae per beaker. The beakers were kept in separate cage for fly emergency (Ahmed and Irfanullah, 2007). Macrocheles muscaedomesticae was found with huge numbers associated with house fly culture reared at laboratory of Economic Entomology and Agricultural Zoology Department, Faculty of Agriculture, Menoufia University, Egypt. Many nymphs were reared in a plastic container (15 cm long, 10 cm wide, 5 cm in depth, the cover had a window 5 x 5 cm screened with fine polyester mesh) and kept in an incubator at 28◦C. One container was used to rear mites fed on house fly adults and eggs and the other fed on house fly larvae. Food was added daily. To study the developmental stages of mite, Petri dishes 5 cm in diameter contains 1 gm of sterilized poultry dung was placed in addition some drops of water was added, then a couple of newly emerges mites were transferred to lay eggs. Ten replicates were used, examined twice daily, kept at 28 ◦C and incubation period was calculated by rearing egg singly until hatching. Other immature stages period were determined by adding either eggs or larvae of house fly and tangle foot was used to prevent mite escaping. Newly emerged females were separated singly to determine its longevity. The same rearing cells were used to determine the number of consumed eggs by mite’s female. Ten eggs were added daily to each female reared singly and ten replicates were used. To study the effect of predatory number on the consumption rate of house fly eggs, 40 eggs were add to 2,5, and 10 females. Daily observation was performed to determine the number of destroyed eggs using microscopic examination. The emerged house fly larvae were removed and replaced with eggs. For house fly larval stage, three counts were used (10, 20 and 25 larvae) for each level of predator numbers (2, 5, and 10 females of M. muscaedomesticae) to determine the consumption rate and the probability of competition between mite individuals. Daily examination was conducted and corrected mortality was calculated. The adult stage of house fly was introduced by (1, 2 and 3 adult flies) for each level of mites (2, 5, 10 and 15 females) experiment was prolonged for five days at 28 ◦C. Control was performed without mites. Mortality percentages of eggs, larvae and adult fly were calculated and corrected using Abbott’s formula 1925.

16

Statistically analyzed was performed using COSTAT 22 according to the method of Snedecor and Cochran (1967).

3. Results and Discussion 3.1. Biology of Macrocheles muscaedomesticae: Table (1) shows the duration of Macrocheles muscaedomesticae, developmental stages and consumption rate when fed on eggs or larvae of Musca domestica at 28 °C. The mean of incubation period of M. muscaedomesticae egg duration 0.7 and 0.8 days when fed on M. domestica eggs or larvae, respectively. The total immatures of M. muscaedomesticae female was ranged between 3.5 and 4.5 with mean value of 4.0 days when fed on fly eggs, meanwhile, the total immature mean value was prolonged to 7.0 days when fed on the larvae of M. domestica. The female longevity was longer when fed on fly eggs ranged between 25.5 to 27 days with mean value of 25.2 days while it lasted only 22.6 days when the mite fed on fly larvae. Table (1): Biological aspects of eggs and larvae at 28◦C.

Biological aspects Incubation period Total immatures ♀ longevity Daily consumption /♀ Total consumption /♀

Macrocheles muscaedomesticae adult females feed on Musca domestica

Feed on M. domestica eggs at 28◦C Total Mean Range S.E

Feed on M. domestica larvae at 28◦C Total Mean Range S.E

3.5

0.7

0.5 : 1.0

0.7 ± 0.22

4.0

0.8

0.5 : 1.0

0.8 ± 0.22

20.0

4.0

3.5 : 4.5

4.0 ± 1.57

35.0

7.0

6.0 : 8.0

7.0 ± 3.58

126.0

25.2

25.5 : 27.0

25.2 ±10.96

113.0

22.6

21.0 : 25.0

22.5 ± 9.84

27.0

5.4

4.0 : 7.0

5.4 ± 1.79

19.0

3.8

3.0 : 5.0

3.8 ± 1.79

655.5

131.1

94.0 :168.0

131.1±42.04

413.5

82.7

60.0 :120.0

82.7 ±37.57

Values are means ± standard error (n = 5).

The daily consumption per female was observed and data showed that mites consumed from 4 to 7 eggs / day with mean number of 5.4 eggs /day/female, while the female of M. muscaedomesticae consumed 3 to 5 larvae /day with mean value of 3.8 larvae /day. The mean total consumption rate of fly eggs was 131.1: eggs or 82.7 larvae /♀. In compareisone with data obtained by Al-Dulaimi, 2002 on the biology of Macrocheles glaber, reared on 28 °C, our results showed that M. muscaedomesticae egg hatched after only 0.7 days while it was 1.34 days for M. glaber. The immature stages was longer for M. muscaedomesticae (4.0 days), while it was only 2.67 days in case of feeding on fly eggs. The female longevity of M. muscaedomesticae was shorter (25.2 days) than M. glaber (27.8 days). 3.2. Effect of prey stage and number and the predatory level on predation rate: 3.2.1. Egg prey: Table (2) illustrates the mortality percentage due to the predation of different levels of predator (2, 5 and 10 individuals). As for egg stage the mortality percentage was 19 % one day after introducing 40 eggs to two predatory individuals, this percentage increased to 100 % five days after introducing with mean of 57.2 %. In case of introducing five predatory individuals, 37 % of eggs was consumed one day after introducing and all eggs were completely destroyed after four days, mean value of 74.9 % mortality. forty eggs of M. domestica were completely destroyed after two days of introducing 10 predatory individuals.

3.2.2. Larvae prey: The larval stage of M. domestica was introduced with three levels of 10, 20 and 25 individuals for each level of predatory mite 2, 5 and 10 adults. Ten larvae of M. domestica were destroyed after three days when exposed to 2 mite individual while the same number of larvae were completely destroyed after two days of the treatment with 10 mite individuals. All larvae were destroyed at the first day at the level of 5 predatory mites. Introducing 20 larvae of fly 17

resulted in mean mortality value of 55.2, 96.0 and 83.3 % and all larvae destroyed after 5 days by two predatory individuals. 2 days only were enough to destroy all larvae when 5 or 10 predatory individuals were applied. Increasing the number to 25 larvae resulted in mean mortality values of 50.5, 76.2 and 75.5 % with 2, 5 and 10 predator, respectively, which approximately the same results recorded before with 20 larvae. Table (2): The effect of different levels of predatory mite Macrocheles musacaedomesticae feeding on Musca domestica eggs and larval stage.

Mortality % of eggs or larvae (day) Level of predator Introducing 40 eggs 2 5 10 Introducing 10 larvae 2 5 10 Introducing 20 larvae 2 5 10 Introducing 25 larvae 2 5 10

1st

2 nd

3rd

4 th

5 th

Mean

19.0 37.0 93.0

37.0 75.0 100

55.0 87.5 -

75.0 100 -

100 -

57.2 74.9 96.5

36.4 100 83.3

83.3 100

100 -

-

-

73.2 100 91.7

13.6 92.0 66.6

22.0 100 100

56.0 -

84.0 -

100 -

55.2 96.0 83.3

12.4 40.2 53.3

27.0 88.3 73.3

36.0 100 100

77.0 -

100 -

50.5 76.2 75.5

3.2.3. Adult prey The predation of Macrocheles muscaedomesticae was observed too in adult stage of Musca domesticate, it was the first record of preying M. muscaedomesticae on the housefly adult, figs a, b and c cleared this predation and how mites can completely destroy the fly. Data in table 3 shows that the level of two or five predatory mites were able to kill the introduced adult fly during the third day with average mortality percentage of 33.3 % while the adult flies in all replicates were destroyed during the fifth day. For the level of 10 predators, the introduced fly was completely killed in all replicates at the second day. Introducing two adult flies for the three levels of predator (2, 5, 10 individuals) recorded 50, 50.03 and 62.5 % mean mortality, with the same adult results of killing the two adult fly four days after introducing respectively. Four levels of mites 2, 5, 10, 15 individuals / 3 adult flies were tested, data proved that the first and second levels killed fly in all replicates after four days, third and fourth levels were able to destroy flies after three days only.

While previous studies used reported the role of Macrocheles muscaedomesticae in controlling Musca domestica eggs and larvae (Geden and Axtell 1988, Geden et al 1988, and Al-Dulaimi 2002), our present study introduced relevant findings on the control of Musca domestica using the same mite M. muscaedomesticae which is the most common and abundant mite in poultry and farm manure. In our study provided evidence that we can control adult fly also by this mite.

18

Fig: a. Macrocheles muscaedomesticae attack housefly, b. large numbers of predators on housefly, c. big hole on ventral of housefly after feeding predators. (These photos were taken by a light microscope by Dr. Hany heikal 2013) Table (3): The effect of different levels of predatory mite Macrocheles musacaedomesticae feeding on Musca domesticate adult stage.

Mortality % of adult (day) Level of predator Mean 1st 2 nd 3rd 4 th 5 th Introducing one adult 2 0 0 33.3 33.3 100 33.32 5 0 0 0 0 100 20.0 10 66.7 100 83.35 Introducing two adult 2 0 16.7 83.3 100 50.0 5 16.7 16.7 66.7 100 50.03 10 0 66.7 83.3 100 62.5 Introducing three adult 2 0 22.2 88.9 100 52.78 5 0 22.2 66.7 100 47.23 10 0 66.7 100 55.57 15 44.4 88.9 100 77.77 However, the reported rates of predation depend on the methods used for evaluation; our study estimated the effect of different levels of both predator and prey. The most suitable data proved that both 5 and 10 levels of predators were able to control egg and larvae stages of fly. The ratio 1 predator: 4 eggs, 1 predator: 2 larvae, 5 predators: 2 adult fly showed promising rate to control house fly on poultry and farm manures.

References [1] [2] [3] [4] [5] [6]

Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18:265-267. Ahmed, S., and M. Irfanullah, 2007. Toxicity of pyrethorids co-administered with sesame oil against housefly Musca domestica L. Int. Journal of Agriculture Biology, 9:782- 784. Al-Dulaimi, S.I., 2002. Predation by the mite macrocheles glaber (müller) (acarina: macrochelidae) on the house fly musca Domestica L. With some notes on its biology. Bull. Iraq nat. Hist. Mus. 9 (4): 7-11. Axtell, R.C., 1961. New records of North American Macrochelidae (Acarina: Mesostigmata) and their predation rates on the house fly. Ann. Ent. Soc. Amer. 54(5): 748. Axtell, R.C., 1963. Effect of Macrochelidae (Acarina: Mesostigmata) on house fly production from dairy cattle manure. Journal of Economic Entomology 56, No.3, pp. 317-321. Axtell, R.C., 1964. Phoretic relationship of some common manure-inhabiting Macrochelidae (Acarina: Mesostigmata) to the house fly. Annals of the Entomological Society of America. 57 (5): 584 - 587. 19

[7]

Axtell, R.C., 1968. Integrated house fly control: populations of fly larvae and predaceous mites, Macrocheles muscadomesticae in poultry manure after larvicide treatment. J. Econ. Entomol., 61(1): 245-249. [8] Axtell, R.C., 1969. Macrochelidae as biological control agents for synanthropic flies, pp. 401-416. In Proceedings, Second International Congress of Acarology. Akademiai Kiado, Budapest. [9] Axtell, R.C., 1970. Integrated fly-control program for caged-poultry houses. J. Econ. Entomol. 63: 400405. [10] Farish, D.J. and R.C. Axtell, 1971. Phoresy redefined and examined in Macrocheles muscaedomesticae (Acarina: Macrochelidae). Acarologia 13: 16-25. [11] Fillipponi, A., 1960. Macrochelidi (Acarina: Mesostigmata) Foreticidi nosch. Risultati parzialidi una indagine ecologica in carso nell agro pontino. Parasitologia. 2(1-2):167-172. [12] Fillipponi, A., 1964. The feasibility of mass production macrochelid mites for field trials against house flies. Bull. WHO. (Geneva), 31: 499-501. [13] Filipponi, A., and G.D. di Delupis, 1963. On the food habits of some macrochelids (Acari: Mesostigmata) associated in the field with synanthropic flies. Riv. Parassito!. 24: 277-288. [14] Filipponi, A., and M.G. Petrelli, 1967. Autoecology and capacity for increase in numbers of Macrocheles muscaedomesticae (Scopoli) (Acari: Mesostigmata). Riv. Parasitol. 28: 129-156. [15] Geden, C.J., and J.G. Stoffolano Jr., 1987. Succession of manure arthropods at a poultry farm in Massachusetts, USA, with observations on Carcinops pumilio (Coleoptera: Histeridae) sex ratios, ovarian condition, and body size. J. Med. Entomol. 24: 214-222. [16] Geden, C.J., and R.C. Axtell, 1988. Predation by Carcinops pumilio (Coleoptera: Histeridae) and Macrocheles muscaedomesticae (Acarina: Macrochelidae )on the housefly (Diptera: Muscidae): functional response effects of temperature, and availability of alternative prey. Environ. Entomol., 17: 739-744. [17] Geden, C.J., and J.G. Stoffolano Jr., 1988. Dispersion patterns of arthropods associated with poultry manure in enclosed houses in Massachusetts: spatial distribution and effects of manure moisture and accumulation time. J. Ent. Sci. 23: 136-148. [18] Geden, C.J., R.E. Stinner, and R.C. Axtell, 1988. Predation by Predators of the House Fly in Poultry Manure: Effects of Predator Density Feeding History Interspecific Interference and Field Conditions. Environmental Entomology Vol. 17(2): 320-329. [19] Ito, Y., 1973. The effects of nematode feeding on the predatory efficiency of house fly eggs and reproduction rate of Macrocheles muscaedomesticae (Acarina: Mesostigmata). Jpn. J. Sanit. Zoo!. 23: 209-213. [20] Krantz, G.W., 1983. Mites as biological control agents of dung-breeding flies, with special reference to the Macrochelidae. In: Biological control of pests by mites, M.A. Hoy, G.L. Cunningham and L. Knutson (eds), pp. 91–98. University of California, Berkeley. [21] O'Donnell, A. E., and R.C. Axtell, 1965. Predation by Fuscoropoda vegetans (Acarina: Uropodidae) on house fly Musca domestica. Ann. Ent. Sco. Amer., 58(3): 403-404. [22] Palacios, S.M., A. Bertoni, Y. Rossi, R. Santander and A. Urzúa, 2009. Efficacy of essential oils from edible plants as insecticides against the house fly, Musca domestica L. Molecules, 14:1938-1947. [23] Peck, J.H., and J.R. Anderson, 1969. Arthropod predators of immature Diptera developing in poultry droppings in northern California Part I. Determination, seasonal abundance and natural cohabitation with prey. J. Med. Entomol. 6: 168-171. [24] Snedecor, G.W., and W.G., Cochran, 1967. “Statistical methods” (6th ed.). Iowa State Univ., Press. Amer. Iowa USA. [25] Willis, R.R., and R.C. Axtell, 1968. Mites predators of the house fly: Acomparison of Fuscoropoda vegetans and Macrocheles muscadomesticae. J. Econ. Entomol., 61(6): 1669-1674. [26] Wilkins, R.M., and M. Khalequzzaman, 1993. Environmental interactions of pesticides: Synergism of permethrin by simazine against housefly. Proc. Brighton crop Protect. Conf., 3B-7: 157-162.

