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Volume No – 4 Issue No – II April, 2016 Journal of Experimental Biology and Agricultural Sciences

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ISSN No. 2320 – 8694 Volume No – 4 Issue No. - II April, 2016 Journal of Experimental Biology and Agricultural Sciences Publisher: HORIZON PUBLISHER INDIA [HPI]

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Dear Readers,

It is with much joy and anticipation that we celebrate the launch of Volume 4, Issue II of Journal of Experimental Biology and Agricultural Sciences (JEBAS). On behalf of the JEBAS Editorial Team, I would like to extend a very warm welcome to the readership of JEBAS. I take this opportunity to thank our authors, editors and anonymous reviewers, all of whom have volunteered to contribute to the success of the journal. I am also grateful to the staff at Horizon Publisher India [HPI] for making JEBAS a reality.

JEBAS is dedicated to the rapid dissemination of high quality research papers on how advances in Biotechnology, Agricultural sciences along with computational algorithm can help us meet the challenges of the 21st century, and to capitalize on the promises ahead. We welcome contributions that can demonstrate near-term practical usefulness, particularly contributions that take a multidisciplinary / convergent approach because many real world problems are complex in nature. JEBAS provides an ideal forum for exchange of information on all of the above topics and more, in various formats: full length and letter length research papers, survey papers, work-in-progress reports on promising developments, case studies and best practice articles written by industry experts.

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Thank you. We hope you will find JEBAS informative.

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INDEX ______

Effects of crude extracts of various pigments isolated from Spirulina platensis and Chlorella vulgaris on the angiogenesis of chick embryo chorioallantoic membrane (cam) Haydee Evangeline H. Biscocho* and Johnny A. Ching [doi: http://dx.doi.org/10.18006/2016.4(2).121.127 ] 121.127

Menstrual syndrome: severity, frequency and symptomatology in adolescent girls of Quetta, Pakistan Faiza Hameed Jan, Zil E Huma, Nabeela Tariq and Naheed Sajjad* [doi: http://dx.doi.org/10.18006/2016.4(2).128.132 ] 128.132

Complementary and alternative medicines among residents in upland cavite, Philippines: their knowledge, patterns of use and attitudes Johnny A Ching*, Rodante G Flores and Maxima J Acelajado [doi: http://dx.doi.org/10.18006/2016.4(2).133.141 ] 133.141

Analysis of long term impact of cyclonic disaster ‘aila’ on soil properties and paddy yield of Sundarbans, West Bengal, India Sisir Kumar Si [doi: http://dx.doi.org/10.18006/2016.4(2).142.148 ] 142.148

Haringhata black chicken shows resistance to pathogenic E. coli by high immune- effector activities Sudip Nandi, Siddhartha N. Joardar*, Indranil Samanta, Bithi Roy, Pradip K. Das, Tapas K. Sar and Sk Sahanawaz Alam [doi: http://dx.doi.org/10.18006/2016.4(2).149.155 ] 149.155

A way of reproductive manipulation and biology of Wolbachia pipientis Habib Ali, Youming Hou*,Baozhen Tang, ZhanghongShi, Bin Huang, Abrar Muhammad and N B Sanda [doi: http://dx.doi.org/10.18006/2016.4(2).156.168 ] 156.168

Modeling the effect of mechanization level index on crop yield approaching system dynamics methodology Behnam Jalalzadeh, Ali Mohammad Borghei* and Morteza Almassi [doi: http://dx.doi.org/10.18006/2016.4(2).169.179 ] 169.179

Reproductive biology of Goodea atripinnis (Jordan, 1880) (cyprinodontiformes: goodeidae) under controlled conditions Joao Roberto Silva-Santos, Ma. Consolación Martínez-Saldaña, Roberto Rico- Martínez, José Luis Gómez-Márquez and José Luis Arredondo-Figueroa* [doi: http://dx.doi.org/10.18006/2016.4(2).180.193 ] 180.193

Effect of papaya and storage time on the quality of the newly developed papaya crackers Habiba Khatun*, Nusrat Jahan, Mojaffor Hosain, Trisna Mardy and Shahinur Rahman [doi: http://dx.doi.org/10.18006/2016.4(2).194.200 ] 194.200

Somatic embryogenesis and callus proliferation in Picrorhiza kurroa Royle ex. Benth Ritu Mahajan*, Nisha Kapoor and Iqbal Singh [doi: http://dx.doi.org/10.18006/2016.4(2).201.209 ] 201.209

Effect of temperature and loading density on drying kinetics of wheat Mojaffor Hosain*, Raihanul Haque, Md. Nazrul Islam, Habiba Khatun and Md. Shams-Ud-Din [doi: http://dx.doi.org/10.18006/2016.4(2).210.217 ] 210.217

Phytochemical analysis, antimicrobial screening and hypoglycemic effect of some selected medicinal plant extract from Oman S Hemadri Reddy*, Aisha Khalid AL-Hinai, Hajer Hafed AL-Yaqoobi and Fatima Jafar AL-Ajmi [doi: http://dx.doi.org/10.18006/2016.4(2).218.224 ] 218.224

Effect of nitrogen level, green and animal manure on the growth attribute of corn crop (Zea mays L.) Khashayar Zakikhani, Ali Kashani* and Farzad Paknejad [doi: http://dx.doi.org/10.18006/2016.4(2).225.231 ] 225.231

Phenotypic characterization of an indigenous Bacillus thuringiensis strain (B.T. LDC 501) expressing cancer cell killing protein Abirami P, Poornima Kkani, Suguna P, Saranya V, Selvanayagam P and Shenbagarathai R* [doi: http://dx.doi.org/10.18006/2016.4(2).232.241 ] 232.241

Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EFFECTS OF CRUDE EXTRACTS OF VARIOUS PIGMENTS ISOLATED FROM Spirulina platensis AND Chlorella vulgaris ON THE ANGIOGENESIS OF CHICK EMBRYO CHORIOALLANTOIC MEMBRANE (CAM)

Haydee Evangeline H. Biscocho* and Johnny A. Ching

Biological Sciences Department, College of Science and Computer StudiesDe La Salle University-Dasmariñas, City of Dasmariñas, Cavite, Philippines

Received – January 15, 2016; Revision – February 12, 2016; Accepted – March 23, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).121.127

KEYWORDS ABSTRACT Angiogenesis This study was conducted to determine the angiogenic effects of different concentrations of S. platensis phytopigments i.e. phycocyanin from Spirulina platensis and chlorophyll from Chlorella vulgaris on the chorioallantoic membrane (CAM) of developing chick embryo. Findings of study revealed that different C. vulgaris concentrations of both crude phytopigments inhibit angiogenesis as shown by the continuous decrease of the vascular density index on the CAM of 10-day old chick embryo. However, Scheffe method Phycocyanin showed that only 200 ppm and 300 ppm of crude phycocyanin and all experimental treatments of crude chlorophyll, including the 150 ppm, were able to significantly inhibit angiogenesis.It was also Chlorophyll established that there is no significant difference in the angiogenic effects between the two phytopigments.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Haydee Evangeline H. Biscocho) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

122 Biscocho and Ching

1 Introduction 2. Materials and methods

Angiogenesis is a process of new blood vessels formation and 2.1 Collection and incubation of test embryos it controlled by certain chemicals produced in the body. Chemicals such as aspartame and serotonin stimulate cells A total of one hundred fifty two-day old fertilized hen’s eggs repair and are also responsible for repairing of damaged blood were obtained from a poultry farm. The eggs were transferred vessels or formation of new ones (Surve et al., 2015; Yesildal to the Biology Research Laboratory of De La Salle University- et al., 2015; Banskota et al., 2016). Further, some chemicals Dasmariñas, Cavite and incubated at 37°C for another eight such as heparin and endostatin are responsible for stopping the days. process of cell repairing and are also responsible for the inhibition of the synthesis of stimulatory chemicals, these 2.2 Preparation of Algal extraction and determination of dry inhibitory chemicals are known as angiogenesis inhibitors weight (Zhao-Na et al., 2015; Jin-Yan et al., 2016). Uncontrolled angiogenesis is a major contributor in a number of diseases Fresh S. platensis and C. vulgaris culture were obtained from such as arthritis, diabetes-related blindness, psoriasis, tumor the Aquaculture Department of the Southeast Asian Fisheries growth and metastasis (Heidenreich et al., 2009; Gayetsky et Development Centre (SEAFDEC) in Binangonan Freshwater al., 2014). Anti-angiogenic compounds would be useful in Station located in the municipality of Binangonan, province of treating diseases caused by uncontrolled angiogenesis. Pro- Rizal. The cultures were placed in an ice chest and transferred angiogenic compounds are also required in wound healing and to the Biology Research Laboratory of De La Salle University- may be useful in minimizing tissue damage following ischemia Dasmariñas, Cavite for the extraction procedure. Each damage from heart attacks or stroke (Mousa & Mohamed, microalgal culture was centrifuged at 4000 × g for 5 min, 2004; Baharara et al., 2014). pelletized, washed and resuspended twice with tap water. After washing, the supernatant was discarded and 40 mL of Scientists and researchers are trying to use gene therapy to distilled water was added to the pelletized microalgae. The simulate this natural process by delivering different substances resuspended algal cells were then placed in a 50°C water bath that may have potential positive or negative effects to for 24 h for autolysis. The autolysed cells were centrifuged at angiogenesis of affected areas (Baharara et al., 2014). Various 10,000 × g for 10 min and the supernatant was collected and studies have been conducted to explore the possibilities of dried in a rotary evaporator (Kightlinger et al., 2014). Two alternate anti-angiogenic substance based on herbal sources. grams for each microalgae were used to determine their dry Microalgae, such as Spirulina and Chlorella, received more weight by using the formula (Boussiba & Richmond, 1979): attention from medical scientists as a nutraceutical and source % dry weight = [(pan weight + dried powder) – pan weight] / of potential pharmaceuticals (Bishop & Zubeck, 2012; AbdEl pan weight (not dried) Baky & El-Baroty, 2013). There are several studies which suggesting the ability of microalgae to inhibit viral replication, 2.3 Phytopigment Assay strengthen both the cellular and humoral arms of the immune system and cause regression and inhibition of cancers (Jensen 2.3.1 Phycocyanin assay et al., 2001; Tokusoglu & üUnal, 2003). Spirulina is a planktonic blue-green alga found in warm water alkaline Using 100 mM phosphate buffer, 40 mg of extracted Spirulina volcanic lakes. It has a dark blue-green color, because it is rich was used for quantitative analysis of crude phycocyanin in a brilliant blue polypeptide called phycocyanin. Hayashi et through a spectrophotometer at 620 nm absorbency level using al. (2006) reported that phycocyanin affects the stem cells the formula (Boussiba & Richmond, 1979): found in bone marrow. Medical scientists reported that Spirulina not only stimulates the immune system but also % crude CPC = [A620 x (10) x (100)] / 3.39 x (mg sample) x enhances the body's ability to generate new blood cells (El-kott (% dry weight) et al., 2007; Kedik et al., 2011). On the other hand, Chlorella is a single-celled green alga, which has highest known levels of Where: 3.39 is extinction coefficient of CPC at 620 nm, 10 is chlorophyll of any plant. Hyo-Jin et al. (2006) reported that total volume and 100 represent 100%. the extract of algae has stimulatory effect on the physical stamina and acts as liver detoxifier, bowel cleanser and as 2.3.2 Chlorophyll assay catalyst for the absorption of essential elements (Hyo-Jin et al., 2006). Chlorella is also considered as one of the most Isolated chlorophyll level was quantified using 85% acetone powerful nutraceuticals (Kyadari et al., 2013). Therefore, and 50 mg of Chlorella extracted by spectrophotometer at 666 present study has been conducted to find out the angiogenic nm and 642 nm against an 85% acetone/water blank potentials of the extract of microalgae S.platensis and C. absorbency level using the formula (Boussiba & Richmond, vulgaris on the chorioallantoic membrane (CAM) of 1979) developing chick embryo.