20

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Histopathological Studies of the Effects of Cyclophosphamide on Liver of Female Albino Mice Intissar Numman Waheed 1+; Khesar Hussein Khalil 2; and Zobayda Adnan Sharafaddin 2 1

Department of Biology, Faculty of Sciences, University of Zakho, Dohuk, Iraq 2

School of Medicine, Faculty of Medical Sciences, University of Duhok

Abstract. Cyclophosphamide (CP) is an alkylating chemotherapeutic agent widely used in cancer treatment but this agent is associated with toxic side effects of varying degree affecting various vital systems. The aim of this study is to evaluate the side effect of CP on liver of the female albino mice. Total 20 adult albino female mice were divided in to four groups (5 females for each group), group 1; 2 and 3were injected intraperitonealy with 75; 150 and 200 mg CP\ Kg (B.W.) respectively. While group 4 (control group) were injected with equivalent volume of normal saline (0.9% NaCl). The results showed that treatment with CP caused many histopathological changes in the liver tissue including hyperplasia, dysplasia, dilated sinusoid containing highly atypical cell (dysplastic cells), accumulation of edematous fluid and fibrinous materials in liver tissue associated with acute inflammatory cells (neutrophils) infiltration and hyperchromatic Kupffer cells. The severity of these changes was affected by the concentration of CP, that’s mean the degree of this harmful effect was elevated with increasing of the concentrations of CP. In conclusion CP caused series of histopathological changes in hepatocytes. However the severity of damages is influenced by the concentration of CP.

Keywords: Cyclophosphamide, Chemotherapeutic agent, Liver, Mice, Histopathology.

1. Introduction Over the last few decades many novel cytotoxic chemotherapeutic agents have been developed which prolong survival of patients with advanced and metastatic tumors [1]. So, chemotherapy of cancer has opened new possibilities and chances for improving the quality and life span. Despite this successful trend, treatment with some of the most effective anticancer drugs caused many of toxic symptoms to normal cells [2]. But all major anticancer drugs such as Cyclophosphamide (CP), doxorubicin, cisplatin, bleomycin etc. are associated with toxic side effects of varying degree affecting various vital systems [3,4,5] Cyclophosphamide is a mustard alkylating chemotherapeutic agent that is commonly used as an anticancer and an immunosuppressant drug for organ transplantation, leukemia, Hodgkin's disease-------etc [2,6,7].CP frequently causes hematopoetic depression, nausea, vomiting, alopecia, hemorrhagic cystitis, water retention, cardiac damage, gonadotrophy and carcinogenicity [8,9].The CP affects also the liver and kidneys, promoting lesions, sometimes irreversible. The renal toxicity is related to nephrotoxic and\ or vesicle toxic effect of the drug [10]. Therefore this study was aimed to evaluate the harmful effect of different concentrations of the CP on the female albino mice liver.

2. Materials and Methods 2.1. Drug (Cyclophosphamide) Cyclophosphamide (Eczacibasi\Baxter Hall-Almanya Baxter Oncology Gmbh) was obtained mainly from Azadi Hospital in Duhok, Iraq and injected intraperitoneal (i.p.) as a single dose.

--------------------------------------------------+ Corresponding author: Tel: +9647700521517 E-mail adress: [email protected]

21

2.2. Experimental animals Twenty adult female albino mice (Mus musculus) (8-10 weeks old with average weight 25-28 gm) of strain Balb/C were obtained from the Animal Breeding House, Faculty of Veterinary, University of Duhok. They were given standard diet as pellets and water ad libitum. These animals were divided into four groups (5 females for each group), group 1; 2 and 3 were injected with 75, 150 and 200 mg CP\ Kg (B.W.) respectively. While group 4 (control group) the females of this group were injected with equivalent volume of normal saline (0.9% NaCl).

2.2. Dissection and histological preparation All animals were dissected after seven days following injection. The livers were removed and cut into smaller pieces and fixed in 10% formalin for 48 hr. After fixation the samples of liver were processed for paraffin wax embedding by dehydrating through ascending concentration of ethanol alcohol and cleared in xylol, infiltrated and embedded in paraffin wax. Sections were cut at 6μm thick with rotary microtome then stained by Harris hematoxylin and eosin (H and E) for histological and histopathological studies and photographing [11].

3. Results 3.1. The effects of CP on the liver weight No significant effect of CP was observed on liver weight (P≤0.351) compared with the control group (table 1). Table (1). The effect of CP on the liver weight.

Groups

N

Mean

Std. Deviation

Std. Error

Controls

5

1.67060

0.230543

0.103102

75 mg CP/kg (B.W) 150 mg CP/kg (B.W)

5 5

1.88180 1.80760

0.132196 0.306380

0.059120 0.137017

200 mg CP/kg (B.W)

5

1.65000

0.213073

0.095289

P-value

0.351 (NS)

NS= none significant according to ANOVA test.

3.2. Histological studies of the liver The liver of control group revealed normal appearance with central vein and surrounding hepatocytes with normal cytoplasm and nuclei, very few lipid droplets, binucleated hepatocytes, and blood sinusoid lumen lined with normal number and structure of Kupffer cells (Fig. 1 A and B). While treatment with CP caused damage to liver tissues but the severity of damages was affected by the concentration of CP, so the degree of this damage was elevated with increasing of the concentrations of CP. Generally the effects of the different concentrations of CP (75; 150 and 200 mg CP\ Kg B.W.) on the liver tissues are presented in (Figure 2, 3, and 4) respectively. From these figures it's appeared that CP caused series of cytological

changes in hepatocytes (Hyperplasia -----Dysplasia------malignant cells).

4. Discussion Cyclophosphamide is one of the most widely used alkylating antineoplastic agents that damage normal cells while killing cancerous cells in vivo [12]. So, the main problem posed by these anticancer drugs is that they target not only the tumor, but also other cells, thus causing the same damage to both abnormal and normal cells [13]. The results of the present study showed that treatment with CP caused many histopathological changes in the liver including hyperplasia, dysplasia, dilated sinusoid containing highly atypical cell (dysplastic cells), accumulation of edematous fluid and fibrinous materials in liver tissue 22

associated with acute inflammatory cells (neutrophils) infiltration and hyperchromatic Kupffer cells. The severity of these changes was affected by the concentration of CP, that’s mean the degree of this harmful effect was elevated with increasing of the concentrations of CP. This result in agreement with the result of the many authors such as DeLeve [14] who reported that CP is a widely prescribed non-cell-cycle specific antineoplastic drug which is known to cause toxic effects including hepatotoxicity. Zorzi et al., [15] their results indicated that chemotherapy induces various changes of the liver parenchyma including steatosis, sinusoidal injury obstruction syndrome. El-Sayyad et al., [16] showed that the chemotherapeutic agents such as cisplatin, doxorubicin and 5-FU caused direct hepatic toxicity and they showed that treatment with these chemotherapeutic agents caused many histopathological and ultrstructural abnormalities in the liver including dissolution of hepatic cords, focal inflammation, hyperplasia, dilated blood sinusoids and necrotic tissues. In 2013 [17], also reported that treated with CP (0.2 ml/100g b.wt/animal/week) for 7 and 35 days altered hepatic and renal GOT, GPT, ACP and ALP levels in Rattus norvegicus. The liver is known to accumulate significant amounts of chemotherapeutic agents and there is pharmacologic evidence that the breakdown of CP into biologically active alkylating compounds takes place principally in the liver [18] but may also occur in other tissues as well [19]. In the parent form CP is inactive and it has to be metabolized to active forms before this drug can exert its cytotoxic effects. CP altered liver function by modulating all liver enzymes [19]. Enzymes are biological molecules that catalyze (i.e., increase the rates of) chemical reactions [20], and the enzymes are specific proteins that increase the rate of chemical reactions in human body. Generally enzymatic breakdown of CP by cytochrome P450 (the cytochrome p450 family is a major subset of all drug-metabolizing enzymes) results in various products with cytotoxic activity. Most important of these are phosphoramide mustard, and acrolein. Phosphoramide mustard is responsible for the anti-tumor effects of CP, while acrolein is responsible for bladder toxicity and is also responsible for causing hemorrhagic cystitis that is often seen with high dose CP treatment [21]. Phosphoramide mustard interacts with DNA producing DNA cross-links through linkage of highly reactive alkyl groups. Crosslink formation makes the DNA dysfunctional by preventing replication and modulation of cell cycle. The cell harboring this damaged DNA in turn will undergo apoptosis and die [22]. In conclusion CP caused series of histopathological changes in hepatocytes. However the severity of damages is influenced by the concentration of CP.

Figure 1: Sections through the liver of control group showing normal appearance of the liver structure. A: the central vein (arrow). B: hepatocytes (arrows), and sinusoid (double head arrow). (A: 80 X) (B: 200 X).

23

Figure (2): Sections through the liver of CP treated female mice (75 mg\Kg B.W. one week. A and B: showing that the parenchyma consist medium sized blood vessels (arrow) and inflammatory cells infiltration (double head arrow). C: shows the accumulation of edematous fluid and fibrinous materials in liver tissue (arrow) associated with acute inflammatory cells (neutrophils) infiltration (double head arrow). D: shows acute inflammatory cells infiltration (arrow) and dilated sinusoid (double head arrow). E: shows increased mitotic activity (double head arrow), hyperchromatic Kupffer cells (arrow). F: Hyperchromatic Kupffer cells (double head arrow) and inflammatory lymphocytes (arrow). G: shows large sized hepatocytes (hyperplastic) hyperchromatic cells (hepatocytoplasia) (arrows). H and I: Foci of inflammatory lymphocytes (arrow). (A,B,D and H: 80 X) (C,E,F,G and I: 200 X).

Figure (3): Sections through the liver of CP treated female mice (150 mg\Kg B.W. one week). A: Shows congested blood vessels (arrow). B: Shows dilated sinusoid (arrow) plus collection of inflammatory cells (double head arrow). C: Shows liver with collection of hugely atypical cells mostly composed of bared nuclei within inflammatory background (double head arrow), plus with singly scattered dysplastic hepatocytes (arrow).D: Dilated sinusoid (arrow) containing highly atypical cell (dysplastic cells) (double head arrows). E: Dilated sinusoid (double head arrow) containing highly atypical cell (dysplastic cells) dysplasia (arrow) plus hyperchromatic cells. F and G: shows highly atypical cell (dysplastic cells) dysplasia (arrow) plus hyperchromatic cells (double head arrow). H: Show feature of hyperplasia (arrow). I: show medium sized vein plus inflammatory cells within the wall (vascularitze hyperchromatic compact hepatocytes) (arrow). (B: 80 X) (A,C,D,E,F,G,H,I: 200 X).

24

Figure (4): Sections through the liver of CP treated female mice (200 mg\Kg B.W. one week). A and B (higher magnification of Figure A): shows hugely dilated sinusoid, focally ruptured containing foamy cells (arrow). C: show aggregate of inflammatory cells (arrow). D: show the aggregation of inflammatory cells (arrow) around the dilated canalliculi (star). E and F: shows variable appearance of dysplastic hepatocytes (arrow).G: shows highly congested sinusoid (arrow) with hyperplastic hepatocytes. H: show congested blood vessel with compact hyperchromatic cells (arrow). I: show dilated sinusoids (arrows). (A: 80 X) (B, C, D, E, F, G, H and I: 200 X).

References [1] [2]

Maor, Y., and S. Malnick, 2013. Liver injury induced by anticancer chemotherapy and radiation therapy. International Journal of Hepatology. 2013. Kumar, RV., PC. Mu, V. Ravikumar, and T. Eevaki, 2007. Inhibitory effects of histone deacetylase inhibitor depsipeptide on benzo (a) pyrene and cyclophosphamide induced genotoxicity in Swiss albino mice. International Journal of Toxicology, 26: p.47-50.

[3]

Hassan, B.A., Z.B. Yusoff, M.A. Hassali, and S.B. Othman, 2011. Association and correlation of different chemotherapeutic regimens and doses with onset and severity of anemia among solid cancer patients. Asian Pacific Journal of Cancer Prevention. , 12: p. 2753-2738.

[4]

Christie, L.A., M.M. Acharya, V.K. Parihar, A. Nguyen, V. Martirosian, and C.L. Limoli, 2012. Impaired cognitive function and hippocampal neurogenesis following cancer chemotherapy. Clinical Cancer Research, 18: p. 1954-1965.

[5]

Pabla, N., and Z. Dong, 2012. Curtailing side effects in chemotherapy: a tale of PKCd in cisplatin treatment. Oncotarget, 3: p. 1055-1074.

[6]

Hosseini, A., S. Zare, F.GH. Pakdel, and A. Ahmadi, 2010. Effects of vitamin E and ginseng extract on fertility changes induced by Cyclophosphamide in rats. Journal of Reproduction and Infertility, 11(4): p. 227-237. Schmidt, M., and H. Koelbl, 2012. Adjuvant chemotherapy in early breast cancer.Minerva Ginecology, 64: p.5365. Bhatia, K., F. Ahamd, H. Rashid, and S.Raisuddin, 2008. Protective effect of S-allyl cysteine against cyclophosphamide-induced bladder hemorrhagic cystitis in mice. Food and Chemical Toxicology., 46: p. 33683374.

[7] [8]

[9]

[10]

Panahi, Y., A. Saadat, A. Sahebkar, F. Hashemian, M. Taghikhani, E. Abolhasani, 2012. Effect of ginger on acute and delayed chemotherapy-induced nausea and vomiting: a pilot, randomized, open-label clinical trial. Integrative Cancer Therapies, 11(3): p. 204-211. Kanno, T.Y. N., L. A. Sensiate, N. A. de Paula, and M. J.S. Salles M, 2009. Toxic effects of different doses of cyclophosphamide on the reproductive parameters of male mice. Brazilian Journal of Pharmaceutical Sciences, 45(2): p. 2013-3019.

25

[11].

Bancroft, J. 1975. Histochemical techniques 2nd edition. Butter worths. London and Boston.

[12]

Chakraborty, P., Sk. U. Hossain, N. Murmu, J.K. Das, S. Pal, and S. Bhattacharya, 2009. Modulation of cyclophosphamide-induced cellular toxicity by diphenylmethyl selenocyanate in vivo, an enzymatic study. Journal of Cancer Molecules, 4(6): p.183-189.

[13]

Granados-Principal, S.; Quiles, J. L.; Ramirez-Tortosa, C. L.; Sanchez-Rovira, P. and Ramirez-Tortosa, M. C. 2010. New advances in molecular mechanisms and the prevention of Adriamycin toxicity by antioxidant nutrients. Food and Chemical Toxicology, 48: p. 1425-1438.

[14]

DeLeve, LD. 1996. Cellular target of cyclophosphamide toxicity in the murine liver: Role of glutathione and site of metabolic activation. Hepatology, 24: p. 830-837.

[15]

Zorzi, D., A. Laurent, T.M. Pawlik, G.Y. Lauwers, J-N.Vauthey, and E.K. Abdalla, 2007. Chemotherapyassociated hepatotoxicity and surgery for colorectal liver metastases. British Journal of Surgery, 94(3): p.274-286.

[16]

[17]

[18]

El-Sayyad, H., M.F. Ismail, F.M. Shalaby, RF. Abou- El-Magd, R. L.Gaur, A. Fernando, M. HG.Raj, and A. Ouhtit, 2009. Histopathological effects of cisplatin, doxorubicin and 5-flurouracil (5-FU) on the liver of male albino rats. International Journal of Biological Science, 5(5): p. 466-473. Chouhan, Sh., N. khan, R. Chauhan, A.K. Raghuwanshi,and V.K. Shrivastava, 2013. Cyclophosphamide induced changes in certain enzymological (GOT, GPT, ACP, and ALP) parameters of adult male Rattus norvegicus. International Journal of Research and Reviews in Pharmacy and Applied Science, 3(1): p. 155-163. Brock, N. and HJ. Hohorst, 1967. Metabolism of cyclophosphamide. Cancer, 20: p.900- 904.

[19]

Abraham, P., K. Indirani, and E. Sugumar, 2007. Effect of cyclophosphamide treatment on selected lysosomal enzymes in the kidney of rats. Experimental and Toxicologic Pathology. 59: p.143–149.

[20]

Grisham Charles, M., and H G. Reginald, 1999. Biochemistry. Philadelphia: Saunders College. Pub pp 426– 427.

[21]

Balu, N., MP. Gamcsik, ME. Colvin, et al. 2002. Modified guanines representing O6-alkylation by the cyclophosphamide metabolites acrolein and chloroacetaldehyde: synthesis, stability, and ab initio studies. Chemical Research in Toxicology, 15: p. 380–387. [22] Zhang, J., Q. Tian, S. Yung, S. Chuen, S. Zhou, W. Duan, and Y. Zhu , 2005. Metabolism and transport of oxazaphosphorines and the clinical implications. Drug Metabolism Reviews, 37: p. 611-703.

26

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

An Evaluation of Resistance to Fusarium Disease in Korean Sesame (Sesamum indicum L.) Germplasm Ramalingam Radhakrishnan1,2, Sang-Mo Kang1, Jae-Man Park1, Soek-Min Lee1, InYoul Baek2 and In-Jung Lee1+ 1

School of Applied Biosciences, Kyungpook National University, Daegu, 702-701, Republic of Korea 2 Legume and Oil Crop Research Division, Department of Functional Crop, National Institute of Crop Science, RDA, Miryang, 672-803, Republic of Korea.

Abstract. Forty one sesame germplasm varieties obtained from National Institute of Crop Science, Korea were screened under greenhouse conditions to identify disease reaction to the pathogen Fusarium. All the sesame varieties were severely infected by Fusarium sp. 40240 and none could be described as immune. Among forty one sesame varieties, Dasak, Namda, Jinki and Mihuek showed a higher rate of germination (14-16 %) and Hansum, Poongnam, Pongan, Nambaek and Milsung expressed a lower rate of germination (less than 2 %) at Fusarium disease infection condition. Yangbaek, Jinbaek, Ansan, Sunbaek, Soonheuk, Yangan and Sangbaek seeds could not germinate in the soil contaminated with Fusarium sp. 40240. The variety identified in the current study may be considered in crop improvement programmes to develop Fusarium disease resistant varieties, however further efforts need to be concentrated on identifying highly resistant genotypes with the inclusion of enormous germplasm in screening procedures.

Keywords: Fusarium, Germplasm, Screening, Sesame

1. Introduction Sesame is widely grown in tropical and subtropical countries. It is rich in nutrients such as oil (4560%), protein (18-28%), and other phytocompounds. The plant growth and yield are affected by biotic factors including pathogens and other environmental factors. The disease-causing soilborne microorganism Fusarium sp. is a major cause of reduction in sesame crop yield [1]. Fusarium sp. is recorded as a causal agent for root rot, vascular wilt, and damping-off diseases in many crop plants [2]. The soil borne disease once noticed in the field cannot be easily controlled by any means and the detection of sesame genotypes resistant to Fusarium wilt disease has been a major research goal for improving cultivation [3]. However, very little study was conducted to known the existence of reliable sources of resistance sesame germplasm against Fusarium diseases [1]. Although, previously we demonstrated the Fusarium induced damaging effects on disease susceptible sesame variety [4,5] but the screening of Korean sesame varieties to Fusarium disease resistance is not studied. The present study is aimed at screening the resistance levels of 41 sesame germplasm varieties against Fusarium disease.

-----------------------------------------------------

Corresponding author. Tel.: + 82-53-950-5708; fax: + 82-53-953-6972. E-mail address: [email protected]. 27

2. Materials and Methods 2.1. Fusarium pretreatment on soil Fusarium sp. 40240 isolate was cultured in potato dextrose broth for 3 weeks. The Fusarium culture filtrate with mycelium was mixed with sterilized baroker soil (peat moss (8-12 % (w/v)), perlite (5-8 % (w/v)), vermiculate (10-14 % (w/v)), coco-peat (65-70 % (w/v)) and zeolite (3-5 % (w/v)) with NH4+ ~150 mg Kg-1; NO3- ~250 mg Kg-1). The pots treated with sterile water served as control.

2.2. Seed sterilization and inoculation to Fusarium infected soil The 41 varieties of sesame seeds (Yangbaek, Jinbaek, Jinju, Hansum, Doobeol, Ansan, Kangbeak, Sunbaek, Suwon, Annam, Osan, Poongsan, Hwangbaek, Seodun, Namsan, Sungbun, Mangeum, Dasak, Hansan, Poongnam, Namda, Pongan, Nambaek, Naman, Kopoom, Pyoungan, Milsung, Yubaek, Yangheuk, Keonhuek, Hwaheuk, Soonheuk, Kangheuk, Jinki, Yoonhuek, Mihuek, Yangan, Dahuek, Manheuk, Chamhwang, Sangbaek) were surfaced sterilized with 0.5% sodium hypochloride solution for 5 min and rinsed in distilled water for 4 times. The surfaced sterilized seeds were sown in Fusarium infected soil (3.7x 105 CFU/ 5 g). The seeds were sown in the Fusarium uninoculated soil as served as control. The plants were grown in a greenhouse under a day/night cycle of 16/8 h and 28/20 °C and 60 % relative humidity. The number of germinated seeds was counted at 10 days after seed inoculation. The experiments were repeated at three times.

3. Results and discussion The pathogenic soil microorganisms reduce seed germination, plant growth and yield. In current study, the infection rate of Fusarium disease was observed in sesame seed germination under Fusarium contaminated soil (Fig. 1 and 2). Forty one Korean sesame varieties screened for resistance to Fusarium wilt showed some percent infection rate and none could be described as immune. Similar results were obtained in Indian sesame accessions by Jyothi et al [1].

Fig.1. Percentage of germination of sesame seeds under Fusarium treatment However, all of the varieties, Mihuek was the most resistant genotype with an average of 16 % germination rate. Dasak, Namda and Jinki were other resistant genotypes expressed 14 %, 15 % and 15 % germination rate, respectively. The Fusarium induced inhibition rate of seed germination was 100 % in Yangbaek, Jinbaek, Ansan, Sunbaek, Soonheuk, Yangan and Sangbaek varieties, which were identified as most Fusarium disease susceptible varieties. The other susceptible varieties such as Hansum, Poongnam, Pongan, Nambaek and Milsung expressed a lower rate of germination (less than 2 %) at Fusarium disease infection condition. The moderate level of disease resistance (10 % - 14 %) was noticed in Osan, Kopoom, Pyoungan, Dahuek and Chamhwang varieties. The results of present study describe the presence of sufficient genetic variation with respect to wilt within the screened germplasm and wide range of infection percent. The number of studies was concentrated to detect the perfect resistant genotypes of sesame against Fusarium wilt disease [6,7,8]. The present research is the 28

first effort to find the Fusarium disease resistant sesame germplasm varieties in Korea. Our results suggest that Mihuek is identified as resistant variety when compared to other sesame varieties.

Fig.2. Sesame (1.Yangbaek, 2.Jinbaek, 3.Jinju, 4.Hansum, 5.Doobeol, 6.Ansan, 7.Kangbeak, 8.Sunbaek, 9.Suwon, 10.Annam, 11.Osan, 12.Poongsan, 13.Hwangbaek, 14.Seodun, 15.Namsan, 16.Sungbun, 17.Mangeum, 18.Dasak, 19.Hansan, 20.Poongnam, 21.Namda, 22.Pongan, 23.Nambaek, 24.Naman, 25.Kopoom, 26.Pyoungan, 27.Milsung, 28.Yubaek, 29.Yangheuk, 30.Keonhuek, 31.Hwaheuk, 32.Soonheuk, 33.Kangheuk, 34.Jinki, 35.Yoonhuek, 36.Mihuek, 37.Yangan, 38.Dahuek, 39.Manheuk, 40.Chamhwang, 41.Sangbaek) seedlings growth on Fusarium uninfected (Upper green portion of tray) and infected soil (Lower portion of tray).

4. Conclusion The identification of disease resistant plant varieties is a major goal for agricultural scientists and plant breeders. We screened the 41 sesame varieties to find the resistance level of Fusarium infection, which showed that only four varieties, Dasak, Namda, Jinki and Mihuek may be used in Fusarium infected soil and possibly resist the disease in some extend. The current study would be helpful to develop mapping population for tagging of wilt resistance genes in sesame.

5. Acknowledgement Authors are thankful to Korea Ministry of Environment for providing financial support through an Eco-Innovation Project.

6. References [1]

Jyothi, B., N. A. Ansari, Y. Vijay, G. Anuradha, A. Sarkar, R. Sudhakar, and E. A. Siddiq, 2011. Assessment of resistance to Fusarium wilt disease in sesame (Sesamum indicum L.) germplasm. Australasian Plant Pathology, 40: p. 471-475.

[2]

Gajbhiye, A., A. R. Rai, S. U. Meshram, and A. B. Dongre, 2010. Isolation, evaluation and characterization of Bacillus subtilis from cotton rhizospheric soil with biocontrol activity against Fusarium oxysporum. World Journal of Microbiology and Biotechnology, 26: p. 1187-1194.