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Effects of crude extracts of various pigments isolated from Spirulina platensis and Chlorella vulgaris on the angiogenesis of chick embryo… 123

% crude CCA = [A666 x (10) x (100)] / 9.93 x (mg sample) x 2.7 Data Collection and Statistical Analysis (% dry weight) Two days after the administration of crude phycocyanin and Where: 9.93 is extinction coefficient of CCA at 666 nm, 10 is chlorophyll to the developing chick embryo, each of the test total volume and 100 represent 100%. embryos was sacrificed and their CAM was prepared for observation of angiogenesis activity. The CAM was spread 2.4 Preparation of different concentrations of crude thinly in a petri dish and observed under stereomicroscope phytopigments (Nikon®C-LED). Formed branch points or collaterals from four randomly selected areas of each CAM were counted and Three different concentrations viz 150 ppm, 200 ppm and 300 tabulated to determine the angiogenic effects of different ppm of S. platensis phycocyanin and C. vulgaris chlorophyll concentrations of phycocyanin from S. platensis and were prepared by double distilled sterile water. The desired chlorophyll from C. vulgaris. concentrations were prepared from the crude extract obtained in the phytopigment assay using the dilution formula, C1V1 = To determine the significant difference in the angiogenic

C2V2, where C1V1 are the initial concentration and volume of effects of different concentrations of crude phycocyanin from crude phytopigment and C2V2 are the desired concentration S. platensis and crude chlorophyll from C. vulgaris on the and the final volume of crude phytopigment. The difference CAM of 10-day old chick embryos, one-way analysis of between the initial and final volume is the dilution factor used variance (ANOVA) was employed. Whenever there is to obtain the desired concentrations. significant difference, Scheffe method was used to compare individual treatment means. 2.5 Preparation of eggs for in situ sample application (West et al., 2000) 3. Results and Discussion

On the 4th day of incubation, the eggs were removed from the Results of study revealed that the crude extract of incubator and were placed in a 37C oven to prepare them in phytopigments from C. vulgaris and S. platensis caused situ sample application. Each egg was swabbed with 70% reduction in the formation and growth of blood vessels (Table alcohol and using a 2-ml syringe, a small hole at the pointed 1). end of the shell was made to puncture the air sac. Using a drill bit, a 0.5-cm square window in the shell was scored. The Averages of 50.07 to 53.56 branch points were formed in the square shell was removed with a sharp pointed forceps to CAM of the tested embryos in the control treatment. Embryos expose the CAM. The small square opening was covered by treated with 150 ppm of crude phycocyanin extracted of S. adhesive tape and each test egg was returned to the incubator platensis formed an average of 51.19 branch points while those horizontally, until treatment application on day 10. treated with 200 ppm and 300 ppm of the crude extract formed an average of 26.13 and 21.27 branch points respectively, a 2.6 In situ sample application (West et al., 2000) decrease of more than half compared to that of the control treatment. Similar results were observed for the embryos At the end of the 10-day incubation, 72 viable eggs were treated with crude chlorophyll extracted of C. vulgaris. An selected, 36 eggs for each phytopigment, to be the test average of 45.04 branch points were formed from the CAM of embryos, 9 for each treatments. In a laminar flow cabinet, test embryos treated with 150 ppm of crude chlorophyll. While through the small square opening of each test egg, 0.3 ml of only an average of 25.54 and 20.54 branch points were formed the different concentrations of phytopigments and distilled for those treated with 200 ppm and 300 ppm of the crude water for the control group were administered. The opening extract, respectively. was resealed and the eggs were returned to the incubator for further two days.

Table 1 Average Number of Formed Branch Points Under the influence of various crude extracts .

Treatments Average number of branch points Applied with phycocyanin Applied with chlorophyll Control 53.56A 50.07A 150 ppm 51.19AX 45.04BY 200 ppm 26.13BX 25.54CX 300 ppm 21.27BX 20.54CX Letters A and B compared the different concentration of each phytopigment while letter X and Y compared the two phytopigments per treatment. Different letters indicate significant difference at 0.05 level

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124 Biscocho and Ching

A B

C D Treated with different concentrations of crude phycocyanin

A B

C D Treated with different concentrations of crude chlorophyll

Plate 1Photomicrographs (Nikon® C-LED 45x) of the CAM of representative embryos treated with different concentrations of crude phytopigments: (A) control, (B) 150 ppm, (C) 200 ppm, and (D) 300 pm.

Continuous reduction in the number of branch points formed Embryos treated with 150 ppm of crude phycocyanin had a was reported with increasing the concentration of the crude percent negative of 95.58 decreased in the angiogenesis or a phytopigments, clearly indicated that both crude extracts had mean difference of -2.37 as compared to the test embryos of angiogenesis inhibition properties and signal the process to the control. While percent negative of 48.79 and 39.71 were stop. Figure 1 shows the vascular density index of each the decrease in the angiogenesis for the test embryos treated treatment as they are compared with the control treatment. with 200 ppm and 300 ppm or a mean difference of -27.43 and -32.29, respectively.

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Effects of crude extracts of various pigments isolated from Spirulina platensis and Chlorella vulgaris on the angiogenesis of chick embryo… 125

Figure 1 Vascular density index of test embryos treated with crude phytopigments as compared with the control.

Embryos treated with 150 ppm of crude chlorophyll had a chlorophyll as angiogenesis inhibitors on the CAM of 1chick percent negative of 90.67 decreased in the angiogenesis or a embryos. Figure 2 shows which of the two phytopigments mean difference of -4.67 as compared to the test embryos of inhibit angiogenesis more effectively. the control. While percent negative of 51.01 and 41.02 were the decrease in the angiogenesis for the test embryos treated All three concentrations of chlorophyll induced greater with 200 ppm and 300 ppm or a mean difference of -24.53 and inhibition to the angiogenesis on the CAM of the test embryos -29.53, respectively. compared to phycocyanin. Embryos treated with 150 ppm crude phycocyanin formed an average of 51.19 compared to These results supported the claims that both phytopigments of only 45.04 branch points formed in the test embryos treated Spirulina and Chlorella are effective in preventing diseases, with crude chlorophyll at the same concentration. Similar such as cancer, brought about by uncontrolled angiogenesis types of trend were observed with the other two concentrations (Tokusoglu & üUnal, 2003; Hyo-Jin et al., 2006; Kedik et al., viz 200ppm and 300ppm. Embryo treated with 200& 300 2011). Previous researches revealed that high concentrations of ppm phycocyanin showed an average of 26.13 & 21.27 branch phycocyanin and chlorophyll inhibited the in vitro growth of points whereas only 25.54 & 20.54 for crude chlorophyll. tumor cell lines, which indicating that some tumor cell lines However, using one-way analysis of variance, there was no are directly sensitive to phycocyanin or chlorophyll. Further, it significant difference between the crude extract of 200 and 300 was reported that these cell lines were inhibited by inhibiting ppm of phycocyanin from S. platensis and the extract of angiogenesis (Jensen et al., 2001; Kyadari et al., 2013; Saini & chlorophyll from C. vulgaris in inhibiting angiogenesis to Sanyal, 2014). The number of branch points formed per CAM of the test embryos. This only shows that both treatment was also compared to determine the significant phytopigments were effective angiogenesis inhibitors. difference between crude extracts of phycocyanin and

Figure 2 Average number of branch points formed in the CAM of test embryos from different experimental treatments.

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126 Biscocho and Ching

Conclusions Archives Microbiology 120: 155-159. Retrieved from http://link.springer.com/article/10.1007%2FBF00409102 Crude extract of phycocyanin and chlorophyll from S. platensis and C.vulgaris respectively, are angiogenesis inhibitors as El-kott AF, Abd-El-Azim AO, Abdeen SH, Azab YA, Ismail shown by the continuous decrease of the vascular density index MF (2007) Protective effect of Spirulina platensis on induced of the CAM of 10-day old chick embryo as their mammary tumour in rats: an immunohistochemical study. concentrations increased. Scheffe method reveals that 150 ppm Egyptian Journal of Experimental Biology 3: 43-47. Retrieved of crude phycocyanin did not significantly inhibit the from http://www.scopemed.org/?mno=188064 angiogenesis on CAM of chick embryo while 200 ppm and 300 ppm inhibit angiogenesis. On the other hand, using the Gayetsky S, Museyko O, Engelke K, Käßer J, Hess A, Schett same statistical method, all experimental treatments of crude G (2014) Characterization and quantification of angiogenesis chlorophyll were able to significantly inhibit the angiogenesis in rheumatoid arthritis in a mouse model using μcT. BMC on the CAM of chick embryos. There is no significant Musculoskeletal Disorders 15:1. DOI: 10.1186/1471-2474-15- difference in the angiogenic effects between crude 298 phycocyanin from S. platensis and crude chlorophyll from C. vulgaris on the CAM of 10-day chick embryo, meaning that Hayashi O, Ono S, Ishii K, Shi Y, Hirahashi T, Katoh T (2006) both phytopigments induced the same effect as angiogenesis Enhancement of proliferation and differentiation in bone inhibitors. marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice. Journal of Applied Phycology 18: 1, 47-56. Conflict Of Interest Retrieved from http://eds.a.ebscohost. com/eds

The authors declare that there is no conflict of interests that Heidenreich R, Röcken M, Ghoreschi K (2009) Angiogenesis could possibly arise. drives psoriasis pathogenesis. International Journal of Experimental Pathology 90: 3, 232-248. DOI: 10.1111/j.1365- Acknowledgements 2613.2009.00669.x

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Boussiba S, Richmond A (1979) Isolation and purification of Kyadari M, Fatma T, Azad R, Velpandian T (2013) Evaluation phycocyanins from blue-green alga Spirulina platensis. of antiangiogenic and antiproliferative potential of the organic extract of green algae Chlorella pyrenoidosa. Indian Journal of

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Effects of crude extracts of various pigments isolated from Spirulina platensis and Chlorella vulgaris on the angiogenesis of chick embryo… 127 Pharmacology 45: 569-574. Retrieved from Isochrisisgalbana. Journal of Food Science 68: 224-226. http://eds.b.ebscohost.com/eds Retrieved from http://eds.b.ebscohost.com/

Mousa SA, Mohamed S (2004) Anti-angiogenic mechanisms West D, Thompson WD, Selis P, Burbridge M (2000) and efficacy of the low molecular weight heparin, tinzaparin: Angiogenesis Assays Using Chick Chorioallantoic Membrane. anti-cancer efficacy. Oncology Report 12:683-688. Retrieved Methods in Molecular Medicine Vol. 46: Angiogenesis from https://www.hoffmancentre.com/assets/files/pdf/ articles Protocols. Humana Press, Inc. Totowa, NJ. Retrieved from http://link.springer.com/protocol/10.1385%2F1-59259-143- Saini MK, Sanyal SN (2014) Targeting angiogenic pathway for 4%3A107 chemoprevention of experimental colon cancer using C- phycocyanin as cyclooxygenase-2 inhibitor. Biochemistry & Yesildal F, Aydin FN, Deveci S, Tekin S, Aydin I, Mammadov Cell Biology 92: 206-218. DOI: 10.1139/bcb-2014-0016 R, Fermanli O, Avcu F, Acikel CH, Ozgurtas T (2015) Aspartame induces angiogenesis in vitro and in vivo models. Surve ZR, Patil SR, Jha SM, Shetty GD, Manekar AP (2015) Human & Experimental Toxicology 34: 3, 260-265. DOI: Angiogenic and antiangiogenic influence of streptozotocin and 10.1177/0960327114537535. insulin on chorioallantoic membrane of chick embryo. Journal of Cell and Tissue Research 15: 4775-4782. Retrieved from Zhao-Na L, Zhong-Fang Y, Guo-Ying M, Ming H, Li-Jun C, http://eds.b.ebscohost.com/ Ya-Li Z, Ming-Xu GE (2015) Augmented anti-angiogenesis activity of polysulfated heparin-endostatin and polyethylene Tokusoglu O, üUnal MK (2003) Biomass nutrient profiles of glycol-endostatin in alkali burn-induced corneal ulcers in three microalgae: Spirulina platensis, Chlorella vulgaris, and rabbits. Experimental & Therapeutic Medicine 10: 3, 889-894. DOI: 10.3892/etm.2015.2602.

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

MENSTRUAL SYNDROME: SEVERITY, FREQUENCY AND SYMPTOMATOLOGY IN ADOLESCENT GIRLS OF QUETTA, PAKISTAN

Faiza Hameed Jan1, Zil E Huma1, Nabeela Tariq1 and Naheed Sajjad2,*

1Department of Zoology, Sardar Bahadur Khan Women’s University, Quetta, Pakistan. 2Department of Biotechnology, Sardar Bahadur Khan Women’s University, Quetta, Pakistan.