[3]

El-Bramawy, M. A. S., 2006. Inheritance of resistance to Fusarium wilt in some crosses under field conditions. Plant Protection Science, 42: p. 99-105.

[4]

Radhakrishnan, R., S.B. Pae, K.B. Shim, and I.Y. Baek, 2013. Penicillium sp. mitigates Fusariuminduced biotic stress in sesame plants. Biotechnology Letters, 35(7) : p. 1073-1078.

[5]

Radhakrishnan, R., K.B. Shim, B.W. Lee, C.D. Hwang, S.B. Pae, C.H. Park, S.U. Kim, C.K. Lee, and I.Y. Baek, 2013. IAA producing Penicillium sp. NICS01 triggers plant growth and suppresses fusarium induced oxidative stress in sesame (Sesamum indicum L.). Journal of Microbiology and Biotechnology, 23(6): p. 856-863.

29

[6]

Dinakaran, D., V. Manoharan, and V. Dharmalingam, 1994. Screening of sesame cultures against major diseases. Sesame and Safflower Newsletter, 9: p. 4-6.

[7]

EL-Shazly, M.S., M.A. El-Ashry, S.M. Ammar, and M.A.S. El-Bramawy, 1999. Evaluation of resistance to Fusarium wilt disease in sesame germplasm. International Journal of Pest Management, 45(3): p. 207-210.

[8]

El-Bramawy, M.A.S., S.E. El-Hendawy, and W.I.A. Shaban, 2008. Assessing the suitability of morphological and phenological traits to screen sesame genotypes for Fusarium wilt and charcoal rot disease resistance. Journal of Plant Protection Research, 48(4): p. 397-410.

30

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Studies on the Bacteriological Quality of Water, Sediment and Fish Samples of Poovar Estuary, South West Coast of India Sekharapilla Premjith 1 1

Department of Zoology, St. Jude’s College, M.S. University, Thoothoor ,Kanyakumari , Tamilnadu, South India. e-mail: [email protected]

Abstract. Estuaries provide an array of natural resource entitlements to rural communities. Poovar estuary is a bar built estuary and its proximity to Kovalan an International tourist destination, makes it one of the study area both for biological and hydrographical studies. The present study on the seasonal variations of bacteriological contaminants showed that maximum bacterial count was during pre monsoon season and minimum during monsoon season. The important bacterial genera encountered were Escherichia coli, Salmonella and Shigella. Among the identified bacterial genera, Escherichia coli were found to be the dominant one. Next in abundance, were Salmonella and Shigella. The bacterial contamination of sediment samples was found to be higher than that in the water samples of Poovar estuary. Fishes also found to be with higher bacterial contamination in their intestine than in their gills and skin.

Key words: Estuary, water, sediments, fish, bacteria

1. Introduction Thiruvananathapuram district in Kerala state of India is predominantly an agricultural land. The river Neyyar that originates in the Western Ghats at an altitude of 1500 m above Mean Sea Level (MSL) is one of the largest rivers and mixes with Arabian Sea, forming an estuary at Poovar. The estuarine environment of Poovar estuary is subjected to pollution through sewage disposal, waste water discharge from tourist resorts and retting activity at Anantha Victoria Marthadan (AVM) canal. The previous studies showed that the residents of Poovar area have the scarcity of good drinking water and sea foods. They are suffering from health hazards due to the consumption of polluted well water and fishes. The main aim of this study is to suggest the management measures to reduce the pollution load of estuarine environment of Poovar estuary.

2. Description of the study area The present study was carried out in the Poovar bar built estuary formed by the confluence of river Neyyar.ry adjacent to the beach resorts of Poovar – Vizhinjam area in Thiruvananthpuram District, Kerala State which has an area of 30.93 hectare and is situated between N. Lat. 8o 18’ 32” to 8o 18’ 6” and E. Long. 77o 4’ 32” to 77o 5’ 14” (Fig. 1). The estuarine environment of Poovar estuary is subjected to pollution through different anthropogenic activities like sewage disposal, coir retting and beach resort effluents.

3. Materials and methods The present study was carried out over a period of twelve months from October 2011 to September 2012, covering pre-monsoon (February – May), the monsoon (June – September) and post monsoon (October – January).Three stations were selected for the present study viz Station I: Poovar Bar mouth (Pozhimugam) Plate 1, Station II: Poovar-Bridge (Plate 11) and Station III: Mavalakadavu (Plate 111) A country boat was used to collect the both surface and bottom water and sediment samples. The collection of samples was done between 6 a.m. to 8 a.m. Water samples were collected separately in sterile glass bottles and the sediment samples were collected in sterile polythene bags and brought to the laboratory in ice box for bacteriological analysis. Fishes Mugil cephalus

31

were collected by cast net. Skin, gill and intestine of fish samples were refrigerated for microbiological analysis. The isolates were maintained on seawater nutrient agar slants and identified up to generic level using the method adopted by Surendran and Gopakumar, (1981); Oliver, (1982).

4. Results and discussion 4.1 Variations of Total variable count (TVC) in Surface water samples Table 1 provided the data for the monthly and seasonal variation of TVC in surface and bottom water samples. Station

Post monsoon Samples

Pre monsoon

Oct

Nov

Dec

Jan

Surface water

30.26

32.10

30.40

31.60

Bottom water

27.20

30.40

28.10

29.13

Surface water

22.40

20.32

23.20

25.46

Bottom water

18.30

20.32

19.40

20.60

Surface water

16.60

18.20

16.40

11.20

Bottom water

16.10

19.20

15.34

9.10

I

II

III

Mean + SD 31.09 + 0.902 28.71 + 1.376 22.85 + 2.124 19.66 + 1.038 15.60 + 3.041 14.94 + 4.233

Monsoon

Feb

Mar

Apr

May

34.20

29.30

35.60

31.43

32.60

23.41

26.03

31.76

27.40

24.60

23.20

26.00

23.14

19.90

20.70

25.43

21.70

14.60

18.80

21.66

20.50

12.10

18.20

20.60

Mean + SD 32.63 + 2.817 28.45 + 4.451 25.30 + 1.807 22.29 + 2.504 19.19 + 3.347 17.85 + 3.99

Jun

Jul

Aug

Sep

22.00

28.16

27.83

24.66

17.46

26.33

22.26

22.00

17.30

19.10

21.66

16.37

13.40

17.67

18.00

12.36

11.53

13.43

12.46

10.16

10.20

11.80

12.40

10.10

Mean + SD 25.66 + 2.907 22.01 + 3.625 18.61 + 2.329 15.36 + 2.895 11.90 + 1.392 11.13 + 1.152

The monthly bacterial counts ranged from 10.16 to 35.60 x 105 CFU/ml and maximum bacterial count was recorded during April at station I and minimum value was recorded during September at station III. Seasonal variations in the pooled data showed the maximum bacterial counts of 32.63 x 10 5 CFU/ml recorded during pre monsoon season at station I and minimum of 11.90 x 105 CFU/ml recorded during monsoon season at station III.

4.2 Variations of TVC in Bottom water samples The maximum value was observed during February at station I (32.60 x 105 CFU/ml) and minimum value was observed during January at station III (9.10 x 105 CFU/ml). The maximum bacterial counts of 28.71 x 105 CFU/ml recorded during post monsoon season at station I and the minimum of 11.13 x 105 CFU/ml recorded during monsoon season at station III.

4.3 Variations of bacterial strains in Surface water Data on monthly and seasonal variation of bacterial stains in surface and bottom water samples is presented in Table 2. Post monsoon Station

Samples

Pre monsoon

Monsoon

Oct

Nov

Dec

Jan

Mean + SD

Feb

Mar

Apr

May

Surface water

97

103

95

91

96.50 + 5.000

104

116

125

114

Bottom water

81

92

83

96

88.00 + 7.164

110

103

112

106

66

74

66

86

70

77

74

88

71

68

58

74

63

72

81

90

60

63

51

55

67

50

64

49

67

58

25

47

64

59

56

61

I

II

III

Surface water Bottom water Surface water Bottom water

73.00 + 9.451 67.75 + 6.946 57.25 + 5.315 49.25 + 18.117

32

Mean + SD 114.75 + 8.616 107.75 + 4.031 77.25 + 7.719 76.50 + 11.619 57.50 + 9.327 60.00 + 3.366

Jun

Jul

Aug

Sep

Mean + SD

109

86

98

84

94.25 + 11.615

97

91

93

71

88.00 + 11.604

71

60

55

62

65

51

69

53

48

40

46

38

41

32

55

30

62.00 + 6.683 59.50 + 8.850 43.00 + 4.761 39.50 + 11.387

Among the total bacterial strains, a maximum of 125 CFU/ml strains were observed in April at station I and a minimum of 38 CFU/ml strains were observed in September at station III. The bacterial strain was maximum during pre monsoon season at station-I (114.75 CFU/ml) and minimum during monsoon season at station-III (43.00 CFU/ml). 4.4 Variations of bacterial strains in Bottom water The highest bacterial strain was detected during April at station I (112 CFU/ml) and lowest was detected during December at station III (25 CFU/ml). The present study reveals that the total bacterial strain was found to be highest in pre monsoon season at station I (107.75 CFU/ml) and lowest in monsoon season at station III (39.50 CFU/ml). Microorganisms like bacteria were widely distributed in the water, sediment and fish samples and they are playing a vital role in aquatic ecosystem. The interactions of the microorganisms with the aquatic biota are unique and diverse. Sediment and water are excellent culture media for the growth of many types of microorganisms. The majority of the bacterial population was found in surface level and their number decreases with depth. In the present study, the data on the seasonal variations showed maximum bacterial count during pre monsoon season and minimum during monsoon season. The same result was observed by Prema (2000) in Rajakkamangalam estuary. The bacterial counts recorded in the present study were of similar magnitudes as given by Zobell (1948) in southern California coast, Civic (1955) in the Adriatic Sea and Kriss (1961) in the Black sea. High bacterial count was recorded during summer by Brown (1964) in the waters of Sydney. Chandrika and Nair (1994) reported maximum counts in the Cochin water during pre-monsoon and post monsoon. Iriberri et al. (1987) noticed higher counts during hot season and lower counts during the cold season. The microorganisms living in particular water and favourable conditions multiply quickly in summer season (Reheinheimer, 1965).

4.5 Variations of TVC in Sediment samples The monthly and seasonal variation of TVC in sediment samples are presented in Table 3. Post monsoon Station

Oct

Nov

Dec

Jan

I

34.27

36.07

33.14

30.46

II

31.96

30.63

32.94

28.35

III

23.46

26.32

24.62

20.27

Pre monsoon Mean + SD 33.49 + 2.350 30.97 + 1.986 23.67 + 2.551

Feb

Mar

Apr

May

36.17

35.68

31.72

38.46

33.63

30.91

28.87

34.08

27.45

23.87

20.94

28.44

33

Monsoon Mean + SD 35.51 + 2.800 31.87 + 2.442 25.18 + 3.438

Jun

Jul

Aug

Sep

37.19

29.33

26.62

28.27

32.36

28.70

24.45

26.16

22.39

20.62

18.87

21.70

Mean + SD 30.35 + 4.692 27.92 + 3.438 20.90 + 1.534

The value ranged between 18.87 x 105 CFU/g and 38.46 x 105 CFU/g. The maximum value was observed in May at station I and minimum value in August at station III. Seasonal variations showed the maximum bacterial counts of 35.51 x 105 CFU/g during monsoon season at station I and the minimum of 20.90 x 105 CFU/g during monsoon season at station III.

4.6 Variations of bacterial strains in Sediment sample The data on bacterial strains in sediment samples recorded at the experimental zones of Poovar estuary is presented in Table 4. Station

Post monsoon Oct

Nov

Dec

Jan

I

110

117

124

115

II

75

84

78

96

III

67

74

61

Pre monsoon Mean + SD 116.50 + 5.802 83.25 + 9.287 66.75 +

65

5.439

Feb

Mar

Apr

103

120

135

128

84

92

96

113

78

67

Monsoon

May

74

Mean + SD 121.50 + 13.772 96.25 + 12.230

61

70.00 + 7.527

Jun

Jul

Aug

Sep

97

114

106

95

84

76

67

71

59

42

58

Mean + SD 103.00 + 8.756 74.50 + 7.325 55.25 +

62

8.995

A maximum of 135 CFU/g strains were isolated and identified at station I in April and a minimum of 42 CFU/g bacterial strains were identified at station III in July. The highest value of sediment bacterial strain was observed from the study area was 121.50 CFU/g during pre monsoon season at station I and lowest value observed was 52.55 CFU/g during monsoon season at station III. The total viable heterotrophic bacterial content in an area depends on the availability of growth supporting organic matter and micronutrients. Due to the abundance of micronutrients and organic matter, high counts of total heterotrophic bacteria were detected in sediment samples. High correlation between bacterial counts and temperature supported the fact that the distribution of nutrients and temperature played a dominant role in the distribution of heterotrophic bacteria. A positive correlation between temperature and heterotrophic bacterial distribution was found by (Nedwell and Floodgate, 1971) in intertidial sediment. In the present study also, there was positive correlation between temperature and heterotrophic bacterial population. In Poovar estuary, high bacterial count was observed during pre-monsoon season at station I. The total heterotrophic bacterial density was found to be higher in the sediment than that of water. Total heterotrophic bacterial population increased probably due to the continuous availability of substrate, nutrients as unconsumed feed, plankton and other organic and inorganic matters in the bottom of the water. In Poovar estuary, the data on the seasonal variations showed maximum bacterial count during pre monsoon season and minimum during monsoon season. Anand et al., (1996) noticed an increasing trend regarding the total heterotrophic bacterial content in estuarine. The change in sediment micro flora depends upon the environment and quality of water. The total heterotrophic bacterial count observed was in the range from 1.82 x 10 6 to 4.72 x 106 CFU/g (Sharmila et al., 1996) and from 9.30 x 105 to 31 x 107 CFU/g (Anand et al., 1996). Simillar range (105 CFU/g) was obtained in the present study also.

4.7 Variations of TVC in Fish (Mugil cephalus) The result on total viable count in fish samples of Poovar estuary ranged from 18.40 x 10 5 CFU/g to 34.10 x 105 CFU/g. The maximum value was noted during April at station I and minimum value was detected during September at station III. (Table 5). Post monsoon Station

Oct

Nov

Dec

Jan

I

28.13

30.26

26.20

23.40

II

25.40

23.72

28.40

21.34

Pre monsoon Mean + SD 27.00 + 2.915 24.72 +

Feb

Mar

Apr

May

27.60

29.20

34.10

31.40

29.78

20.54

28.44

30.07

34

Monsoon Mean + SD 30.58 + 2.819 27.21 +

Jun

Jul

Aug

Sep

25.64

27.45

21.17

20.60

20.67

24.91

19.47

21.32

Mean + SD 23.72 + 3.358 21.59 +

III

20.31

24.45

22.74

23.10

2.968

4.501

2.340

22.65 +

25.66 +

20.92 +

1.724

21.71

24.60

29.80

26.54

3.398

19.62

26.04

19.60

18.40

3.464

The average bacterial counts recorded with the maximum of 30.58 x 105 CFU/g during premonsoon season at station I and a minimum of 20.92 x 105 CFU/g during monsoon season at station III.

4.8 Variations of bacterial strains in Fish (Mugil Cephalus) A maximum of 120 CFU/g strains were isolated and identified in the fish samples during the month April at station I and a minimum of 33 CFU/g strains were isolated during the month of December at station III. The total bacterial strains of fish samples recorded with maximum of 107 CFU/g in pre monsoon season at station I and recorded minimum of 43.50 CFU/g in monsoon season at station III.The eutrophication status of the estuary were related to microbial activities. The estuarine waters uptake the inorganic nutrients and developed pico planktonic microbial communities. So the inorganic nutrient concentrations were low but microbial activities were high. The higher bacterial load of fish may due to its bottom dwelling behavior. The viable bacterial counts in pond fishes are higher than those reported from the fishes collected from the sea. Gramnegative bacteria are predominant in the marine environment, usually constituting the majority in the normal micro flora of wild Prawns. Fluctuations of temperature, salinity, pH, and dissolved oxygen, high levels of ammonia, nitrate, nitrite, organics, heavy metals, pesticides and nutritional factors induce stress in fishes. Stress either lowers the resistance of fish or enhances the population and pathogen city of the pathogen. The bacterial population in sediment was found to be higher than in water. In the fish samples total heterotrophic bacterial count was higher than the water samples. The distribution of heterotrophic bacteria studied in water, sediment and fish samples were in the order water > fish > sediment.

5. Summary and conclusion In the present study, the data obtained on the seasonal variations of bacteriological contaminants showed that maximum bacterial count was during pre-monsoon season and minimum during monsoon season. The important bacterial genera encountered were Escherichia coli, Salmonella and Shigella. Among the identified bacterial genera, Escherichia coli were found to be the dominant one. Next in abundance, were Salmonella and Shigella. The bacterial contamination of sediment samples was found to be higher than that in the water samples of Poovar estuary. Fishes also found to be with higher bacterial contamination in their intestine than in their gills and skin. The data obtained from this study could therefore serve as base line information for future environmental impact assessment of the effluents discharged from tourist resorts in this region. Man requires a clean ecologically well balanced environment to promote healthy living. Society produces a vast array of waste products and the water provides an effective means of disposing many of these. It is the responsibility of the society to ensure minimum damage to the water resources.

References [1] Anand,T.P. Pattersen Edwaed, J.K. and Ayyakkannu,K. 1966. Monotoring of a shrimp culture system with special reference to Vibrio and fungi. Indian J. Mar. Sci. ,25, 253-258. [2] Brown,A.D. 1964 Sesonal variations in heterotrophic bacterial population in water off Sudney. Aust.J. Mar. Fresh wat. Res. 15 (10) : 73 -76. [3] Chandrika,V and Nair. P.V.R. 1994. Seasonal variations of aerobic , heterotriphicbacteria in cochin waters. J. Mar. Biol,Ass. India.36 (1&2):81 -95. [4] Civic,V., 1955. Distribution of bacteria in the waters of the mid Adriatic sea. Ref.M.V. Hyar Cruises , SplitYogoslavia, 4 (1).

35

[5] Iriberri,J. Marian Unanue, Isabel Barcia and Luis Egea ,1987. Seasonal variations in population density and hetero trophic activity of attached and free living bacteria in coastal waters. Appl. Environ. Microbial. 53: 2308-2314. [6]

Kriss , A.F. 1961. Marine Microbiology .Deep sea. Oliver and Boy Ltd. Edinburg.

[7] Nedwell and Floodgate, 1971. Seasonal selection in temperature of hetertrophic bacteria in intertidal sediment. Mar.Biol.. 11: 306- 310. [8] Oliver, J.D. 1982.Instruments and methods, taxonomic scheme for the identification of marine bacteria. Deep sea .res.29:795- 798. [9] Prema,P. 2000 .Studies in the microbial ecology of Rajakkamangalam estuary. Ph.D Thesis. Manonmanium Sundaranar university,214 pp. [10] Reheinheimer,G. 1965. Mikro biologische Unter Scuchungen in der. Elbe. Zwischen Schnaken burg and Cuxhaven. Arch. Hydrobiol., 29 suppl. Elbe.Awstuar.2: 181-251. [11] Sharmila,R.Jawahar Abraham,T and Sundara Raj, V. , 1996. Bacterial flora of semi intensive pond reared penaeus indicus J. Aqua. Trop. 11:193-203. [12] Surendran and Gopakumar, P.K. and Gopakumar , K. 1981, Selection of bacterial flora in the chlorotetracycline treated oil sardine ( sardinella longiceps)Indian mackerel (Rastrelliger kanagurta) and prawn (Metapenaeus dobsoni) during ice storage. Fish.Technol.,18: 133-140. [13]

ZoBell, C.E. 1948.Sulfate reducing bacteria in marine sediments. J. Mar.Res..,7:602 – 617.

36

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Selection of Mutant Lines of Maize for Yield and Early Maturity Based on Line x Tester Analysis Jajang Supriatna1 and Dedi Ruswandi2,* 1

2

Maize Development Cluster, University of Padjadjaran Lab. Of Plant Breeding, University of Padjadjaran, Bandung, Indonesia Email address: [email protected]

Abstract. Line x tester analysis is one of the important methods in the hybrid formation. It can estimate the value of combining ability and heterosis which is required to determine the best combination of crosses between parental lines. The objectives of this research were to obtain the best mutant pure line(s) that will be used as hybrid parent based on yield component and early maturity. The experiment was conducted at Arjasari, Indonesia. The experiments were conducted in two continuous steps. The first step, from March to July 2012, was conducted to form line x tester hybrids. The second step, from February to May 2013, was to evaluate mutant inbred x tester hybrids. The results showed that 16 potential mutant maize inbreds can be used as hybrid parents based on the value of GCA. Two pair crosses can be used as potential cross pair based on the value of SCA. 37 hybrids exceeded the value of parents based on high parent heterosis value. These mutant lines could be considered for breeding program for high yield and early maturity.

Keywords: maize, mutant lines, line x tester, yield components, early maturity,

1. Introduction Pure-line hybrid is a biological concept which has greatly contributed in agricultural practices. Many crops are planted as hybrids to increase yield over open-pollinated varieties, especially maize. In the formation of hybrid, pure-line plays an important role. Pure-line is the basic genetic material to make hybrids. Pure-line is genetically homozygous, a condition achieved through a series of self-pollinations [1]. The concept of the use of pure-line was proposed by Shull [2,3]. Their observation showed that when the maize plants are self-polinated, their vigor and yield decreased. Also, when two pure-lines are crossed, vigor and yield of the F1 hybrid were greater than the two parents (heterosis). This concept led to modern maize seed industries. The formation of inbred-lines need sources of genetic materials. Mutation treatment is one method that can potentially produce new genetic sources that are useful in plant breeding programs. Mutations induce changes in genetic structure at the level of genome, chromosome, DNA, or genes, so that the result of the mutation allows to obtain new genetic sources [4]. The main advantages of the mutation breeding can improve important traits, without changing most of the genetic construction so as to maintain the good traits [5]. Plant characters improved by mutation breeding include: grain yield, sterility handicap genetic, growth habit, tolerance to environmental stress, disease and pest resistance and quality [6]. The method used to test early generation is line x tester. Line x tester method was developed by Kempthorne (7) which provides reliable information on the combining ability effects of parents and heterosis phenomenon in applied breeding programs. This method is important for plant breeders to discard undesirable materials while generating inbred lines for the production of hybrids [8]. Line x tester is a cross pattern by crossing various lines with multiple tester [9]. The use of testers aims to dissect different traits and show the diversity in the germplasm. Combining ability analysis provide information on the mechanisms of genetic inheritance of controlling quantitative traits and allow breeders to select parents that potentially produce progeny that have the character expected [9]. Two types of combining abilities are considered i.e. general combining ability (GCA) and specific combining ability (SCA). GCA value is the average performance of the genotype in a series of 37

cross combinations [10]. GCA can assume the involvement of additive gene activity in eliciting a character [11,12]. while the value of SCA is the performance of the combination of specific inbred in particular cross [10]. GCA value can expect non-additive gene activity [11,12]. Heterosis is a phenomenon in which F1 hybrid has superior performance over its parents [13]. Heterosis role to select cross combination that can produce F1 that have exceeded the performance of the parents so that yield and other traits can be improved. Genetic hypotheses are amongst the oldest but still most prevailing explanations for heterosis were dominance, overdominance and epistasis hypothesis [14]. Dominance hypothesis assumes heterosis obtained by interaction of dominant genes that contribute to enhance the trait, overdominance hypothesis assumes heterosis obtained by overdominant gene action at many loci and epistasis hypothesis assumes heterosis obtained by epistatic interactions between non-allelic genes. The objectives of the experiment were to estimate the combining ability and heterosis of new mutant (M5DR) and to select new superior hybrid combination(s). Information from this research will be useful to determine the lines that potentially serve as parent in the formation of hybrid cultivars that have high yield and early maturity characters that can be released to the maize farmers.

2. Materials and Methods Genetic Material Forty-six mutant lines (M5DR) and three tester (DR4, DR6 and DR 8) lines were developed at Plant Breeding Laboratory of the Faculty of Agriculture, Universitas Padjadjaran, Indonesia. Mutant lines are generated from DR lines that were mutated by using gamma rays at a dose of 200 Gy.These mutated seeds of DR lines were then selfed and selected based on pedigree method for five generations to generate M5DR lines. DR lines developed from crosses Downy Mildew Resistant (DMR) Maize and Quality Protein Maize (QPM). To form line x tester hybrid, 46 M5DR were used as female parental and 3 testers (DR4, DR6 and DR8) as male parental. Crosses produced 138 hybrid combinations that were evaluated in the second season. Experimental Design The experiment was conducted at Arjasari, Indonesia. The experiments consisted of two steps. The first step, from March to July 2012, was intended to form mutant line x tester hybrids. The second step, from February to May 2013, was to evaluate mutant inbred x tester hybrids. The experimental design used in the evaluation of hybrids was a randomized complete block with 187 treatments (138 F1, 46 M5DR and three testers) and two replications. Each treatment was a single row with 15 planting holes. The characters observed in the research were days to anthesis, days to harvest,1000 grain weight, and grain weight per plot. Estimation of Combining ability Estimation of General combining ability (GCA) used the formula according to Singh and Chaudary, [9]., namely:

xi ... x...  tr ltr x. j . x.. b. g t   lr ltr a. g i 

Where : gi : GCA value of lines gt : GCA value of tester x... : grand total li xt xi... :



1 : number of lines t : number of testers r : number of replications t j xl X.j. :



Estimation of Specific combining ability (SCA) used the formula according to Singh and Chaudary, [9]. namely:

S ij 

xij .. r



xi.. x. j . x...   tr lr ltr 38

Where : Sij : SCA value xi... :  li xt x...