Received – February 21, 2016; Revision – March 08, 2016; Accepted – March 27, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).128.132

KEYWORDS ABSTRACT Dysmenorrhea The purpose of the study was to investigate the prevalence of menstrual characteristics, its frequency, Menstruation age of menarche, symptomatology and factors associated with menstrual cycle. A total of 450respondentswere studied in 5 different government girls’ schools of Quetta, Pakistan. Data was Menarche collected by close-ended questionnaire. Analysis of data was carried out by statistical software SPSS version 20 and significant association between different variables was analyzed by chi-square test. Nausea Significant association was reported between food and nausea, amount of blood flow and weakness and vice versa. The study revealed that the mean duration of blood flow was 5-7 days. It has been analyzed Quetta that there is a statistically significant relationship between type of pain and amount of flow (p<0.05). Among the studied group respondents, about 88.89% respondents reported dysmenorrhea. The study concludes that dysmenorrhea was prevalent and no significant association was found between dysmenorrhea and regularity of menstruation. Majority of the girls avoid physical activities like sports and exercise.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Naheed Sajjad) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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129 Faiza et al

1 Introduction al., 2005). The infection can be caused due to the use of unclean or dirty cloth napkins that result in the harboring of The transition period from childhood to adulthood is called microorganisms in vagina. Improperly dried pads also provide adolescence which is characterized by marked differentiation favorable condition to the microbes in vaginal tract to cause in endocrinal, emotional and mental growth. The adolescent infection (Paul, 2007). In India and Pakistan 98.5% girls of period is a preparatory period for girls in which they get both urban and rural areas used old cloth material for pads prepared physically and psychologically for safe motherhood. (Moaweds, 2001).Different prohibitions regarding Being the “direct reproducers” of future generation, the healthy menstruation has been observed such as, entry in the holy adolescent girl is not only responsible for her own health but places, taking bath, changing cloths, combing hairs and for the health of the future population too (Agarwal & restriction on particular food such as, potato, rice, onion, Agarwal, 2010). sugarcane, curd, milk and lassi etc. (Kaplowitz, 2006). Due to the importance of this syndrome, a study was designed to The distinctive feature of female puberty that signals the investigate the frequency, severity, symptomology about reproductive maturity is called menarche (El-Gilanya et al., menstrual syndrome in adolescent school girls in Quetta, 2005). Menstruation shows a remarkable range of variation, Pakistan. most of the women experience it at the age of 10-16 years. The age of menarche varies from population to population 2 Materials and Methods depending upon nutritional, geographical and environmental factors (Kaplowitz, 2006). Adolescent females suffer from A cross-sectional study was designed to explore information various common problems such as premenstrual syndrome, from 450 randomly selected adolescent girls. The respondents amenorrhea and dysmenorrhea (Diaz et al., 2006). The social ranging from 12 to 16 years age, from 5 different government and educational life of 5-10% of the girls is interrupted by schools of Quetta, Baluchistan, Pakistan were briefed about severe dysmenorrhea in their teens (Dawn, 1990). close-ended questionnaires after taking their consent. Prior to data collection, pilot study was also conducted in order to According to the study conducted by Shersha et al. (1991) evaluate feasibility. The questionnaires comprised information among 1600 women in Karachi, the prevalence of regarding the menarche, dysmenorrhea, food preferences, premenstrual syndrome (PMS) was 33%.Within first 2 years of mood fluctuations, type of napkins used, menstrual hygiene, the menarche; the girls face comparatively more problems than effect of physical activities, bathing etc symptoms which were those who have crossed this duration (Ziv et al., 1999). commonly faced by girls during menstruation period. The Reproductive tract infection (RTI) is also associated with collected data was analyzed by statistical software SPSS menstrual hygiene. The unclean and improperly stored napkins version 20. Associations of variable factors were calculated by used during menstruation can cause problems (El-Gilanya et chi-square test.

Figure 1 Source of menstrual guidance. Figure 2 Variety of aids used as absorbents for menstrual bleeding.

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Menstrual Syndrome: Severity, Frequency and Symptomatology in Adolescent Girls of Quetta, Pakistan. 130 Table 1Association between different variables analyzed by chi-square test.

Association between two variables p-value Type of pain and amount of blood flow 0.000 (p<0.05) Amount of blood flow and weakness 0.000(p<0.05) Regularity of menstruation and dysmenorrhea 0.463 (p>0.05) Food and nausea 0.003 (p<0.05)

3 Results and Discussion Further, it was reported that severe abdomen pain was Among the studied 450 respondents, the mean age of menarche experienced by 44% girls who were having heavy menstrual was 12.5 years. Results of present study are in agreement with flow, mild pain was experienced by12% girls having the study conducted by Dasgupta & Sarkar (2008) and reported comparatively light flow and the remaining (44%) individuals the age of menarche was 12.8 years. Similar types of results had a moderate pain and moderate blood flow. Association were also reported by Khanna et al. (2005). Results of present between type of pain and amount of flow was found significant study revealed the effect of guidance on appropriate (p<0.05) (Table 1). knowledge about personal hygiene for menarche and menstruation (Figure 1), mothers were found to be the main Current study revealed that the mean duration of blood flow in source of guidance (55.11% cases), sisters (30.22%), and 48% cases was 5-7 days. The studies conducted by Zegeye et friends (12.22%) while only 2.44% get informed by relatives. al. (2009) and Ali et al. (2011) found the mean duration of The results were similar to Dasgupta & Sarkar (2008) who menstrual blood flow was 4±1.2 days. Current results showed reported mothers to be the first informants followed by friends that weakness was also associated with amount of flow (p< and relatives. Similar findings were reported by Khanna et al. 0.05)(Table 1). The study of Roberts et al. (2003) also (2005) in Rajasthan, India and El-Gilanya et al. (2005) in reported heavy bleeding as cause of weakness among girls. Egypt. The outcomes of the present analysis indicate that, The results of this study suggested equal use of cloth (48%) dysmenorrhea was prevalent in most cases (88.89%) (Figure and branded sanitary pads (48%) while the remaining 4% 3).Similar results was obtained by Zegeye et al. (2009), they preferred the cotton as absorbent (Figure 2). The observations reported the prevalence of dysmenorrhea in 72% cases. Result of Zegeye et al. (2009) were similar to the findings of present suggested that 73% girls showed premenstrual symptoms. No study where as the findings of Moaweds (2001) were association was found between dysmenorrhea and regularity contradictory to the findings of this study, these researchers (p>0.005) (Table 1). Al-Kindi & Al-Bulushi (2011) also reported that98.5% of the Pakistan and Indian girls were used reported that there is no significant association between to wear old cloth materials as sanitary napkin for blood dysmenorrhea and regularity of cycle. absorption.

Figure 3 Prevalence of dysmenorrhea Figure 4 Physical and psychological changes during menstruation.

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131 Faiza et al

Figure 5 Psychological effects of menstruation. Figure 6 Participation of respondents in physical activities (during menstruation).

Variable physical and psychological changes were also reported between food and nausea, amount of flow and experienced during menstruation, 76.22% of the subjects weakness, type of pain and amount of flow. The mean duration suffered physical changes such as enlargement of hips, weight of blood flow was 5-7 days. Depression and anxiety were also gain, swelling in legs etc. and 23.78% of them underwent found among the girls who remained psychologically disturbed psychological changes like depression and anxiety (Figure 4). during this period. Similar findings were also reported by Parsons & Sammers (1978), Jarrett et al. (1995), George & Bhaduri (2002), Lee et Acknowledgement al. (2006). These researchers suggested that physical and psychological changes during menstruation were associated Authors would like to thanks all the subjects who participated with dysmenorrhea. The findings of the present study in the study. regarding psychological behavior indicated that 46.89% subjects complained depression, 8.89% anxiety and the rest of Conflict of Interest 44.22% remained normal during menstruation (Figure 5). A statistically significant association between nausea and food Authors would hereby like to declare that there is no conflict of was observed (p<0.05) (Table 1). Similarly, Wentz (1988) interests that could possibly arise. noted that, during menstruation dysmenorrhea was also the cause of sickness, headache, nausea, diarrhea and vomiting. References Regarding the food habit during period, present survey concludes that 74% respondents prefer variety of food, Agarwal AK, Agarwal A (2010) A study of Dysmenorrhea vegetables and meat. Only 11% preferred to drink milk. during Menstruation in Adolescent Girls. Indian Journal of Dasgupta & Sarkar (2008) reported that 50% girls avoid Community Medicine 35:159-164 DOI: 10.4103/0970- certain foods during menstruation; in this manner their study is 0218.62586 contradictory to the findings of present study. Ali A, Rayis DA, Mamoun M, Adam I (2011) Age at Although, the condition of majority of participants remained menarche and menstrual cycle pattern among schoolgirls in normal but due to taboos, the majority (61.11%) was hesitated Kassala in eastern Sudan. Journal of Public Health and to perform any physical activity, only 38.89% girls took partin Epidemiology 3:111-114 activities like sports and exercise (Figure 6). Lee et al. (2006) reported that physical activity has no relationship with Al-Kindi R, Al-Bulushi A (2011) Prevalence and Impact of menstrual disorders. Similar results have been shown by Dysmenorrhoea among Omani High School Students. Sultan Parsons & Sammers (1978). Qaboos University Medical Journal 11: 485-491 PMCID: PMC3206751 Conclusion Dasgupta A, Sarkar M (2008)Menstrual Hygiene: How It is concluded from the study that dysmenorrhea was strongly Hygienic is the Adolescent Girl? Indian Journal of Community prevalent. The majority of girls avoid physical activities like Medicine 33:77-80 DOI: 10.4103/0970-0218.40872 sports and exercise. Significant association (p<0.05) was also

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Menstrual Syndrome: Severity, Frequency and Symptomatology in Adolescent Girls of Quetta, Pakistan. 132 Dawn CS (1990) Textbook of Gynaecology and Contraception. Moaweds S (2001) Indigenous practices of Saudi girls in 10th edition. Dawn Books, Calcutta, India Riyadh during their menstrual period. Eastern Mediterranean Health Journal 7:197-203PMID: 12596970 Diaz A, Laufer MR, Breech L (2006) Menstruation in girls and adolescents: Using the menstrual cycle as a vital sign. Parsons L, Sammers S (1978) Gynecology. 2nd edition. Pediatrics 118: 2245-2250 Philadelphia: WB. Saunder's Company, 42–5

El-Gilanya A, Badawib K, EL-Fedawyb S (2005)Menstrual Paul D (2007) A report of an ICMR funded research project: hygiene among adolescent school girls in Mansoura, Egypt. Knowledge and practices of adolescent girls regarding Reproductive Health Matters 13:147–152 PMID: 16291496 reproductive health with special emphasis on hygiene during menstruation. New Delhi: National Institute of Public George A, Bhaduri A (2002) Dysmenorrhea among adolescent Cooperation and Child Development (NIPCCD) girls – symptoms experienced during menstruation. Health Promotion and Education17:4. Roberts TA, Glen J, Kreipe RE (2003) Disordered eating and menstrual dysfunction in adolescent female athletes Jarrett M, Heitkemper MM, Shaver JF (1995) Symptoms and participating in school-sponsored sports. Clinical Pediatrics self-care strategies in women with and without dysmenorrhea. (Philadelphia) 42:561-564 Health Care for Women International16:167-78 PMID: 7759347 Shersha, Syed, John JM, Sadiqa J (1991) Prevalence of premenstrual syndrome in Pakistani women. Journal of the Kaplowitz P (2006) Pubertal development in girls: Secular Pakistan Medical Association 41:101-103 trends. Current Opinion in Obstetrics and Gynecology 18:487- 491PMID: 16932041 Wentz AC (1988) Dysmenorrhoea, Premenstrual syndrome and related disorders. Novak's textbook of Gynaecology. Khanna A, Goyal RS, Bhawsar R (2005) Menstrual practices Baltimore: Williams and Wilkins, 240–60 and reproductive problems: a study of adolescent girls in Rajasthan. Journal of Health Management 7:91–107 Zegeye DT, Megabiaw B, Mulu A (2009) Age at menarche and menstrual pattern of secondary school adolescents in Lee LK, Chen PCY, Lee KK, Kaur J (2006) Menstruation northwest Ethiopia. BMC Women’s Health 9: 29 DOI: among adolescent girls in Malaysia: a cross-sectional school 10.1186/1472-6874-9-29 survey. Singapore Medical Journal 47:869–874 PMID: 16990962. Ziv A, Boulet JR, Slap GB (1999) Utilization of physician offices by adolescents in the United States. Pediatrics 104:35- 42

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

COMPLEMENTARY AND ALTERNATIVE MEDICINES AMONG RESIDENTS IN UPLAND CAVITE, PHILIPPINES: THEIR KNOWLEDGE, PATTERNS OF USE AND ATTITUDES

Johnny A Ching1,2,*, Rodante G Flores2,3 and Maxima J Acelajado2

1Biological Sciences Department, College of Science and Computer Studies, De La Salle University-Dasmariñas, Cavite, Philippines 2Graduate Studies Department, College of Science and Computer Studies, De La Salle University-Dasmariñas, Cavite, Philippines 3PATTS College of Aeronautics, Paranaque, Metro Manila, Philippines

Received – February 03, 2016; Revision – February 17, 2016; Accepted – March 30, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).133.141

KEYWORDS ABSTRACT Complementary and alternative medicine The study was aimed to determining the knowledge, patterns of use, and attitude towards the use of complementary and alternative medicines (CAM) by herbalists (n=38) and lay people (n=582) from the Herbal medicines municipalities of Indang, Maragondon, Amadeo, Magallanes, and Alfonso and the city of Tagaytay, all located in the upland portion of the province of Cavite. Data pertinent to the afore-said aims were Upland Cavite gathered through questionnaires and focus group discussions. Nine forms of CAM were documented as used by the interviewees. Leaf extract (decoction) and traditional massage (hilot)was the most prevalent types of CAM across all studied areas, contributing 71% (n= 440) and 54% (n= 335), respectively. Furthermore, their widespread utilization was mainly due to the recommendations from family members (31%, n=192) and doctors (35%, n= 217). Amongst the various ailments associated with the use of CAM, common colds (70%, n= 434), cough (69%, n=428), and fever (61%, n= 378) were the most treated. Overall, a generally positive attitude towards the use of all forms of CAM recorded in this study was shown by most participants in all the surveyed municipalities/city. Therefore, present study concludes that CAM plays a major health care role amongst the interviewees.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Johnny A Ching) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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134 Ching et al

1 Introduction 2 Materials and Methods

The practice of complementary and alternative medicines 2.1 Study area and data collection (CAM) is very common in many countries of the world, including where modern biomedical healthcare is predominant The study was carried out in Indang, Maragondon, Amadeo, and readily available (Shaikh et al., 2008). CAM is a group of Magallanes, Alfonso and Tagaytay city located in the upland diverse medical and healthcare systems, therapies, and portion of the province of Cavite. A total of 620 respondents products that are not presently considered to be part of modern (38 herbalists and 582 lay people/users of CAM), average of medicine. A therapy is generally considered to be as 100 participants from each municipality and city, were complementary when it is used in combination with purposely selected. conventional treatments while it is considered as alternative when it is used in place of the modern treatment (Koh et al., Data were collected using a pre-tested questionnaires and focus 2004). Recognizing that there can be some overlapping group discussions (Erasto et al., 2005). The protocol was amongst the therapies, the National Center for Complementary approved by the Research Ethics Committee under the and Integrative Health (NCCIH) (2012)formerly the National University Research Office (URO) of De La Salle University– Center for Complementary and Alternative Medicine Dasmariñas, and informed consent was obtained from all the (NCCAM) of the US Department of Health and Human respondents, who were willing to take part in the study, prior to Services categorized CAM practices into two subgroups: the data collection. Self-administered questionnaires and group natural products or mind and body practices. Natural products discussion as adapted from previous studies (Furnham, 2000; include use of a variety of biologically-based products such as Xue et al., 2007; Shaikh et al., 2008) but with a sight herbs or botanicals, vitamins and minerals, and probiotics. modification was aligned to gather information relating to the These products are widely-marketed, readily available as respondents’ knowledge, patterns of use, and attitude towards dietary supplements. Mind and body practices includes a large the use of CAM. and diverse group of practices or procedures usually administered by a trained practitioner such as massage therapy, The questionnaires were distributed to each of the respondents. acupuncture, yoga, qi gong, and meditation. Those who needed assistance in terms of understanding and filling-up the questionnaire were assisted by their family Literature-based evidences exist indicating that the practice of relatives. The completed questionnaires were retrieved by the CAM is common in most developing countries including India researchers three weeks after the distribution, and (Singh et al., 2004; Jadhav et al., 2011; Roy et al., 2015), subsequently, a face-to-face interview and focus group Malaysia (Lee et al., 2007; Sivadasan et al., 2014; Silvanathan discussion with respondents was conducted to gather other & Low 2015), Africa (Matheka & Demaio 2013; McFarlane, relevant information that might not have captured by 2015), and Mexico (Valenzuela-Antelo et al., 2011; Marshik et respondents. al., 2016). This is therefore suggests that CAM is an important aspect of public health care in these countries. Studies also 2.2 Data analysis highlighted that there is an increasing prevalence of CAM use among Western physicians practicing in developed countries Comparison technique and descriptive statistics such as (Bensoussan et al., 2004; Koh et al., 2004; Wolf et al., 2006; percentage/frequencies was used to analyze certain data Stange et al., 2008). The apparent reversal of trend from collected across all the studied municipalities/city. modern to traditional medicine is partly attributed to the Furthermore, Likert scale was used to evaluate the attitude of multifaceted factors including the fact that synthetic drugs the respondents towards CAM, with 5 representing very strong have always shown adverse reactions and other undesirable and 1 representing very weak responses. side effects (Erasto& Majinda, 2011). Results and Discussion In addition, the high cost of administering modern drugs, which is beyond the reach of many people in the low income A total of 620 respondents with ages ranging from 16 to 68 group and of those living in the rural areas is also responsible years old from the selected city and municipalities in upland for this trend (Erasto et al., 2005). Regardless of the earlier Cavite disclosed that they utilize CAM, thus suggesting that eluded studies reporting on the significant of CAM in various this sort of therapy is common amongst all age brackets in the countries, there are still dearth of information relating to the study areas. There was almost an equal ratio between male utilization of this therapy, particularly the knowledge, patterns (48%; n = 300) and female (52%, n= 320) respondents and of use, and attitude towards its use by residents in the upland most of them attained only up to secondary education. This portions of the province of Cavite, Philippines. The present finding therefore, buttresses the common knowledge that most study was therefore initiated as an attempt to contribute of the users of CAM obtained lower educational qualifications. towards this gap in knowledge.

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Complementary and alternative medicines among residents in upland cavite, Philippines: their knowledge, patterns of use and attitudes. 135 Table 1 Different form of complementary and alternative medicines used by residents (in percent) of selected city and municipalities representing upland Cavite.

Forms of CAM Municipalities/City

Alfonso Amadeo Indang Maragondon Mendez Tagaytay n = 116 n = 98 n = 100 n = 100 n = 94 n = 112 Leaf extract/decoction 64 78 78 81 68 56 Traditional massage 47 68 68 45 48 46 Herbal tablets 33 8 55 24 7 11 Rituals - 43 - 29 6 1 Plastering of leaves 3 4 - 22 2 1 Western massage (spa) - - - 11 - - Prayers and murmuring - - - 13 - - Yoga and meditation - - - 5 - - Qigong - - - 6 - -

3.1 Forms of CAM used by the Respondents noted are plant derives. On the other hand high preferences of herbal medicine in a form of tablet by interviewees from Table 1 shows the various forms of CAM being used by the Indang compared to those residing in other studied areas, is respondents from the upland areas of the province of Cavite. suggestive of the respondents’ preference for readily available Drinking of decoction or leaf extracts of medicinal plants and medicines in pack rather than for raw herbal medicines, which traditional massage are the two most popular forms of CAM in requires preparation. Figure 1 shows an over-all graphic all sampling areas. Other forms of CAM being used to some comparison of the most common forms of CAM used by the extent include drinking herbal tablets, performing different residents of upland Cavite. It is clear from the graph that rituals and plastering of leaves. drinking of leaf extract or decoction and traditional massage represent more than 70% of the combined responses in almost Respondents from Maragondon showed more varied forms of all surveyed areas. This finding is somewhat in line that CAM compared to other places in the upland area, such as previously reported by Salibay et al. (2007) and Arsenio et al. going to modern spa and practicing yoga and meditation. This (2011) in the Philippines. Furthermore, Shaikh et al. (2008) might be due attributed to the diversity of medical knowledge who also conducted a similar study in Pakistan reported possessed by inhabitants from Maragondon as opposed to other comparable finding to present study. On contrary a study municipalities. The opposite of this might be true amongst the carried out in Australia reported acupuncture as predominant participants from Indang who reported the least forms of CAM (Bensoussan et al., 2004). This disparity might be CAM. On one hand the least noted types of CAM amid attributed to the variation in knowledge or preferred form of participants to some extent indicate that they rely on medicinal CAM amongst the Australians and Filipinos. plants. This is ascribed to the fact that most sorts of CAM they

Figure 1 Over-all forms of CAM used by the residents of upland Cavite.

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136 Ching et al

Table 2 Reasons given by the residents (in percent) for using CAM of selected city and municipalities representing upland Cavite.

Reasons for using CAM Municipalities/City

Alfonso Amadeo Indang Maragondon Mendez Tagaytay n = 116 n = 98 n = 100 n = 100 n = 94 n = 112 Recommended by medical practitioner 21 59 44 - 23 42 Recommended by friends or family 45 20 61 41 1 13 To treat health problems 28 27 21 22 33 16 Natural and therefore safer 1 8 - 38 5 1 For maintenance of general health 3 3 2 5 8 1 Dissatisfaction with conventional medicine - 3 - 6 - - To relieve stress - 4 - 8 - -

However, the least utilization of other forms of CAM in this Indang, and Maragondon were persuaded by family members study was expected. For instance, limited use of Western for using different forms of CAM. Given that most of the massage might be due to its high cost which is way beyond the respondent are old people, this finding suggest the practice of reach of many participants in this study. Yoga and meditation CAM amid participants in this municipalities is an old tradition might be least utilised due to the fact that its time consuming. passed from one generation to another. On contrary, people Surprisingly, prayer and murmuring was expected to be questioned in the Tagaytay City and municipality of Amadeo featured amongst the top used CAM, as most of the Filipinos use CAM as ordered by doctors, thus suggesting that most of are religious. inhabitants in this areas reliant on western health care services for their well-being. The results obtained from Mendez showed 3.2 Reasons for using CAM that participants use CAM from probable positive past experiences. Overall the various reasons as outlined in Table 2, Based from the interview and survey conducted, there were put forth by participants for utilizing CAM is obviously eight primary reasons why the residents in the upland areas of associated with either tradition or trust that they have in the Cavite tend to practice CAM. Table 2 below shows the various person who recommended them. The over-all reasons for using reasons given by the respondents. Six major reasons were CAM by the residents of upland Cavite are graphically identified by the respondents why they prefer to use CAM presented in figure 2. (Table 2). Respondents from the municipalities of Alfonso,

Figure 2 Over-all reasons for using CAM by the residents of upland Cavite.

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Complementary and alternative medicines among residents in upland cavite, Philippines: their knowledge, patterns of use and attitudes. 137 Table 3 Ailments associated with the usage of CAM as given by the residents (in percent) of selected city and municipalities representing upland Cavite.

Disease associated for using Municipalities/City traditional medicine

Alfonso Amadeo Indang Maragondon Mendez Tagaytay n = 116 n = 98 n = 100 n = 100 n = 94 n = 112 Common colds 64 78 77 91 60 49 Cough 64 73 77 89 61 50 Fever 64 71 77 59 50 42 Flu 10 41 8 26 2 5 High blood pressure 4 9 13 2 2 4 Muscle pains 3 12 7 4 9 4 Diabetes 2 9 2 - 9 4 Abdominal pains - 3 3 12 - 1 Asthma 2 - 4 - 3 - Skin diseases 1 1 2 1 2 1 UTI 1 5 - - 1 - Diarrhea 1 - 1 - - -

One of the reasons given by respondents in this study for 3.3 Ailments Associated with usage of CAM practicing CAM, particularly leaf extract/decoction, traditional massage and herbal tablets is due to both the abundant and The top three ailments identified by the respondents associated availability of plants resource, a similar finding which is with CAM were common colds, cough, and fever. This finding common among Filipino families. This fact is to some extent are in harmony with those previously noted by Arsenio et al. supported by more than 40 medicinal plant species (Appendix (2011) in Philippines, and Shaikh et al. (2008) in Pakistan, 1) reported by participants the across all the studied areas. both reporting colds, cough and fever are being amongst the Other pressing reasons for the use of herbal medicine, reported top ailments treated with CAM. On contrary, Strange et al. in this study which are comparable to previous studies (2008) reported hypertension and cancer as highly treated (Furnham, 2000; Kuo et al., 2004; Salibay et al., 2009) include disorders in Germany. This might be an indication that these their high potency and safeness as compared to synthetic diseases are prevalent in Germany. medicines.

Figure 3 Over-all ailments associated with the use of CAM by the residents of upland Cavite.

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138 Ching et al

Table 5 Attitude towards CAM by the residents (in percent) of selected city and municipalities representing upland Cavite.

Attitude Towards CAM Municipalities/City

Cavite

Alfonso Amadeo Indang Maragondon Mendez Tagaytay Upland Very Strong 18 6 0 12 10 0 8.2 Strong 39 58 44 52 28 25 33.4 Fair 33 13 56 34 56 87 57.6 Weak 0 3 0 2 0 0 1.0 Very Weak 0 3 0 0 0 0 0.5 Over-all Scale 3.94 3.74 3.56 3.74 3.51 3.22 3.62 Interpretation Strong Strong Strong Strong Strong Fair Strong

Sicknesses which were less treated with the different forms of conviction of the efficacy of different forms of these therapies CAM in present study comprised of complicated diseases such for preventive and treatment of some common ailments. as diabetes, hypertension, and kidney disorders. Use of CAM as therapies for these complicated ailments might be attributed Conclusions to the patients’ hope for total cure, this might be especially true since some of such illnesses notably diabetes and hypertension, Leaf extract/decoction and traditional massage (hilot) emerged are chronic in their nature (Table 3). In general different as the most common forms of complementary and alternative ailments treated by respondents with CAM in the present medicines used by the respondents. These therapies were both might be an indication of their extent of prevalent in the ranked first and second, respectively, in all municipalities/city studied area. representing upland Cavite. The present study further found that the questioned participants were mainly persuaded by their It is quite interesting to note that all respondents were family members and doctors to utilise CAM. unanimous in their choice of top three diseases in which CAM is used. These are the three most common illnesses which The principal afflictions treated with CAM in upland Cavite normally are non-life threatening, whereas those illnesses encompassed common colds, cough, and fever. Overall, which can be considered more serious such as heart problem remedies used for treatment of these ailments were mainly and anemia seemed to have not been treated with CAM as prepared from herbal medicines. The four municipalities shown in figure 3. representing upland Cavite rated the use of CAM as strong while Tagaytay City rated it with fair. In general, survey 3.4Attitude towards CAM revealed a positive attitude towards CAM among residents of the study sites with an over-all rating of 3.62 interpreted as Participants residing in the municipalities of Alfonso, Amadeo, strong. Maragondon, and Mendez in upland Cavite responded positively on the use of CAM, having a mean range of 3.51 to Conflict of interest 3.94 and with a descriptive interpretation of strong. This is to some extent is a reflection that the practice of CAM amongst Authors would hereby like to declare that there is no conflict of the people inhabiting these areas is rooted in their culture, thus interests that could possibly arise. future studies similar to ours, should target such areas for rich data collection. However, the opposite of these can be said Acknowledgments with regards to participants from the city of Tagaytay who rated the use of CAM as fair, with a mean rating of 3.22. The researchers would like to thank De La Salle University– Table 6 below summarizes the attitude of the respondents Dasmariñas through the University Research Office, for the towards the use of CAM. funding of this research. Furthermore we are indebted to the residents of Tagaytay City and municipalities of Indang, Table 5 shows that, generally respondents from upland Cavite Maragondon, Amadeo, Magallanes, and Alfonso for their strongly agreed on the use of CAM, with a mean rating of 3.62. willingness and cooperationduring data collection. This suggests that attitudes of the public regarding CAM therapies have remarkably become positive. Furthermore, is References show that this type of therapies is becoming increasingly acceptable amongst general public in the areas studied. The Arsenio JJ, Medecilo M, Mercado ET, Salibay ET Jr, Valera strong attitude displayed by the respondents in the present AH (2011) The Vegetation of Mt. Maculot, Cuenca Batangas, study towards the use of CAM might be ascribed to the strong

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Complementary and alternative medicines among residents in upland cavite, Philippines: their knowledge, patterns of use and attitudes. 139 Philippines. International Journal of Environmental Science Kenyan perspective. Pan African Medical Journal 15. DOI: and Development 2: 274-278. 10.11604/pamj.2013.15.110.2925.