: grand total

1

: number of lines

t r

: number of testers : number of replications

X.j. :  t j xl Sij : ∑ cross line i x tester j

Analysis of High-Parent Heterosis (HPH) HPH values were calculated based on parental that have the best performance. The formula according to Fehr [13], is as follows:

HPH (%) = where : HPH (%) F1 HP

: HPH value : value of F1 : value of the best parent

3. Results and Discussions Selected parental mutant lines based on Combining ability The combining ability effect varies for all lines. Combining ability of all the lines were calculated to show the performance of different lines regarding different testers. The results showed that significant GCA value was observed for early maturity and yield traits, whereas SCA was significant only for the yield traits. This indicated the importance of additive and non-additive genes to expression of early maturity, while on the yield traits are strongly influenced by the additive genes. In line with this result, additive and nonadditive gene action controlling early maturity was also reported by Estakhr et al. [15], whereas additive gene action controlling yield trait was reported by Bertoia [16]. Combining ability effects can be considered as the numerical values assigned to the parents in relation to their performance in cross-combinations. Based on the analysis of combining ability, lines were obtained that can potentially be used as a cross parent based on certain characters. The expected value of combining ability for days to anthesis and days to harvest character is negative. Negative value of combining ability on days to anthesis character showed that a line has the potential to produce progeny that have earlier maturity age. The expected value of combining ability for yield characters such 1000 grains weight and grains weight per plot is positive. Positive value of combining ability on yield characters showed that line has the potential to produce progeny that have the character higher yield components. Based on GCA value and significance test based the t-test, 16 lines were selected. The list of 5 best lines based on each character is shown in Table 1. Data indicates 11 lines selected on the days to anthesis and days to anthesis. Similarity of lines were selected on the both character revealed that there is correlation between the GCA value of days to anthesis with days to harvest. This indicated that GCA value of days to anthesis can be used as an indicator of the level of ability of line to produce progeny that have early maturity age. M5DR 18.3.1 is the best line based on the value of GCA, with an average value of 60.67 for days to anthesis and 95.33 for days to harvest. On the yield component indicates that 6 lines selected based on 1000 grains weight and 4 lines based on grains weight per plot. 4 lines selected on the Grains weight per plot including on lines were selected based on 1000 grains weight. M5DR 3.1.2 is a line that has GCA highest on 1000 grains weight with 59.50 and average performance 216.65g, whereas on grains weight per plot was M5DR 18.4.1 with GCA value 913.00 and average performance 2.65 kg. Based on SCA and significance test based the t-test, two cross pairs were selected. The list of five best cross pairs based on each character is shown in Table 2. On days to anthesis selected one cross pair, that is M5DR 16.2.1 x DR4, whereas on days to harvest selected two pair crosses, those are M5DR 16.2.1 x DR4 and DR6 M5DR x 8.6.3. On yield component no significant cross pair to SCA value. 39

Table 1. Five best lines on each character, GCA values, average values of lines and the best tester used in the cross. No.

Days to anthesis

1

Line M5DR 18.3.1 GCA -2.51** Mean 60.67 days 2 Line M5BR 153.4.1 GCA -2.45** Mean 60 days 3 Line M5DR 14.3.8 GCA -2.45** Mean 60.17 days 4 Line M5DR 14.3.1 GCA -2.45** Mean 59 days The best tester Tester DR4 GCA -1.86** Mean 64.00 days

Days to harvest

1000 grains weight

Grains weight per plot

Line M5DR 18.3.1 GCA -2.55** Mean 95.33 days Line M5BR 153.6.1 GCA -2.33** Mean 102.34 days Line M5DR 14.3.1 GCA -2.28** Mean 91 days Line M5BR 153.4.1 GCA -2.28** Mean 103.84 days

Line M5DR 3.1.2 GCA 59.50** Mean 216.65 g Line M5DR 16.1.1 GCA 45.63** Mean 223.35 Line M5DR 18.4.1 GCA 40.06* Mean 306.65 Line M5DR 4.8.8 GCA 34.51* Mean 181.65

Line M5DR 18.4.1 GCA 913.00** Mean 2.65 kg Line M5DR 4.8.8 GCA 658.00* Mean 2.36 kg Line M5DR 9.1.3 GCA 611.35* Mean 3.23 kg Line M5DR 3.1.2 GCA 561.30* Mean 2.72 kg

Tester DR4 GCA -2.18** Mean 103.34 days

Tester DR8 GCA 6.87 Mean 241.65 g

Tester DR8 GCA 170.07* Mean 2.37 kg

Notes * significant at the 0.05 probability level ** significant at the 0.01 probability level

Table 2. Five best cross pairs on each character and SCA values. No.

Days to anthesis

Days to harvest

1000 grains weight

Grains weight per plot

1

M5DR 16.2.1 x DR4 SCA -2.03** M5DR 8.5.2 x DR8 SCA -1.33 M5DR 8.6.3 x DR6 SCA -1.31 M5DR 14.3.8 x DR4 SCA -1.19 M5DR 5.5.1 x DR6 SCA -1.03

M5DR 16.2.1 x DR4 SCA -1.77** M5DR 8.6.3 x DR6 SCA -1.45* M5DR 8.5.2 x DR8 SCA -1.12 M5DR 10.2.2 x DR8 SCA -1.12 M5DR 1.2.12 x DR4 SCA -1.1

M5DR 16.1.1 x DR8 SCA 48.68 M5DR 18.3.1 x DR4 SCA 47.77 M5DR 9.4.1 x DR6 SCA 43.43 M5DR 7.4.1 x DR4 SCA 41.12 M5DR 3.6.1 x DR8 SCA 39.80

M5DR 16.2.1 x DR4 SCA 898.80 M5BR 153.6.1 x DR6 SCA 832.97 M5DR 1.6.3 x DR8 SCA 679.93 M5DR 7.4.1 x DR4 SCA 677.15 M5DR 18.3.1 x DR4 SCA 662.15

2 3 4 5

Notes: * significant at the 0.05 probability level ** significant at the 0.01 probability level

Selected Hybrid(s) based on High-Parent Heterosis (HPH) HPH value used to determine whether the progeny from crosses have characters exceed the highest parental [13]. HPH values expected for days to anthesis and days to harvest character is negative while HPH value expected to 1000 grains weight and grain weight per plot was positive. Based on HPH value and significance test based the t-test, 37 hybrids were selected. The list of five best hybrids based on each character is shown in Table 3. On days to anthesis selected 24 cross pair whereas on days to harvest selected 37 pair crosses. On yield component no significant cross pair to HPH value.

Table 3. Five best hybrids for each character based on HPH value. No. 1 2 3 4

Days to anthesis M5DR 16.2.1 x DR4 HPH -10.16%** M5DR 14.3.8 x DR4 HPH -9.63%** M5DR 18.3.1 x DR4 HPH -9.38%** M5BR 153.4.1 x DR4

Days to harvest M5DR 16.2.1 x DR4 HPH -9.92%** M5DR 14.3.8 x DR4 HPH -9.40%** M5DR 18.3.1 x DR4 HPH -9.14%** M5BR 153.4.1 x DR4 40

1000 grains weight M5DR 16.1.1 x DR8 SCA 48.68 M5DR 18.3.1 x DR4 SCA 47.77 M5DR 9.4.1 x DR6 SCA 43.43 M5DR 7.4.1 x DR4

Grains weight per plot M5DR 16.2.1 x DR4 SCA 898.80 M5BR 153.6.1 x DR6 SCA 832.97 M5DR 1.6.3 x DR8 SCA 679.93 M5DR 7.4.1 x DR4

5

HPH -9.12%** M5DR 14.3.1 x DR4 HPH -8.86%**

HPH 8.88%** M5DR 14.3.1 x DR4 HPH -8.62%**

SCA 41.12 M5DR 3.6.1 x DR8 SCA 39.80

SCA 677.15 M5DR 18.3.1 x DR4 SCA 662.15

Notes * significant at the 0.05 probability level ** significant at the 0.01 probability level

4. Summary and conclusion Line x tester analysis is an effective method for the selection of lines that could potentially be used as parental hybrids. There are 16 potential mutant maize inbreds that can be used as hybrid parent based on the value of GCA. Thus, two crosses pair can be used as potential hybrids based on the value of SCA. Furthermore, 37 hybrids exceeded the value of parent based on high parent heterosis value. These mutant lines could be considered for breeding program for high yield and early maturity.

5. Acknowledgement The authors would like to put into words their appreciation to the Directorate General Higher Education, Ministry of Culture and Education, Republic Indonesia and Universitas Padjadjaran for research funding through PUPTN granted to the 2 nd author.

6. References [1] Darwin, C.R. (1876). The Effects of Cross and Self Fertilization in the Vegetable Kingdom. (London: John Murray). [2] Shull G.H., 1908 The composition of a field of maize. Am. Breed. Assoc. Rep. 4: 296-301. [3] Shull G.H., 1909 A pure line method of corn breeding. Am. Breed. Assoc. Rep. 5: 51-59. [4] Mba, C. 2013. Induced Mutations Unleash the Potentials of Plant Genetic Resources for Food and Agriculture. Journal Agronomy 2013, 3, 200-231; doi:10.3390/agronomy3010200 [5] Sigurbjornsson,B. 1977. Introduction: Mutations in Plant Breeding Programs. In: Manual on Mutation Breeding, Second Edition, IAEA, Vienna, 1977, pp. 219 [6] Novak, F.J. and H. Burner. 1992. Plant breeding: Induced mutation technology for crop improvement. IAEA Bulletin, 4/1992 [7] Kempthorne, O. 1957. An introduction to genetic statistics. Jonh Wiley and Sons, New York: 468-472 [8] Ali, F., Irfan A.S., Hidayat U.R., Mohammad N., Durrishahwar, Muhammad Y.K., Ihteram U., and Jianbing Y. 2012. Australian Journal of Crop Science 6(3):455-462 [9] Singh, H,K. and B,D, Chaudhary. 1979. Biometrical Methods in Quantitative Genetic Analysis, Kalyani Publisher, Ludhiana, New Delhi, P, 191-200. [10] Poehlman, J, M, and D, A, Sleeper. 2006. Breeding Field Crops Fifth Edition, Iowa State University Press, Ames, Iowa 50014. [11] Falconer, S.P. (1967). Introduction to quantitative genetics. New York: The Ronald Press Company. [12] Sprague, G.F. & Tatum, L.A. (1942). General vs specific combining ability in single crosses of corn. J. American Soc. Agron., 34, 923-932. [13] Fehr, W, R. 1987. Principle of Cultivar Development, Theory and Technique, Vol 1, Macmillan Publishing Company, New York. [14] Reif, J.C., A.R. Hallauer, A.E. Melchinger. 2005. Heterosis and Heterotic Patterns In Maize. Maydica 50 (2005): 215-223 [15] Estakhr, A., Bahram. H. 2012. Combining Ability and Gene Action for Maturity and Agronomic Traits in Different Heterotic Groups of Maize Inbred Lines and Their Diallel Crosses. Crop Science. Biotech. 15 (3) : 219 229 [16] Bertoia, L., C. Lopez, and R. Burak. 2006. Biplot Analysis of Forage Combining Ability in Maize Landraces. Crop Science. 46:1346-1353 41

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

The Potential of Ornamental Plant, Sansevieria trifasciata to Inhibit the Growth of Harmful Algal Bloom Species Ima Amirah Mohd Suberi 1, Normawaty Mohammad Noor 1,2, Deny Susanti Darnis 1, Yukinori Mukai 1,2 and Gires Usup 3 1

Department of Marine Science, Kuliyyah of Science, International Islamic University, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia. 2 Institute of Oceanography and Maritime Studies, International Islamic University, Kg. Cherok Paloh, 26160, Kuantan, Pahang, Malaysia 3 Faculty Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.

Abstract. Malaysia is one of the countries that have been affected by harmful algal bloom (HAB); a phenomena that caused human health problems such as seafood poisoning and loss to aquaculture industries. In Peninsular Malaysia, several HAB problems have been reported since 1991. Many studies have shown that compounds extracted from plants have a promising result to mitigate HAB problems. Unfortunately, this type of study is very limited in Malaysia. Therefore in this study, the potential of ornamental plant; S. trifasciata to inhibit the growth of HAB species i.e. A. tamiyavanichii and A. tamarense was evaluated. The crude extracts of this plant which were obtained from fresh and dried materials were tested on these HAB species at different concentrations (0.5, 1, 1.5, 2 and 2.5 mg/mL). Result showed that all concentrations tested inhibited the growth of both HAB species. The highest removal efficiencies for A. tamiyavanichii using fresh and dried plants were 47.33% at concentration of 0.5 mg/mL and 37.53% at concentration of 1.0 mg/mL, respectively. For A. tamarense, the highest removal efficiency for both fresh and dried plants were 51.59% and 54.83%, respectively, at concentration of 1.5 mg/mL. After 24 hours, all cells in every treatment died whereas cells in control were still active. This shows that S. trifasciata have the potential to be used to mitigate HAB but further studies are needed to refine the findings.

Keywords: Harmful algal bloom (HAB), S. trifasciata, ethanol crude extract, removal efficiency.

1. Introduction HAB happens due proliferation of microalgae species in marine and/or freshwater environment reaching high cell density. Over the last decade, this event had been increasingly reported in various countries including in Malaysia. Most of the red tide events documented were caused by dinoflagellates [1]. The genus Alexandrium consists of more than 20 species and many of the species have been identified as toxic species [2]. These dinoflagellates species can cause problem known as Paralytic Shellfish Poisoning (PSP) [3]. In Malaysia, several HAB cases have been reported in Malacca and Kelantan caused by A. tamiyavanichii and A. minutum, respectively [4]. Snake plant is an ornamental plant and approximately 70 distinct species have been identified [5]. This plant is a succulent plant and originally found in tropical central Africa and drier part of eastern and southern Africa. One common species is Sansevieria trifasciata which commonly known as mother-in-law’s tongue. Studies showed that this plant has potential to be used for medicinal, fodder, soil conservation and fibre [6]. ______________________________________________ +

Normawaty Mohammad Noor. Tel.: +09 5716400; fax: +09 5716781 Email address: [email protected] 42

In literature, many mitigation ways have been reported in order to reduce the occurrences of harmful algal bloom. Clay for example, has been used in Korea to control HAB [7]. Others used barley straw but this method only effective for a specific area [8]. In Malaysia, very limited study has been carried out to mitigate HAB problem particularly using ornamental plant. Therefore, this study was performed to evaluate the potential of ornamental plant, S. trifasciata to inhibit the growth of two toxic species of Alexandrium isolated from Malaysia.

2. Materials and methods 2.1. Harmful algal bloom cultures Toxic HAB, A. tamiyavanichii and A. tamarense were obtained from Universiti Kebangsaan Malaysia laboratory. These microalgae were maintained in ES-DK medium enriched with f/2 vitamin and placed at 26˚C in a 12:12 L:D (light and dark) cycle and 5000 lux of light intensity.

2.2. Extraction of S. trifasciata plant Plants were obtained from the private nursery and grown in the IIUM nursery. Then, the fresh plant samples were washed, cut into smaller size, and weighted. Same processes were applied to dried plant samples but need to be dried in the oven at 80˚C for 24 hours before grind using mortar or grinder. The fresh and dried samples then were extracted using 95% ethanol. Plant samples (fresh and dried) were soaked in 95% ethanol at room temperature for 9 days. The samples were stirred daily and replaced with fresh 95% ethanol every 3 days. The collected crude extracts were filtered through 0.45 µm Whattman filter using vacuum pump and concentrated with rotary evaporator to remove plant extract solvent.

2.3. Test of crude extract on selected HAB species Crude extracts of S. trifasciata were tested on A. tamiyavanichii and A. tamarense using different concentrations which were 0 (control), 0.5, 1, 1.5, 2 and 2.5 mg/mL. For HABs cultures, 5000 cells/mL of each species in 20 mL medium were prepared in Petri dish and kept for 24 hours for acclimatization. Three replicates were prepared. The cells were observed under light microscope for any morphological changes such as cell disruption and times of morphological changes were recorded. The number of survived algal were counted using a Sedgwick rafter chamber at 30 minutes, 5 hours, 10 hours and 24 hours. The removal efficiency was calculated using equation suggested by Padilla et al. 2010 [9]: Removal efficiency (RE) = [1-(cell counted in the treatment/cell counted in the control)] ×100%

3. Results and discussion 3.1. Test of crude extract on selected HAB species Figures 1 and 2 showed the average of cell removal efficiencies of ethanol crude extract of S. trifasciata (fresh and dried) tested at 4 different concentrations on two selected HAB species i.e. A. tamiyavanichii and A. tamarense. Results showed that both species react differently to each concentration tested. A. tamiyavanichii tested with 0.5 mg/mL of crude extract from fresh plant showed the highest removal efficiency which was 47.33% whereas the lowest removal efficiency was 20.9% when tested with 2 mg/mL of crude extract. Compared to A. tamarense, the highest removal efficiency was 51.59% when tested with 1.5 mg/mL of fresh plant crude extract and the lowest result was 36.88% when exposed with 0.5 mg/mL of crude extract. For extracts from dried plants, A. tamiyavanichii showed the highest removal efficiency when tested with 1 mg/mL (37.53%) of crude extract whereas the lowest removal efficiency was resulted from 2.5 mg/mL (25.57%) of crude extract. Nevertheless, A. tamarense showed better removal efficiency at 1.5 mg/mL which was 54.83% and the lowest removal efficiency was 35.18% at concentration of 0.5 mg/mL of crude extract. For both extractions i.e from dried and fresh plants, the removal efficiencies for each species were not 43

significantly different. This indicates that these two methods can be used to extract the crude compounds. The inhibitory rates for both crude extracts did not show positive correlation between concentration tested and number of cell inhibited. Study using crude extract from garlic reported that the higher concentration used, the stronger the inhibitory rate [10]. This indicates that the higher concentration of crude extracts may need to be considered in this study. Based on morphology, cells exposed to all concentration of both crude extracts did not showed significant morphological changes after 30 minutes. However, the cells tend to swim weakly after 5 and 10 hours. After 24 hours, all cell died and some of the cells ruptured whereas in 0 hours (control), the cells were actively swam. A study has shown that algicidal actinomycete inhibited the growth of A. tamarense by causing the cell to lyse and therefore released the cellular component as the exposure time increased [11].

Fig. 1: Removal efficiency (%) of HABs using ethanol crude extract (fresh sample).

Fig. 2: Removal efficiency (%) of HABs using ethanol crude extract (dried sample).

44

4. Conclusion This study indicates that S. trifasciata has the potential to inhibit the growth of HAB species particularly A. tamiyavanichii and A. tamarense. However, the concentration of crude extract used may need to be higher than 2.5 mg/mL in order to obtain better result.

5. Acknowledgements We would like to express our appreciation to Kuliyyah of Science, International Islamic University Malaysia for the facilities provided. This study was partially supported by FRGS grant from Ministry of Higher Education, Malaysia

6. References [1]

Sunda, W., Granelli, E. and Gabler, C. J. 2006. Positive feedback and the development and persistence of ecosystem disruptive algal blooms. Journal of Phycology, 42: p. 963-974.

[2]

Lim, P. T. and Ogata, T. 2005. Salinity effect on growth and toxin production of four tropical Alexandrium species (Dinophyceae). Toxicon, 45: p. 699-710.

[3]

Ken, F., Patricia, M. G., Zhou, M. and Raine, R. 2010. Regional background and challenges for advancing the understanding of Asian HABs. Global Ecology and Oceanography of Harmful Algal Bloom in Asia: A regional comparative programme. IOC and SCOR, Paris and Newark, Delaware. p.1-68.

[4]

Lim, P. T., Leaw, C. P. and Usup, G. 2003. Identification of Alexandrium halim (Avaceae) using EPIfluorescence microscopy. Annals of microscopy, 3: p. 102-107.

[5]

Allan, L. K. and Thomas, L. R. 1988. Leaf Anatomy in Sansevieria (Avaceae). America Journal Botany, 75(5): p. 616-633.

[6]

Khalumba, M. L., Mbugua, P. K. and Kung'u, J. B. 2005. Uses and conservation of some highland species of the genus Sansevieria Thunb in Kenya. African Crop Science Conference Proceeding, 7: p. 527-532.

[7]

Young-Ju, L., Joong-Ki, C., Eun-Ki, K., Seok-Hyun, Y. and Eun-Ji, Y. 2008. Field experiments on mitigation of harmful algal blooms using a Sophorolipid-Yellow clay mixture and effects on marine plankton. Harmful algae, 7(2): p. 154-162.

[8]

Ferrier, M. D., Butler, Sr., B. R., Terlizzi, D. E. and Lacouture, R. V. 2005. The effects of barley straw (Hardeum vulgare) on the growth of freshwater algae. Bioresource Technology. 96(16): p. 1788-17985.

[9]

Padilla, L. V., Maria, L. S. D. and Azanza, R. V. 2010. Exploring the potential of clay in mitigating Pyrodinium bahamense var. compressum and other harmful algal species in the Philippines. Journal of Applied Phycology, 22: p. 761-768.

[10]

Zhou, H., Pan, G., Chen, H. and Yuan, X. 2006. Removal of cyanobacterial blooms in Taihu Lake using local soils II. Effective removal of Mycrocystis aeruginosa using local soils and sediments modified by chitosan. Environment Pollution, 141(2): p. 201-205.

[11]

Bai, S. J., Liping, P. H., Jiangqiang, Q. S., Yun, T. and Tianling, L. Z. 2011. Algicidal effects of a novel marine actinomycete on the toxic dinoflagellate Alexandrium tamarense. Curr Microbiology, 62: p. 1774-1781.

45

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Brain Regional and Hypophyseal Protein Profiles of Boars Fed Dietary Fumonisin B1 Francis Ayodeji Gbore Department of Environmental Biology and Fisheries, Adekunle Ajasin University, Akungba-Akoko, Nigeria

Abstract. The impacts of dietary fumonisin B1 (FB1), a mycotoxin produced mainly by Fusarium verticillioides and F. proliferatum, on protein profiles of brain regions and hypophyses were studied in 24 male Large White weanling pigs randomly divided into four groups (n = 6) in a completely randomized design. Each group of the animals with six replicates, received one of the four diets containing approximately 0.2, 5.0, 10.0 and 15.0 mg FB 1/kg constituting the Control, Diet 1, Diet 2 and Diet 3, respectively, in a 6-month feeding experiment. At the end of the feeding experiment, all the animals were slaughtered and the brains and the hypophyses were carefully dissected out to determine the total protein (TP) concentrations in the regional brain and hypophyses. Results showed that TP concentrations decreased significantly (P < 0.05) in the cerebellum, hypothalamus and the medulla oblongata as the dietary FB1 concentration increased. The TP concentrations in these brain regions and hypophyses of the animals on Diets 1, 2 and 3 ranged from 42.1 - 105.6, 30.5 - 96.2 and 26.3 - 92.3 % of the Controls respectively. Chronic dietary exposure to FB1 at concentrations above 5.0 mg/kg is a potential health risk that may significantly interfere with protein metabolism that may not be lethal to growing pigs, but a potential health risk that may produce adverse physiological response in the animals.

Keywords: Brain, fumonisin B1, hypophysis, pigs, protein.

1. Introduction Mycotoxins are natural contaminants of cereals and other food commodities throughout the world and they significantly impact human and animal health. Animals, as well as humans, are exposed to mycotoxins through consumption of contaminated diets, which can be considered the gateway to cases of natural intoxication by these fungal secondary metabolites [1, 2]. The economic consequences of mycotoxin contamination are profound, and exposure of people and livestock to mycotoxin-contaminated foods is particularly a serious problem in the tropics [3]. Fumonisins are mycotoxins produced by Fusarium moulds, most notably Fusarium verticillioides (=F. moniliforme) and F. proliferatum. Mycotoxins are natural contaminants of cereals and other food commodities throughout the world and they significantly impact human and animal health. The economic consequences of mycotoxin contamination are profound, and exposure of human and livestock to mycotoxin-contaminated foods is particularly a serious problem in the tropics [3]. Fumonisins occur as contaminants of agricultural products, particularly maize worldwide [4] and have been documented to cause various physiological responses in humans and animals. The mycotoxins have been implicated as a causative agent in several animal and human diseases. Also, significantly altered concentrations of neurotransmitters in brain regions of starter pigs fed a blend of grains naturally contaminated with Fusarium mycotoxins have been reported [5]. An estimation of the proteins in body tissues may be utilized as an assessment of the nutritive state of the animal. The nutritive state may be dependent not only on the proper and adequate intake of protein building materials in the diet but may also be a reflection of the nutritive state existing within the animal body, reflecting alterations in metabolism [6]. Fumonisins inhibit sphingolipids metabolism in tissues, leading to an accumulation of sphingoid bases -sphinganine and sphingosine, which are intermediates in sphingolipid biosynthesis [7]. Alterations in the amounts of any of these by fumonisins could potentially result in a variety of biological and pathological effects and a variety of biological activities for sphingolipids have been reported [8]. It was therefore hypothesized that fumonisin could alter protein synthesis in the brain since the brain contains high levels of sphingolipids and proteins in the nervous system are known to occur in complexes with 46

lipids. Based on the reviewed physiological effects of fumonisin on animals, this study was designed to determine the effects of chronic exposure to dietary FB1 on protein profiles in brain regions and hypophyses of pubertal boars.

2.

Materials and Methods

2.1

Fumonisin B1 production and diets

Maize grits in 500 g quantities soaked with 200 ml of distilled water were placed into autoclavable polypropylene bags and autoclaved for 1h at 121 oC and 120 kPa. The autoclaved maize grits were then cultured with a toxigenic strain of F. verticillioides (MRC 286) to produce FB1 as previously described [9]. The uncultured maize grits were used to formulate the Control diet, while three other diets were formulated with the cultured maize grits substituted for the uncultured maize grits at various proportions. Samples of homogenously mixed diets were quantified in replicates for FB1 and other common Fusarium mycotoxins using mycotoxin quantitative CD-ELISA test kits (Neogen, Lansing, MI, USA). The concentration of FB1 in the Control diet was 0.2 mg/kg while the concentrations of FB1 in the other three diets were adjusted to 5.0, 10.0, and 15.0 mg/kg, constituting Diets 1, 2, and 3, respectively. Table 1. Compositions (%) of the test diets for the various physiological phases

Ingredient Maize* Soybean meal Palm kernel cake Wheat offal Rice husk Fish meal Fixed ingredients** Nutritional levels Crude fibre (%) Crude protein (%) DE*** (kcal/kg)

Physiological phase Weanlin Pre-pubertal g 40.00 30.00 20.00 15.00 20.00 25.00 14.00 14.30 11.00 3.00 2.00 2.70 2.70 5.35 20.38 2700.00

9.82 17.97 2270.00

Pubertal 20.00 8.50 45.00 5.00 17.80 1.00 2.70 10.83 15.30 2240.00

*Mixture of Fusarium-cultured and non-cultured maize in various proportions to achieve desired dietary FB 1 levels for each treatment. **Contained Dicalcium phosphate (1.50), Oyster shell (0.50), Salt (0.45) Minerals-vitamins premix (0.20), Methionine (0.01) and Lysine (0.04). ***Calculated Digestible Energy values.