Bensoussan A, Myers SP, Wu SM, O’Connor K (2004) Mc Farlane C (2015) South Africa: The Rise of Traditional Naturopathic and Western herbal medicine practice in Medicine. Insights on Africa 7: 60 – 70. doi: Australia: A workforce survey. Complementary Therapies in 10.1177/0975087814554070. Medicine 12: 17 – 27. DOI: http://dx.doi.org/10.1016/j.ctim.2004.01.001. Roy V, Gupta M, Ghosh RK (2015) Perception, attitude and usage of complementary and alternative medicine among Erasto P, Majinda RRT (2011) Bioactive proanthocyanidins doctors and patients in a tertiary care hospital in India. Indian from the root bark of Cassia abbreviata. International Journal Journal of Pharmacology 47: 2, 137-142. DOI: 10.4103/0253- of Biological and Chemical Sciences 5(5): 2170-2179. doi: 7613.153418. http://dx.doi.org/10.4314/ijbcs.v5i5.36 Salibay CC, Aoanan G, Aspra MET, Unabia T, Perrido E Erasto P, Adebola PO, Grierson DS, Afolayan AJ (2005) An (2007) Tenured migrants’ resource utilization of Mts. ethnobotanical study of plants used for the treatment of Palaypalay/MataasnaGulod National Park, Luzon Island, diabetes in the Eastern Cape Province, South Africa. African Philippines. Journal of Science and Technology 1: 62-72. Journal of Biotechnology 4:1458 – 1460. Salibay CC, Aoanan G, Aspra MET, Unabia T, Perrido E Furnham A (2000) Attitudes towards homoeopathy in (2009) The socio-cultural practices and beliefs of the teunured particular and beliefs about complementary medicines in migrants and their impact on Mts. Palaypalay/Mataasna Gulod general. Psychology, Health and Medicine 5: 327 – 342. forest resources. Journal of Science and Technology 3:34-46. DOI:10.1080/713690198. Shaikh SH, Malik F, James H, Abdul H (2008) Trends in the Jadhav MP, Jadhav PM, Shelke P, Sharma Y, Nadkar M use of complementary and alternative medicine in Pakistan: a (2011) Assessment of use of complementary alternative population-based survey. Journal of Alternative and medicine and its impact on quality of life in the patients Complementary Medicine15: 545 – 550. doi: attending rheumatology clinic in a tertiary care centre in India. 10.1089/acm.2008.0232. Indian Journal of Medical Sciences 65: 50-57. DOI: 10.4103/0019-5359.103961. Silvanathan S, Low BS (2015) Current public awareness on the safety of traditional and complementary medicines in Koh HL, Ng HL, Teo HH (2004) A survey of knowledge, Malaysia. European Journal of Integrative Medicine 7 :184- attitudes and usage of complementary and alternative medicine 189. DOI: 10.1016/j.eujim.2014.12.003 in Singapore. Asia-Pacific Biotech News 8 : 1266 – 1270. DOI: 10.1142/S0219030304002162. Singh V, Raidoo DM, Harries CS (2004) The prevalence, patterns of usage and people’s attitude towards complementary Kuo GM, Hawley ST, Weiss LT, Balkrishnan R, Volk RJ and alternative medicine (CAM) among Indian community in (2004) Factors associated with herbal use among urban Chatsworth, South Africa. BMC Complementary and multiethnic primary care patients: A cross-sectional survey. Alternative Medicine 4:1-7. DOI: 10.1186/1472-6882-4-3. BMC Complementary and Alternative Medicine 4:18. DOI: 10.1186/1472-6882-4-18. Sivadasan S, Ali AN, Lin LW, Balakrishnan D, Ramachandran S, Dhanaraj SA (2014) Use of complementary and alternative Lee PY, Taha ABA, Lin K, Ghazali SR, Almashoor SHSA medicine in the population of Kedah Darul Aman, Malaysia. (2007) Usage of complementary and alternative medicine International Journal of Pharmaceutical Sciences and Research among primary care clinic attendees, Kuching, Sarawak, 5:4, 1263 – 1273. DOI: 10.13040/IJPSR.0975-8232.5(4).1263- Malaysia, January – April 2004. Asia Pacific Journal of Family 63 Medicine 6:1-6. Stange R, Amhof R, Moebus S (2008) Complementary and Marshik PL, Kharat AA, Jakeman B, Borrego ME, Dodd MA, alternative medicine: Attitudes and patterns of use by German Bachyrycz A, Anderson JR, Bond R, Deming P, Mercier R, physicians in a national survey. Journal of Alternative and Nawarskas JJ, Ray G, Salazar K (2016) Complementary and Complementary Medicine 14: 1255-1251. doi: Alternative Medicine and Therapy Use in a Diverse New 10.1089/acm.2008.0306. Mexican Population. Journal of Alternative & Complementary Medicine22: 1, 45-51. DOI: 10.1089/acm.2014.0378. Valenzuela-Antelo O, Garibay-Escobar A, Velázquez- Contreras C, Navarro-Navarro M, Contreras LR, Corral OL, Matheka DM, Demaio AR (2013) Complementary and Lozano-Taylor J (2011) Use of Complementary and alternative medicine use among diabetic patients in Africa: A Alternative Medicine in a Region of Northwest Mexico.

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Journal of Alternative & Complementary Medicine 17: 787 - Xue CL, Zhang AL, Lin V, Da Costa C, Story DF (2007) 788. DOI: 10.1089/acm.2011.0234 Complementary and alternative medicine use in Australia: A national population-based survey. The Journal of Alternative Wolf U, Maxion-Bergemann S, Bornhoft G, Matthiessen PF, and Complementary Medicine 13: 643 – 650. Wolf M (2006) Use of complementary medicine in doi:10.1089/acm.2006.6355. Switzerland. Forsch Komplementarmed 13:S4-S6.

Appendix 1. Herbal plants used by residents of upland Cavite to treat common ailments.

Botanical Family Scientific name Common name Part/s used Method of Preparation

Fever Annonaceae Annona muricata L. Guyabano Leaves Decoction Asteracee Artemisia vulgaris L. Maria Leaves Decoction Fabacecae Tamarindus indica L. Sampalok Fruits Eaten Leaves/stems Bath Lythraceae Lagerstroemia speciosa (L.) Pers. Banaba Leaves Poultice Orchidaceae Grammatophyllum scriptum L. Blume. Tawa-tawa Leaves Decoction Oxalidaceae Averrhoa carambola L. Balimbing Leaves Decoction Verbenaceae Vitex negundo L. Lagundi Leaves Decoction Common colds Asteraceae Blumea balsamifera (L.) DC. Sambong Leaves Decoction Moringaceae Moringa oleifera Lam. Malunggay Leaves Extraction Orchidaceae Grammatophyllum scriptum L. Blume. Tawa-tawa Leaves Decoction Phyllantaceae Phyllantrus niruri L. Sampa-sampalukan Leaves Decoction Cough and flu Asteraceae Artemisia vulgaris L. Maria Leaves Decoction Blumea balsamifera (L.) DC. Sambong Leaves Decoction Annonaceae Annona muricata L. Guyabano Leaves Decoction Bignoniaceae Tabebuia avellanedae Lorentz ex Taheebo Leaves Decoction Griseb. Crassulaceae Bryophyllum pinnatum (Lam.) Oken. Kataka-taka Leaves Decoction Fabaceae Gliricidia sepium (Jacq.) Kunth ex Kakawati/ malabe Leaves/fruits Decoction Steud. Euphorbiaceae Croton tigilium L. Gasi. (Sul.) Tuba Leaves Decoction Lamiaceae Origanum vulgare L. Oregano Leaves Decoction Lythraceae Lagerstroemia speciosa (L.) Pers. Banaba Leaves Decoction Mackinlayaceae Centella asiatica (L.) Urb. Takip-suso Leaves Decoction Orchidaceae Grammatophyllum scriptum L. Blume. Tawa-tawa Leaves Decoction Piperaceae Piper betle L. Ikmo Leaves Poultice Rutaceae Citrus microcarpa Bunge. Kalamansi/ Leaves Decoction Kalumunding Fruit Eaten Klaba Leaves Extraction Verbenaceae Vitexnegundo L. Lagundi Leaves Decoction

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Complementary and alternative medicines among residents in upland cavite, Philippines: their knowledge, patterns of use and attitudes. 141

Wound Myrtaceae Psidium guajava L. Bayabas Leaves Washing as antiseptic Solanaceae Polanisia iconsandra (L.) Wight and Sili-silihan Leaves Poultice Arn. Fabaceae Cassia alata L. Akapulko Leaves Extraction Mimosa pudica L. Makahiya Leaves Poultice Urinary tract infection (UTI) Asteraceae Blumea balsamifera (L.) DC. Sambong Leaves Decoction Annonaceae Annona muricata L. Guyabano Leaves Decoction Fabaceae Cassia alata L. Akapulko Leaves Decoction Lythraceae Lagerstroemia speciosa (L.) Pers. Banaba Leaves Decoction Stems Decoction Poaceae Imperata cylindrica (L.) Beauv. Kogon Roots Decoction Zea mays L. Corn/mais Silk Decoction Lamiaceae Orthosiph onaristatus (Blume) Miq. Balbaspusa Leaves Decoction Piperaceae Pepperomia pellucida L. Pansit-pansitan Leaves Eaten as raw

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

ANALYSIS OF LONG TERM IMPACT OF CYCLONIC DISASTER ‘AILA’ ON SOIL PROPERTIES AND PADDY YIELD OF SUNDARBANS, WEST BENGAL, INDIA

Sisir Kumar Si

Vivekananda Institute of Biotechnology, Sri Ramakrishna Ashram, P.O.-Nimpith Ashram, South 24-Parganas (Sundarbans), West Bengal, 743338

Received – November 06, 2015; Revision – November 28, 2015; Accepted – April 04, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).142.148

KEYWORDS ABSTRACT

Aila A field study was conducted to evaluate the long term impact of cyclonic disaster ‘Aila” on soil properties and paddy yield. Study was conducted during four consecutive rabi season started from 2009- Cyclone 10 onwards after the exposure of Aila at three highly Aila affected regions of Sundarbans viz Achintanagar (Patharprotima block), Kuemuri (Patharprotima block) and Shyamnagar (Kultali block) Land degradation villages. Results of the study were compared to the adjoined non Aila affected area after soil test based Paddy crop fertilizer application (STNPK). Results of study revealed that Aila degraded the quality of paddy cultivation land by increasing EC 3 dSm-1 , ESP 18 and 2 to 2.7 times available iron over non Aila Fertilizer affected area along with completely failure of paddy production (average yield 0.45 t/ha) in 2009-10 season. Results of next consecutive three seasons revealed that soil pH, EC, ESP is improved and consequently paddy yield also increased naturally as compare to the yield of previous years in Aila affected area as well as yield of non Aila affected area in three villages. After third year of Aila

occurrence, it was reported that paddy yield increase average 14% on application of extra 25%P2O5, average 8% on application of extra 25% K2O and average 15% on application of extra 25% NPK over STNPK (Soil test based NPK dose)treatment. These fertilizer treatments ultimately made yield gap between Alia affected and non Aila affected area after third year of Aila occurrence. This is perhaps due

to extra P2O5 fulfilled the P requirement of paddy in Fe rich Aila affected soils and at the same time

extra K2O increased availability of K to paddy by maintaining the activity ratio of K / √( Ca +Mg) in saline environment of Aila affected areas.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Si SK) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

143 Si SK

1 Introduction The nature’s fury ‘Aila’ in the form of massive sea tidal waves on 25th May, 2009 created devastation not only in terms of Salinity is an important index of low soil quality reducing crop animal lives and loss of infrastructure in coastal Sundarban, production. The amount of salt that is to accumulate is a but it also caused complete submergence of adjoining function of water quality (Ayers & Westcot, 1985) and agricultural fields and rendered the soil and water resources evapotranspiration (Abrol, 1982). In arid and semi-arid salt affected. The cultivated land in coastal Sundarban is regions, high rates of evapotranspiration and low rainfall can situated in low lying areas where drainage is often difficult due result in large amounts of salt accumulation near the soil to very low slope gradient. Sea water ingression at the time of surface. Whereas irrigated agriculture using saline water and Aila led to stagnation situations in the agricultural land of ingression of sea water in land can lead to salt accumulation in Sundarban and this situation varies according to areas; in some soil profile, reduction in yield and deterioration in soil area it was purely temporary and receded after temporary resource. Several researchers such as Feizi, 1993 and Garcia- stagnation while in some are it stayed for long time and land Sanchez et al., 2003 reported advantageous effects of leaching remained stagnation with sea water for long time. by irrigation as well as rain water on improvement of soil salinity and crop yield. It was reported that this type of cyclones completely affect the agricultural production and in the passage of next few years Soil of Sundarban has been affected by different types of salt; farmers faced poor soil health conditions and this thing characterization and distribution of soil in Sundarban coastal hampered crop production. Hence, there was an emergent need area was earlier described by Bandyapadhyay & Sarkar, 1987; to assess the changes of soil characteristics and search suitable Bandyapadhyay et al., 2001 and Bandyapadhyay et al., 2003. ways to reverse the degradation processes through optimal soil These soils are highly acidic as well as saline in nature (Yadav management measures. Therefore, present study has been et al., 1979) and it was formed from the gangetic alluvium due undertaken to find out the impact of Aila on the characteristic to the accumulation of sulphide materials of inter-tidal of Achintanagar (Patharprotima block), Kuemuri sediments. Acidic and saline natures are characteristics of (Patharprotima block) and Shyamnagar (Kultali block) area such soils and play a critical role in the nutrient uptake, which soil and rice yield. Furthermore, study also investigated to needs to be assessed for judicious and efficient management of effect of fertilization (types and dose of fertilizers) on the sustainable agriculture. retention and regaining of the soil quality and in improvement of the rice production.

Fig1: Study area map of Sundarbans, W.B., India

3 2 1.Achintanagar and 2. Kuemuri (Patharprotima) 3.Shyamnagar (Kultali) 1

Figure 1 Study area map of sunderbans, W.B. India.

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Analysis of long term impact of Cyclonic Disaster ‘Aila’ on soil properties and paddy yield of Sundarbans, West Bengal, India. 144 2 Materials and Methods occurrence, to study the yield gap between Aila affected and non Aila affected areas, selected farmer’s fields were divided With a view to the above objectives, collection and analysis of into five equal plots of approximate size 133m2 for application soil samples along with field studies of boro paddy (Lal of five fertilizer treatments viz soil test based recommended Minikit, WGL 20471) were undertaken from selected Aila 100% NPK dosed for boro paddy ( STNPK) ; STNPK + Extra affected and non Aila affected area of three villages in 25% nitrogen ( STNPK+ 25% N); STNPK + Extra 25% 0 0 Sundarbans (lies between longitude 88 10’E – 89 51’E , phosphate( STNPK+ 25% P2O5); STNPK + Extra 25% 0 0 latitude 21 31’N – 22 30’ N) viz. Achintanagar (Patharprotima potash( STNPK+ 25% K2O) and STNPK+ Extra 25% block), Kuemuri (Patharprotima block) and Shyamnagar nitrogen, phosphate and potash( STNPK+ 25% NPK) as (Kultali block) in south 24 Parganas district, West Bengal recommended by Ali, 2005 and boro paddy yields of the five (Figure. 1). Ten farmers of each Aila affected area and plots of each farmers of Aila affected and non affected area of adjoined non Aila affected area in the aforesaid three villages three villages are recorded. were selected for present study. Surface soil samples (0-15 cm) were collected from farmers’ fields of each village The data are presented statistically and relative productivity covering entirely three different Aila affected areas and percentage of crops is calculated as adjoined non Aila affected areas. Soil samples were air-dried, Average Paddy ground and pass through 2mm sieve. Soil properties viz., pH ------X 100 (1:2), EC (1:2) soil: water ratio and organic carbon were Maximum average paddy yield of non Aila affected areas determined as par the procedure giving by Jackson, 1973 and Walkley & Black’s method (Black , 1965). The ESP value was 3 Results and Discussion calculated by determination of cation exchange capacity (CEC) The value of selected soil characteristics viz. pH, EC, OC, ESP of the soil by extracting with neutral 1N NH4OAc solution and exchangeable sodium by flame photometer using standard and available Fe in Aila affected and non Aila affected laboratory methods (Page, 1986). Achintanagar, Kuemuri and Shyamnagar villages of Sundarban was recorded for 2009-10 and three consecutive years after Available Fe was determined by Atomic Absorption Aila, results of these characteristics are presented in table 1. Spectrometric (AAS) technique using DTPA extraction. Yield The table 1 revealed that the overall soil pH of non Aila of boro paddy (2009-10) of selected farmers were recorded affected Achintanagar is acidic in nature with an average pH separately. Similarly next consecutive three years of Aila value 5.24 where as nature of soil pH of Aila affected occurrence, soil test based fertilizer application and same Achintanagar is neutral with an average pH value 6.93. It may agronomic management, analytical soil data as well as paddy be due to accumulation of carbonate and bicarbonate salt from yield (Lal Minikit) were recorded. After the third year of Aila sea water at the time of Aila occurrence.

Table 1 Soil properties of Achintanagar, Kuemuri and Shamnagar village in Sundarban after Aila (value given in table is mean of ten replicates)

Time interval pH EC dSm-1 ESP OC(%) Fe(mg/kg) Achintanagar Non Aila Affected area 5.24±0.49 1.23±0.46 10.40±3.82 0.40±0.13 37.97±25.23 Aila Affected area 6.93±1.04 11.02±2.56 30.72±7.86 0.46±0.20 101.64±167.82 1st. Year after Aila 6.7±0.83 2.40±0.69 17.37±6.78 0.46±0.19 102.29±171.47 2nd. Year after Aila 6.51±0.77 137±0.52 12.49±4.75 0.44±0.19 99.72±164.79 3rd. Year after Aila 6.55±0.69 1.30±0.38 12.00±3.00 0.42±0.16 100.51±161.41 Kuemuri Non Aila Affected area 5.36±0.65 1.36±0.68 8.81±2.89 0.43±0.11 46.69±26.64 Aila Affected area 6.63±1.05 12.13±3.99 31.75±8.74 0.57±0.23 126.42±207.63 1st. Year after Aila 6.47±0.93 2.61±0.96 17.26±7.52 0.55±0.20 124.21±204.23 2nd. Year after Aila 6.34±0.69 1.66±0.91 12.46±6.17 0.53±0.21 123.21±204.32 3rd. Year after Aila 6.39±0.71 1.57±0.79 11.95±3.49 0.49±0.16 121.35±201.68 Shyamnagar Non Aila Affected area 5.37±0.55 1.19±0.89 11.69±1.89 0.43±0.13 32.66±22.97 Aila Affected area 6.98±0.93 10.33±4.38 40.71±8.09 0.45±0.20 59.10±84.92 1st. Year after Aila 6.71±0.72 2.19±1.17 24.59±5.72 0.44±0.19 58.61±82.52 2nd. Year after Aila 6.51±0.52 1.43±1.12 18.66±5.19 0.42±0.18 56.84±80.01 3rd. Year after Aila 6.60±0.48 1.34±0.80 17.59±3.90 0.39±0.15 55.73±76.89 CD(P= 0.05 0.127 0.496 1.96 … 17.22 Value given in table is mean of ten replicates; value give after ± represent SD

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145 Si SK

It is also reported that after first year, in successive years of 3% samples are at injury level (>3 dSm-1 ) in non Aila affected Aila, the soil pH has slowly come down. The soil pH of Aila areas whereas in Aila affected areas 100% samples are at affected Kuemuri and Shyamnagar was also showing similar injury level with respect to EC values (Ali, 2005). After first, trends to Achintanagar with respect to non Aila affected areas. second and third year of Aila the injury level have become The average soil EC of non Aila affected Achintanagar is 1.23 30%, 10% and 3% samples respectively. On encountering the dSm-1 and the average values of ESP is 10.40 (Table 1). While whole three villages, the status of soil pH are 53% samples are the average EC 11.02 dSm-1 and ESP 30.72 is attained after strong acidic (pH<5.5), 37% samples are moderately acidic Aila occurrence at Achintanagar. With passage of first, second (pH 5.5- 6.0) and 10% slightly acidic (pH 6.0-6.5) in non Aila and third year after Aila, the mean EC value come down in 2.4 affected area, whereas in Aila affected areas the status of soil dSm-1, 1.37 dSm-1 and 1.30 dSm- 1 respectively Whereas the pH are 30% alkaline (pH>7.5) 26% neutral (pH 6.5-7.5),20% mean ESP value come down in 17.37, 12.49 and 12.00 slightly acidic (pH 6.0-6.5),20% moderate acidity (pH 5.5-6.0) respectively at Achintanagar. The reason of improvement of and rest are strong acidic. After first year of Aila occurrence soil salinity on passage of time is the effect of salt leaching i.e. the pH status are almost unchanged but in second and third desalinization by rainwater infiltration (Mostafazdeh-Fard et year about 40% and 54% samples have neutral (pH 6.5-7.5) al., 2008). The same trend of EC and ESP values are observed and rest soils are slightly to moderate acidity (pH 5.5-6.5). in Kuemuri and Shamnagar village at different time intervals There are no soils in alkaline range (pH>7.5) in second and with respect to non Aila affected area. third year after Aila.

The average OC percentage of the non Aila affected On the other hand, 73% collected samples shows presence of Achintanagar soils is recorded 0.40 whereas in Aila affected low OC while 27% samples shows the presence of medium OC area, it is 0.46. It is also found a decreasing tendency of means in non Aila affected areas, whereas in Aila affected areas, the OC values after passing of first, second and third year may be status of OC had became 53% low, 27% medium and 20% due to effects of rain water leaching as well as land use for high. These changes in the status of OC may be due to paddy (Kolahchi & Jalali ,2007). Similar patterns are also deposition of fresh organic matter during the flood. After first observed in others two Aila affected areas (Table 1). Analysis year of Aila occurrence the OC status are unchanged but in of the results presented in the table 1, suggested that about second year only 13% OC are high level (OC % >0.75) and in 40% samples are in safe limit (ESP <10) and 60% samples are third year the OC status are almost equal to non Aila affected moderate safe limit (ESP 10-18) in non Aila affected area of areas i.e. 64% low and 36% medium. Higher analytical values three localities. Whereas after Aila only 3% samples are of OC leads the blackish coloration of soils in some fields after moderately safe limits (ESP 10-18) and the rest are in higher Aila which may be resulted by the dissolution and dispersion ESP (ESP>18) which are unsafe for cultivation (Richards , of organic matter at high pH and ESP(Table 1), which is also 1954). The passage of first, second and third year after Aila the agreement of the observation of Bandyapadhyay et al. 64%, 40% and 23% samples became unsafe (ESP >18) (2001). respectively in the aforesaid areas. It is also found that about

Table 2 Yield of boro paddy at farmers’ field in Sundarban after Aila

Time interval Mean ±SD Relative productivity (%) Achintanagar Non Aila Affected area 5.18±0.23 100.0 Aila Affected area 0.48±0.37 8.80 1st. Year after Aila 2.89±038 55.74 2nd. Year after Aila 3.58±0.41 69.20 3rd. Year after Aila 3.66±0.43 70.63 Kuemuri Non Aila Affected area 4.91±0.26 94.90 Aila Affected area 0.48±0.27 9.33 1st. Year after Aila 2.75±0.33 53.01 2nd. Year after Aila 3.43±0.41 66.22 3rd. Year after Aila 3.49±0.50 67.44 Shyamnagar Non Aila Affected area 5.04±0.26 97.40 Aila Affected area 0.38±0.26 7.30 1st. Year after Aila 3.00±0.41 57.94 2nd. Year after Aila 3.51±047 67.73 3rd. Year after Aila 3.55±0.50 68.54 CD(P= 0.05) 0.09 Value given in table is mean of ten replicates

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Analysis of long term impact of Cyclonic Disaster ‘Aila’ on soil properties and paddy yield of Sundarbans, West Bengal, India. 146 Table 3 Yield of boro paddy on different fertilizer treatments at farmers’ field in Sundarban after third years’ of Aila occurrence

Different treatments Mean ±SD Relative productivity (%) Achintanagar STNPK 3.66±0.43 70.63 STNPK+ 25% N 3.71±0.46 71.73

STNPK+ 25% P2O5 4.29±0.36 82.85

STNPK+ 25% K2O 3.93±0.45 75.84 STNPK+ 25% NPK 4.42±0.46 85.40 Kuemuri STNPK 3.49±0.50 67.44 STNPK+ 25% N 3.52±0.36 67.90

STNPK+ 25% P2O5 4.39±0.35 84.70

STNPK+ 25% K2O 4.06±0.45 78.33 STNPK+ 25% NPK 4.52±0.41 87.20 Shyamnagar STNPK 3.55±0.50 68.54 STNPK+ 25% N 3.60±0.45 69.52

STNPK+ 25% P2O5 4.26±0.40 82.33

STNPK+ 25% K2O 3.96±0.35 76.54 STNPK+ 25% NPK 4.37±0.53 84.36 CD(P= 0.05) 0.09

Whereas: STNPK (100% NPK dosed for boro paddy); STNPK+ 25% N (STNPK + Extra 25% nitrogen); STNPK+ 25% P2O5(STNPK +

Extra 25% phosphate);STNPK+ 25% K2O(STNPK + Extra 25% potash)and STNPK+ 25% NPK(STNPK + Extra 25% nitrogen, phosphate and potash).