2.2

Animals and feeding

The feeding experiment was carried out at the Animal Physiology Unit of the Teaching and Research Farm, University of Ibadan, Ibadan, Nigeria (7o20'N, 3o50'E, 200 m above sea level with an average daytime temperature of 24 - 25 oC and relative humidity 80 - 85 %). The study was approved by the local Institutional Animal Ethics Committee and performed in accordance with “Guide for the care and use of Laboratory Animals” [10]. After a 2-week physiological adjustment period, 24 clinically normal male Large White weanling pigs of about 8 - 9 weeks of age averaging 6.94 + 0.26 kg housed in individual concrete floor indoor pens were randomly divided into four groups, with six animals per group. Each group received one of the four diets. The feeding experiment, which lasted 6 months, was divided into 3 physiological phases [weanling (starter), prepubertal (grower) and pubertal (finisher)]. The animals were fed their respective diets ad libitum daily. The gross compositions of the experimental diets, fed during weanling, pre-pubertal and pubertal’s phases for 6, 10 and 8 weeks, respectively, are shown in Table 1.

2.3

Brain tissue protein determination

At the end of the experiment, all the animals were stunned mechanically prior to bleeding, quickly decapitated and the brains and hypophyses immediately removed, freed of all adhering meninges and blood vessels and dissected on ice-cold porcelain tile into the pons, cerebellum, amygdala, hippocampus, 47

hypothalamus, cerebral cortex, mid brain, medulla oblongata, adenohypophysis and neurohypophysis as previously described [11]. The brain and hypophyseal total protein concentrations were determined by Biuret method according to Reinhold [12] immediately after the brain and hypophyseal samples from each animal was homogenized (1%, w/v) with a Potter-Elvehjem homogenizer in 0.1 M ice-cold phosphate buffer containing 0.1% Triton X100 (Sigma-Aldrich, St. Louis, MO, USA).

2.4

Statistical analysis

The experimental design used was complete randomized. All data obtained were subjected to one-way statistical analysis of variance (ANOVA) procedure of SAS [13]. The significant treatment means were compared at 5% probability level using the Duncan multiple range test option of the same software.

3.

Results and Discussion

The effect of varied dietary FB1 on total protein in the brain regions and hypophyses of pubertal boars are shown in Table 2. Results showed significant (P < 0.05) decline in the total protein concentrations in the cerebellum, hypothalamus and the medulla oblongata with an increase in the dietary FB1. The total protein concentrations in the pons, hippocampus, midbrain and the hypophyses tended to decline with an increase in the dietary FB1 concentrations while the concentrations in the amygdala did not follow any particular trend. The total protein concentrations in the brain regions and hypophyses of the animals on diet 3 ranged from 26.32 – 92.31 % of those on the Control diet. Table 2. Total protein (g/dl) in the brain regions and hypophyses of pubertal boars fed dietary FB 1

Brain Region Pons Cerebellum Amygdala Hippocampus Hypothalamus Cerebral cortex Mid Brain Medulla oblongata Adenohypophysis Neurohypophysis ab

Control 0.2 mg FB1 0.52 0.60a 0.30 0.55 0.95a 0.21b 0.38 0.90a 0.47 0.26

Diet 1 5 mg FB1 0.45 0.45ab 0.42 0.35 0.40b 0.45a 0.35 0.95a 0.45 0.25

Diet 2 10 mg FB1 0.37 0.25b 0.30 0.37 0.29b 0.25ab 0.31 0.40b 0.31 0.25

Diet 3 15 mg FB1 0.25 0.24b 0.29 0.35 0.25b 0.30ab 0.35 0.55b 0.30 0.24

s.e. 0.031 0.010 0.015 0.014 0.021 0.007 0.006 0.006 0.005 0.004

Means on the same row with different superscripts differ significantly (P5.0 mg/ kg dietary FB1. Sharma and Bahadur [16] reported that neurosecretory cell number and total protein concentration in the brain are related to reproductive phenomenon. The reported reduced sperm production [17], delayed sexual maturity [18], and inferior semen qualities [19] by the growing pigs fed increased concentrations of dietary FB1 might be secondary effects of the altered total protein in these brain regions of the animals.

4.

Summary and conclusion

The impacts of dietary FB1, a mycotoxin produced mainly by Fusarium verticillioides and F. proliferatum, on protein profiles of brain regions and hypophyses were studied in weanling pigs. The study revealed that dietary FB1 significantly reduced total protein in the cerebellum, hypothalamus and the medulla oblongata as the dietary FB1 concentration increased. This study suggests that dietary FB1 has an adverse influence on protein metabolism in animals chronically exposed to feeds containing >5.0 mg FB1/kg. Feed contaminated with F. verticillioides that will yield above 5.0 mg FB1/kg for a 6-month period may not be lethal to growing pigs, but a potential health risk that may produce adverse physiological response in growing pigs.

References [1] Gutema, T., C. Munimbazi, and L.B. Bullerman, 2000. Occurrence of fumonisins and moniliformin in corn and cornbased food products of U.S. origin. Journal of Food Protection, 63: p. 1732-1737. [2] Hennigen. M.R., S. Sanchez, N.M Di Benedetto, A. Longhi, J.E. Torroba, and L.M. Valente Soares, 2000. Fumonisin levels in commercial corn products in Buenos Aires, Argentina. Food Additives and Contaminants, 17: p. 55-58. [3] Reddy, B.N., and C.R.Raghavender, 2008. Outbreaks of fusarial-toxicoses in India. Cereal Research Communications, 36 (Suppl. B): p. 321-325. [4] Nelson, P.E., R.D. Plattner, D.D. Shackelford, and A.E. Desjardins, 1991. Production of fumonisins by Fusarium moniliforme strains from various substrates and geographic areas. Applied and Environmental Microbiology, 57: p. 2410-2412. [5] Swamy, H.V.L.N., T.K. Smith, E.J. MacDonald, H.J. Boermans, and E.J. Squires, 2002. Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on swine performance, brain regional neurochemistry, and serum chemistry and the efficacy of a polymeric glucomannan mycotoxin adsorbent. Journal of Animal Science, 80: p. 3257-3267. [6] Gbore, F.A., and G.N. Egbunike, 2009. Toxicological evaluation of dietary fumonisin B 1 on serum biochemistry of growing pigs. Journal of Central European Agriculture, 10 (3): p. 255-262. [7] Wang, E., W.P. Norred, C.W. Bacon, R.T. Riley, and A.H. Merrill Jr., 1991. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. Journal of Biological Chemistry, 266: p. 14486-14490. [8] Wang, E., P.F. Ross, T.M. Wilson, R.T. Riley, and A.H. Merrill Jr., 1992. Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins, mycotoxins produced by Fusarium moniliforme. Journal of Nutrition, 122: p. 1706-1716. [9] Nelson, P.E., J.H. Juba, P.F. Ross, and L.G. Rice, 1994. Fumonisin production by Fusarium species on solid substrates. Journal of AOAC International, 77: p. 522-524. [10] NRC, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press Washington, USA. [11] Egbunike, G.N., 1981. Regional distribution of acetylcholinesterase activity in the brain and hypophyses of crossbred European boars reared in the humid tropics. Acta Anatomica, 110: p. 248-252. [12] Reinhold, J.G., 1953. Manual determination of total serum proteins, albumin and globulin fractions by Biuret method. In: Standard Methods of Clinical Chemistry, M. Reiner (editor). Academic Press, New York. [13] SAS, 2001. SAS/STAT User’s Guide. Version 9.2 for windows. SAS Institute Inc., SAS Campus Drive, Cary, North Carolina, USA. [14] Adejumo, D.O., and G.N. Egbunike, 2004. Changes in acetylcholinesterase activities in the developing and aging pig brain and hypophyses. International Journal of Agricultural and Rural Development, 5: p. 46-53. [15] Adejumo, D.O., and G.N. Egbunike, 2002. Regional variation in acetylcholinesterase activity and total protein in the brain and hypophyses of Large White boars managed under a hot humid environment. ASSET Series A, 2: p. 49-53. [16] Sharma, P.K., and J. Bahadur, 1982. Age-related changes in the total protein in the brain of Periplaneta americana (L.). Mechanism of Ageing and Development, 20: p. 49-52. 49

[17] Gbore, F.A., and G.N. Egbunike, 2008. Testicular and epididymal sperm reserves and sperm production of pubertal boars fed dietary fumonisin B1. Animal Reproduction Science, 105: p. 392-397. [18] Gbore, F.A., 2009. Growth performance and puberty attainment in growing pigs fed dietary fumonisin B 1. Journal of Animal Physiology and Animal Nutrition, 93: p. 761-767. [19] Gbore, F.A., 2009. Reproductive organ weights and semen quality of pubertal boars fed dietary fumonisin B 1. Animal, 3: p. 1133-1137.

50

3rd International Conference on Applied Life Sciences (ICALS2014) Malaysia, 18-20 September, 2014

Cannibalistic Behaviour of African Catfish Juveniles, Clarias gariepinus under Different Light Wavelengths and Intensities Firdaus Sallehudin 1 and Yukinori Mukai 1 Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia, Pahang, Malaysia

Abstract. The African catfish shows strong cannibalism during larval and juvenile stages. Environmental factor such as light illumination affect the cannibalistic behaviour of the fish, but the effect of light wavelengths towards African catfish behaviour is still unknown. Thus, the aim of this study was to investigate the effect of different light wavelengths and intensities towards the behaviour of African catfish juveniles. In this study, the juvenile’s behaviour was examined under five light wavelengths (white, blue, green, yellow and red) and four light intensities (0.0017, 0.021, 0.19, and 1.40 µmoles/m²/s). The results of the present study showed that the juveniles of African catfish had low biting activity under light intensities of 0.0017 and 0.021 µmoles/m²/s for white, blue, and yellow light wavelengths. Under light intensities of 0.19 and 1.40 µmoles/m²/s for blue and red light wavelengths, the juveniles of African catfish showed low biting activity. Only blue light wavelength showed low biting activity for all conditions of light intensities.

Keywords: African catfish, cannibalistic behaviour, light intensities, light wavelengths,

1. Introduction The African catfish (Clarias gariepinus) is one of the highly demanded freshwater fish species throughout the world for its abilities in resisting diseases and in withstanding poor water quality. This catfish species can also survive under high stocking densities. Furthermore, it has a fast growth rate and good quality meat [1]. However, this species shows strong cannibalism especially during larvae and juveniles rearing, which can result in low survival rates in hatcheries. Several biotic and abiotic factors are known to constitute as stressors of fish in the aquatic environment. These environmental conditions such as temperature, water salinity, oxygen availability, water velocity, and light availability could influence its survival as well as its growth [2, 3]. Modification of light illumination can be used to increase the survival of fish. It can be adjusted in three aspects which are quality (light wavelength), quantity (light intensity), and periodicity (duration of light) [4]. Previous study finds that survival rate of African catfish larvae can be increased if there are reared under dark condition [5]. In another study, Moshood et al. (2012) [6] state that the growth rate of African catfish is increased when they are reared continuously under dark conditions. Furthermore, the cannibalism behaviour of these larvae is decreased when they're reared under dark and dim conditions [7]. Although the previous studies have demonstrated that the African catfish is sensitive towards certain light periodicity and intensity, but the data on the issue of light wavelength towards fish behaviour are still scarce. Fish responsiveness to light differs from species to species. Defining optimum light conditions based on species is necessary when culturing fish in an artificial environment. Thus, the aim of the present study was to investigate the aggressive behaviour of African catfish under the different light wavelengths and light intensities to obtain the optimum light condition needed for rearing this fish species.

*Corresponding author. Tel.: + 060198206125 Fax.:+065716781

E-mail address: [email protected]. 51

2. Materials and Methods 2.1.

Juvenile Specimen

The experiments were conducted at the laboratory of Institute of Oceanography and Maritime Studies, (INOCEM). The juveniles of African catfish with the total length of 4 – 5 cm were obtained from the private fish farm in Kuantan. The juveniles were reared in 250 L polyethylene tanks with sufficient aeration, and the water temperature was at 28.0 - 30.0 °C. The juveniles were then fed with commercial pelleted diets given twice daily.

2.2.

Illuminating Method and Experimental Design

The experiments were conducted in the dark room. Light emitting diodes (LED) (Wayjun Technology, Shenzen, China) were used as the source of light, while the light intensities were adjusted by using a dimmer and neutral-density filters (NDx8, HOYA, Tokyo, Japan). Light intensities were measured by using a reflection spectrophotometer (Ocean Optic spectrometer, Model USB4000-UV-VIS). A CCD camera (WAT – 232/NTSC (colour & IR), Watec. Co. Ltd.) was used to record fish behaviour, while an infrared lamp with 850 NM filter (IR85, Fuji film, Tokyo, Japan) was used to record the fish behaviour under dark and dim conditions. The CCD camera images were recorded with a DVD recorder (LG DVD Recorder DR 166H). The behavioural experiment was conducted under 0.0017, 0.021, 0.19, and 1.40 µmoles/m²/s for five light wavelengths which are white, red, blue, green, and yellow. 3 L of fresh water was filled into a glass basin (20 cm in diameter). The water depth was 7.1 cm for all the experiments, and the water temperature was maintained at 28 °C. Then, 5 tails of fish were placed in the glass basin. Before the fish behaviour was recorded, they were left for 30 minutes under each light condition in the dark room for adaptation and to remove handling stress. After this adjustment period, juvenile behaviour was recorded for 30 minutes by using a CCD camera and a DVD recorder.

2.3.

Fish Attack Activity

Attack activities were considered when there was a contact between an attacking individual to another individual. The number of attack activities per individual per minute was calculated by dividing the total number of attack activities in 30 minutes for one individual with 30 minutes. No. of attack activities per individual = Total number of attack activities in 30 minutes 5 fish No. of attack activities per individual per minute = No. of attack activities per individual 30 minutes

2.4.

Statistical Analysis

Data were analysed using two-way analysis of variance (ANOVA) with light spectrum and light intensity as factors. Where p values were significant (p