The average DTPA-Fe content in three non Aila affected there found a yield gap between Aila affected areas and non Achintanagar; Kuemuri and Shyamnagar villages are 37.97, Aila affected areas after third year (Table 2). It may be due to 46.69 and 32.66 mg/kg (Table 1). Whereas in these three Aila the high concentration of iron and comparatively higher value affected villages the average Fe content increases remarkably of EC and ESP in compare to non Aila affected areas (Table 1) and their values are 101.64, 126.42 and 59.10 mg/kg has still hampered the paddy production in Aila affected areas. respectively. There is no remarkable change in average value of Fe with passage of first, second and third year after Aila. Based on five fertilizer treatments i.e. STNPK , STNPK+ 25%

N, STNPK+ 25% P2O5, STNPK+ 25% K2O and STNPK+ 25% The yield of boro paddy and their average value along with NPK in Aila affected areas of three Villages, the average their relative productivity percentage in Aila affected and non paddy yield in rabi season, after third year of Aila are recorded Aila affected areas of Achintanagar; Kuemuri and Shyamnagar in table 3. The Paddy yield of Aila affected Achintanagar, villages in consecutive four seasons are presented in table 2. Kuemuri and Shyamnagar villages are remarkably increased of Considering total non Aila affected areas of three villages, the an average 4.29, 4.39 and 4.26 t/ha by application of STNPK+ average yield of boro paddy lie between 4.91 and 5.18 t/ha, 25% P2O5, 3.93, 4.06 and 3.96 t/ha by application of STNPK+ whereas considering total Aila affected areas of three villages 25% K2O, 4.42, 4.52 and 4.37 t/ha by application of STNPK+ lie between 0.38 and 0.48 t/ha. Next successive three boro 25% NPK over the average yield 3.66, 3.49 and 3.55 t/ha based seasons the average yield of total Aila affected areas of three on STNPK treatment in three respective villages. On the other villages have increased naturally and their average values lie hand,, STNPK+ 25% N treatment increases slightly paddy between 2.75 and 3.00 t/ha in first year, 3.43 and 3.58 t/ha in yield of an average 3.71, 3.52 and 3.60 t/ha with respect to second year, 3.49 and 3.66 t/ha in third year (Table 2) STNPK treatment in three respective villages. respectively. Table 3 also shows that the relative productivity percentage

Table 2 shows that the relative productivity percentage in three 82.85, 84.70, 82.33 on treatment STNPK+ 25% P2O5 and Aila affected villages and these are 8.80, 9.33 and 7.30. It is 85.40, 87.20, 84.36 on treatment STNPK+ 25% NPK in Aila also observed that relative productivity percentage of boro affected areas of Achintanagar, Kuemuri and Shyamnagar paddy in total three Aila affected areas of three villages have villages are very close to non Aila affected areas of respective been increased (ranged from 53.08 to 57.94) after first year of three villages and simultaneously it is agreement with the same Aila occurrence. After passage of second and third years, the land suitability class for paddy according to Sys et al. (1993). range became 66.22 to 69.20 and 67.44 to 70.63 respectively. From the results of five fertilizer treatments (Table 3), it may Through the average paddy yield in third year increased be said that recommended phosphate are less available to slightly in compare to the yield of second year after Aila, yet paddy perhaps due to high concentration of Fe in Aila affected

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147 Si SK soils (Adhikari & Si, 1994) which fixed most of recommended Bandyapadhyay AK, Sarkar D (1987) Occurrence of acid phosphate. When extra phosphate over recommended dose is saline soils in coastal area in Sundarban area of West Bengal. applied then paddy response and yield is increased. On the Journal Indian Society of Soil Science 35: 42-44. other hand, saline environment of Aila affected soils with high amount of Ca and Mg decrease the activity ratio K / √ (Ca + Bandyapadhyay BK, Maji B, Sen HS, Tyagi NK (2003) Mg).So, plant face problem to uptake available K (Joshi, Coastal saline soils of West Bengal, Their nature, distribution

1992). Extra K over recommended dose (STNPK+ 25% K2O) and characteristics, CSSRI Regional Research Station, Bull no. lead increases the activity ratio K / √ (Ca + Mg) and 1 West Bengal, India, 62. consequently increases the uptake of available K as well as significantly paddy yield. Bandyapadhyay BK, Maji B, Sen HS, Yadav JSP (2001) Saline and alkaline soils and their management, ISCAR Conclusions Monograph no. 1, Indian Society of Coastal Agricultural. Research, West Bengal, India. Present study shows that cyclone Aila affected the lands of Sundarban by making various types of degradation like high Black CA (1965) Methods of soil analysis, Part 1 & 2, salinity, alkalinity and Fe content and it resulted poor crop American Society of Agronomy, Inc. Madison, Wisconsin, performance. Result of study indicated that poor growth of USA. paddy is due to high salinity (>3 dSm-1 ) and high alkalinity (ESP>18) which gradually reduced by leaching with rain water Feizi M (1993) Investigation of the effects of water quality and in passage of consecutive three years. The yield of boro paddy quantity on soil desalinization of Rudasht region of Isfahan is further improved by application of extra phosphate and extra province, Journal Soil Water 8:16-34. potash over soil test based fertilizer dose that enhance phosphorous and potassium availability to paddy in Fe richer Garcia-Sanchez F, Carvajal M, Porras I, Botia P, Martinez V saline environment. (2003) Effects of salinity and rate of irrigation on yield, fruit quality and mineral composition of lemon. European Journal Acknowledgement Agronomy 19: 427-437. doi:10.1016/S1161-0301(02)00138-7.

The author is grateful to Swami Sadananda Maharaj, Joshi DC (1992) Relationship between the quantity and Chairman, Sri R.K. Ashram, Nimpith, Dr. B. K. Datta, intensity parameters of labile potassium in arid sols of Indian Director, Vivekananda Institute of Biotechnology, Nimpith and desert. Journal Indian Society of Soil Science 40: 431-442 Prof. A. K. Sarkar, Former Professor and Head (Soil Science) and Dean Faculty of Agriculture, Birsa Agricultural Jackson ML (1973) Soil chemical analysis. Prentice Hall of University, Ranchi, Jharkhand for necessary help and India Pvt. Ltd., New Delhi. continuous encouragement. Kolahchi Z, Jalali M (2007) Effect of water quality on the Conflict of interest leaching of potassium from sandy soil. Journal Arid Environment 68:624-639. doi:10.1016/j.jaridenv.2006.06.010. Authors would hereby like to declare that there is no conflict of interests that could possibly arise. Mostafazadeh-Fard B, Haydarpour M, Aghakhani A, Feizi M (2008) Effects of leaching on soil desalinization for wheat crop References in an arid region. Journal Plant Soil Environment 54:20-29

Abrol IP (1982) Reclamation and management of salt affected Page AL (1986) Methods of soil analysis, Part II: Soil soils. Review of soil research in India, Part II, Trans. 12th chemical analysis. American Society of Agronomy, Inc. International Congress Soil Science, New Delhi. Madison, Wisconsin, USA.

Ali SK (2005) Fertilizer recommendation for principal crops Richards LA (1954) Diagnosis and improvement of saline- and cropping sequences of West Bengal, Booklet no. 1, alkaline soils, United State Department of Agriculture, Department of Agriculture, Government of West Bengal, 4-5. Agriculture Hand Book, 60.

Ayers RS, Westcot DW (1985) Water quality for agriculture, Sys Ir C, VanRanst E, Debaveye Ir J, Beernaert F (1993) Land irrigation and drainage, Paper no. 29, FAO, Rome. Evaluation, Part II, Crop requirements, Agricultural publications no 7, General administration for development Adhikari M, Si SK (1994) Distribution of inorganic cooperation place du champ de mars 5 bte 57-1050, Brussels, phosphorous fractions in some soils of West Bengal. Journal Belgium, 117-124. Indian Society of Soil Science 42:459-461.

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Analysis of long term impact of Cyclonic Disaster ‘Aila’ on soil properties and paddy yield of Sundarbans, West Bengal, India. 148 Yadav JSP, Bandyapadhyay AK, Rao KVGK, Sinha TW, Biswas CR (1979) Bulletin Central Soil Salinity Research Institute 5: 34.

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

HARINGHATA BLACK CHICKEN SHOWS RESISTANCE TO PATHOGENIC E. coli BY HIGH IMMUNE-EFFECTOR ACTIVITIES

Sudip Nandi1, Siddhartha N. Joardar1,*, Indranil Samanta1, Bithi Roy1, Pradip K. Das2, Tapas K. Sar3 and Sk Sahanawaz Alam1

1Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Belgachia, Kolkata-700037,West Bengal, India 2Department of Veterinary Physiology (RKVY Laboratory), West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Belgachia, Kolkata-700037,West Bengal, India 3Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Belgachia, Kolkata-700037,West Bengal, India

Received – January 25, 2016; Revision – February 21, 2016; Accepted – April 11, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).149.155

KEYWORDS ABSTRACT

Broiler To substantiate the popular notion regarding disease-resistance potentiality of native breed of poultry against pathogens, immune responses in one native breed of India (Haringhata Black, HB), one of best Disease resistance adapted exotic breed of backyard system (Rhode Island Red, RIR) and commercial broiler birds against E. coli virulent E. coli were assessed upon experimental inoculation. Immunological parameters measured were superoxide production by heterophils, macrophage phagocytic activity, in vitro lymphoproliferation and Haringhata black antibody production against the antigens of E. coli. HB and RIR birds showed more cellular immune responses than that of broiler. Moreover, anti- E. coli antibody production was observed highest in HB Poultry than RIR and broiler as assessed by ELISA. Clinical manifestations in form of diarrhoea, roughned feather, rise of temperature as observed in broiler and RIR were not detected in HB birds. It is Rhode Island Red concluded that high immune responses shown by HB birds against virulent E. coli might be the driving factor of disease resistance as opposed to broiler and RIR birds where clinical symptoms were discernible.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Siddhartha N. Joardar) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

150 Nandi et al

1 Introduction observation up to 21 days. Neither the sensitized group nor the control group of birds in the present study for each category The poultry and farm animals are considered as major reservoir (HB, RIR, Broiler) received any kind of antibiotic during the of E. coli and Salmonella throughout the world without any study period. clinical syndrome (Girlich et al., 2007; Carattoli, 2008; Watson et al., 2012; Samanta et al., 2014a). However, mode of disease 2.4 Antigen resistance generated in native breeds of poultry against E. coli and its underlying immunological mechanism is not yet known The somatic soluble antigen of the E. coli isolate (SK-3) was (Samanta et al., 2014b). Therefore, it is quite expedient to extracted by ultrasonication on ice at 150W with repeating study immune response patterns in native breeds of poultry to duty cycles and 0.5 sec pulse pressure for two min with 30 sec explore their scale of disease resistance against pathogenic E. interval (five times) using an ultrasonicator (Hielscher coli (Samanta et al., 2015). Further, reports on antigenic Ultrasonics GmBH, Germany). The soluble sonicated extract characteristics of the target pathogen are scarce that might be was centrifuged at 10,000 g for 30 min at 4°C and the useful while considering immunodiagnostic and/ or supernatant was collected as antigen as described earlier (Choi immunoprophylactic preparations against such pathogen (Roy et al., 1989). The somatic soluble antigen was kept at -20ºC for et al., 2015). With this background the present study was further use. conducted to assess cellular and humoral immune responses upon experimental inoculation of pathogenic E. coli in one 2.5 Protein Estimation native breed (Haringhata Black, HB), one of best adapted exotic breed of backyard system (Rhode Island Red, RIR), and Protein concentration of somatic soluble antigen was estimated commercial broiler birds in West Bengal, a major backyard using commercially available protein estimation kit (Merck and native breed rearing state in India. Biosciences, India). The absorbance was measured at 660 nm by UV-VIS Spectrophotometer (TechComp, Taiwan). 2 Materials and Methods 2.6 Leucocyte functional assay 2.1 Experimental bird To measure non-specific immune response of poultry birds Rhode Island Red, Haringhata black and Broiler birds (3 against pathogenic E. coli, oxidative radical production by months old) were used as experimental birds procured from the heterophils was determined by the nitroblue tetrazolium (NBT) University Farm at Mohanpur, Nadia. Each variety of birds (SRL, India) reduction assay at different days post inoculation was divided into two groups viz. control group & sensitized (DPI) period (Siwicki et al., 1998). group, and each group contained 6 birds. 2.7 Isolation of PBMC 2.1 Experimental design Heparinised blood was diluted with PBS at 1:1 ratio. For All the experimental birds were given normal feed (Amrit isolation of peripheral blood mononuclear cell (PBMC), feedTM, India) containing maize, soyabean, ground nut cake, diluted blood was layered onto HiSepR (Himedia, India) at the mineral mixture, vitamin up to the end of the experiment with ratio of 2:3 (2 part of HiSep and 3 parts of cell suspension) and ad libitum feed and water. The period of observation was centrifuged for 30 min at 400g (Chung & Secombs, 1988). started from the day of inoculation (0-day) with subsequent Using density gradient centrifugation, mononuclear cells from post inoculation periods, viz. 0, 2nd, 7th, 14th and 21st days after blood of poultry of the three varieties were isolated at different inoculation. The Institute Ethics Committee of West Bengal DPI as earlier. Enumeration of viable cells was done using University of Animal and Fishery Sciences, India approved Trypan blue dye exclusion method. In most of the cases > 90 this study. % of the cells were viable.

2.3 Sensitization of experimental bird 2.8 Phagocytosis assay

After 7 days of acclimatization in experimental cages of the In vitro phagocytosis made by blood monocytes of the Department, all the birds of one group (sensitized) of each inoculated poultry birds was examined in different DPI as per variety were inoculated (I/P) with virulent field isolate of Yoshida et al. (1993). The Phagocytic activity (PA) was ESBL producing E. coli [strain- SK-3, serogroup - O62, determined as per Findlay & Munday (2000) using the genotype- bla TEM (+ve), bla CTX-M (-ve), bla SHV (-ve)] bacteria following formula- (109 CFU/ml dose). The field isolate (SK-3) was obtained from Number of phagocytic cell a local broiler (29 days old) which was suffering from PA = ------X 100 (expressed as %) diarrhoea, fever, roughened feather and was un-responding to Total number of phagocytes higher group of cephalosporin antibiotics. Another group of birds were kept as control. The birds were maintained for

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Haringhata black chicken shows resistance to pathogenic E. coli by high immune-effector activities 151 2.9 Stock solutions for ConA and bacterial antigen 3 Results and Discussion

Stock solution of concanavalin A (Con A) was prepared at the Present study was aimed to detect changes in immune effector concentration of 1mg/ml of the growth medium (RPMI-1640), activities in three breeds of poultry viz. Haringhata Black, a filtered through a sterile membrane filter (0.22µ) and stored at native breed of India, Rhode Island Red, a backyard breed and -20⁰C for future use. Stock solution of bacterial somatic broiler bird upon experimental inoculation of pathogenic E. soluble antigen was prepared at the concentration of 40µg/ml coli isolate. The study revealed the comparative role of with the growth medium (RPMI). immune effector activities in three different poultry breeds by which they can resist pathogenic E. coli. The E. coli strain 2.10 Lymphoproliferation Assay (LPA) (SK-3) was selected for experimental inoculation because it was isolated from a local diseased broiler. Further, the isolate 6 PBMC (2 X 10 cells /ml) were suspended in RPMI-1640 and possessed bla TEM gene which is one of the major ESBL genes 100 µl of cell suspension was dispended into wells of 96-well produced by E. coli (bla TEM, bla SHV, bla CTX-M) and the bacteria tissue culture plates. The final volume of each well was made of reservoir poultry chiefly harbours bla TEM (Olsen et al., up to 200 µl with somatic antigen (20µg/ml), Con A (10 2014). µg/ml) (Sigma-Aldrich, USA) in positive control well and growth medium in negative control well. Finally, the plate was The broiler birds inoculated with pathogenic E. coli showed ⁰ incubated at 37 C for 72 hr under 5% CO2 tension. The the clinical syndrome such as high fever, roughened feather colorimetric 4,5- dimethylthiazol-2,5- diphenyltetrazolium and diarrhea within 7 days. In RIR birds intensity of diarrhea bromide (MTT) (Sigma-Aldrich, USA) assay described by was less than the broiler birds. HB chickens didn’t show any Daly et al. (1995) was used to determine the proliferation of observable clinical sign and symptom. After 30 days, blood lymphocytes in response of somatic antigen. sensitized broiler birds died of the infection, although both of the sensitized HB and RIR birds survived. Probably the higher 2.11 Indirect Enzyme Linked Immunosobent Assay (i-ELISA) disease resistance potential in HB birds was responsible for the development of less severe clinical manifestation which was To detect anti- E. coli antibody in serum samples of RIR, HB investigated in present study. and broiler birds (collected at 21 DPI), plate ELISA was performed based on the principle of indirect-ELISA as per Super oxide anion produced by heterophils in E. coli Mockett et al. (1987) with some modifications. inoculated Haringhata Black, Rhode Island Red and Broiler birds was significantly (p<0.05) higher during first 15th DPI, 2.12 Statistical analysis however, lower in 21st DPI in all the variety in comparison to the control birds (Table-1). Higher super oxide anion The results of leukocyte functional assay, phagocytosis assay production by leucocytes of all the sensitized birds was and lymphoproliferation assay were expressed as the corroborated with earlier works where significantly enhanced mean±standard error of mean (SEM) and analysed using SPSS super oxide anion production was reported in treated chicken 21 (SPSS Inc. Chicago, USA). and turkey than control groups (Lowry et al., 2005; He et al., 2008; Paul et al., 2012).

Table 1 Assessment of super oxide production by blood leukocytes of E. coli inoculated (sensitized) and control birds at different days post inoculation by NBT reduction test.

Types of bird Status of bird 0-day 2nd day 7th day 14th day 21st day HB Sensitized 0.426±0.009b 1.892 ±0.005a 1.745±0.007 a 1.085±0.183a 0.490±0.003c Control 0.413±0.003b 0.399±0.002b 0.406±0.001b 0.418±0.009b 0.421±0.005b RIR Sensitized 0.321±0.004b 1.576 ±0.003a 1.559±0.005a 1.168±0.040a 0.315±0.008c Control 0.318±0.004b 0.347 ±0.006b 0.325±0.007 b 0.321±0.008b 0.319±0.011b Broiler Sensitized 0.495±0.022b 1.128±0.001a 1.221±0.004a 1.022±0.029a 0.530±0.005c Control 0.487±0.010 b 0.469±0.004b 0.413±0.003b 0.492±0.010 0.471±0.010b HB-Haringata Black, RIR-Rhode Island Red b Results shown are mean of six observations ± SEM The mean bearing different superscript in the same row and column differs significantly (p<0.05)

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152 Nandi et al

Table 2 Phagocytic activity of PBMC of E. coli inoculated (sensitized) and control birds at different days post inoculation

Types of bird Status of bird 0-day 2nd day 7th day 14th day 21st day HB Sensitized 15±0.032c 22±0.023b 21±0.023b 19±0.024b 27±0.033a Control 15±0.023c 16±0.021c 14±0.026 c 14±0.031 c 16±0.028c RIR Sensitized 18±0.024c 24±0.022b 25±0.021b 24±0.025b 30±0.031a Control 17±0.026 c 15±0.030c 16±0.032c 15±0.032c 16±0.027c Broiler Sensitized 13±0.023b 19±0.022a 18±0.028a 17±0.028a 13±0.023b Control 12±0.031b 11±0.033b 12±0.032b 11±0.034b 14±0.024b HB-Haringata Black, RIR-Rhode Island Red Results shown are mean of six observations ± SEM The mean bearing different superscript in the same row and column differ significantly (p<0.05)

The phagocytic activity (PA) of PBMC of E. coli inoculated significantly (p<0.05) differed on 14th DPI. An increase in and control HB, RIR and Broiler birds are presented in Table - stimulation index was noticed on 14th DPI (0.363 & 0.301) but 2. The sensitized broiler birds showed little higher PA values that decreased at 21st DPI (0.218 & 0.183). Similar significant (19±0.022) at 2nd DPI which was decreased (13±0.023) in 21st enhanced proliferation of blood leucocytes was detected in DPI. Whereas, sensitized HB and RIR birds showed higher PI treated broiler chicken (Lee et al., 2007). Moreover, in vitro values at 2nd (22±0.023 and 24±0.022), 7th (21±0.023 and lymphocyte proliferation of peripheral blood leucocytes (PBL) 25±0.021), 14th (19±0.024 and 24±0.025) and 21st (27±0.033 was reported in treated broiler chicken that gave 100% and 30±0.031) DPI than the control birds. In short, higher protection in IBD virus challenge (Tayade et al., 2006). In the phagocytic activity of HB and RIR PBMC was observed than present study, high specific cellular stimulation (in form of the broiler birds and it was maintained up to 21st DPI. Similar lymphocyte proliferation) was noted up to 21st DPI in HB birds significant higher phagocytic potential of macrophages in than RIR and broiler that might be related to its enhanced treated chicken was reported by previous workers against E. immune potential. coli (Qureshi, 1998). The seroreactivity expressed as O.D. values were 0.82±0.021 Lymphocyte proliferation upon conA and antigen stimulation in sensitized HB birds, 0.74±0.032 in sensitized RIR and of blood leucocytes of experimental birds are expressed as 0.47±0.017 in sensitized broiler birds (Figure 1) with stimulation index (Table -3). Sensitized HB birds showed experimental sera at 1:400 dilution, respectively. Whereas, the similar SI values on the first day (0.413 & 0.359) with that of O.D. values were detected from 0.583±0.032; 0.534±0.022 and control birds (0.401 & 0.343) but it significantly (p<0.05) 0.433±0.025, respectively for the same range of dilution with differed on 14th and 21st DPI. An increase in stimulation index the sera of control birds. The humoral immune -effector was noticed on 14th day (0.478 & 0.462) and 21st day (0.522 & activity measured by indirect ELISA to detect anti- E. coli 0.517) post inoculation. Sensitized RIR birds showed similar antibody in serum samples of RIR, HB, and broiler birds SI values on the first day (0.413 & 0.359) with that of control showed enhanced seroreactivity in all the sensitized than the birds (0.439 & 0.285) but it significantly (p<0.05) differed on control birds. Similar enhanced seroreactivity in E. coli 14th and 21st DPI. An increase in stimulation index was sensitized birds were observed by Mukherjee (2006) and Mitra noticed on 14th (0.478 & 0.327) and 21st DPI (0.534 & 0.473). (2007) using i-ELISA. In the present study, highest Sensitized broilers showed similar SI values (0.237 & 0.192) seroreactivity was noted in HB birds compared to RIR and with that of control (0.194 & 0.183) on 0-day but it brolier birds indicating more immune-potential of the breed.

Table 3 Lymphoproliferation assay of PBMC of E. coli inoculated (sensitized) and control birds at different days post inoculation

Types of Status of 0-day 7th day 14th day 21st day bird bird ConA Antigen ConA Antigen ConA Antigen ConA Antigen HB Sensitized 0.413±0.009b 0.359±0.016c 0.471±0.011b 0.402±0.021b 0.478±0.013b 0.462±0.021b 0.522±0.006a 0.517±0.019a Control 0.401±0.006b 0.343±0.008c 0.400±0.009c 0.384±0.006c 0.434±0.021b 0.384±0.006c 0.422±0.008b 0.351±0.010c RIR Sensitized 0.439±0.006b 0.285±0.009c 0.421±0.014b 0.327±0.013c 0.478±0.011b 0.327±0.013c 0.534±0.010a 0.473±0.003a Control 0.421±0.010b 0.273±0.003c 0.422±0.010b 0.265±0.003c 0.425±0.013b 0.265±0.003c 0.429±0.011b 0.281±0.006c Broiler Sensitized 0.237±0.010c 0.192±0.004d 0.269±0.002c 0.301±0.006c 0.363±0.010c 0.301±0.006c 0.218±0.010c 0.183±0.003d Control 0.194±0.007d 0.183±0.005d 0.231±0.005c 0.175±0.006d 0.241±0.005c 0.178±0.006d 0.243±0.007c 0.201±0.005d HB-Haringata Black, RIR-Rhode Island Red Results shown are mean of six observations ± SEM The mean bearing different superscript in the same row and column differ significantly (p<0.05)

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Haringhata black chicken shows resistance to pathogenic E. coli by high immune-effector activities 153

Figure 1 Assessment of anti-E. coli O62 antibodies in poultry birds by enzyme linked immunosorbent assay (Serum dilution-1:400; Results shown are mean of six observations ± SEM) .

Thus, the present study showed enhanced cellular and humoral Acknowledgements immune effector activities in native and backyard poultry breed than the test broiler birds against pathogenic E. coli. This work was supported by the grant (BT/164/NE/TBP/2011) Similarly, Munir et al. (2010) detected a virulent Newcastle of Twinning Programm for NE, sponsored by The Department disease virus in backyard birds without any clinical syndrome of Biotechnology, Ministry of Science and Technology, probably due to enhanced immune effector activities which Government of India, New Delhi. Authors are thankful to the was unexplored in the conducted study. Further, in a Vice Chancellor, West Bengal University of Animal and comparative study between a native chicken (Erlang Fishery Sciences, Kolkata for providing necessary mountainous chicken) and commercial broiler in China, native infrastructure ficilities. No financial interests or benefits are chicken breed showed higher resistance against Marek’s arising from the direct applications of the present study. disease virus than the broiler due to difference in expression pattern of transcription factor (IRF-3) and interferon (IFN-훽) Conflict of Interests genes associated with genetic background (Feng et al., 2013). Emam et al. (2014) also detected the possible role of genetic Authors would hereby like to declare that there is no conflict of background in different immune response (in vitro blood interests that could possibly arise. mononuclear cell proliferation) between native chicken and broiler birds against sheep red blood cell and Brucella antigen. References

Conclusions Carattoli A (2008) Animal reservoirs for extended-spectrum beta-lactamase producers. Clinical Microbiology and Infection The present study detected dynamic changes in cellular and 14:117-123. DOI: 10.1111/j.1469-0691.2007.01851.x humoral immune responses in all the poultry breeds/strain used upon exposure to pathogenic E. coli during study period. Choi KH, Maheswaran SK, Felice LJ (1989) Characterization However, HB birds showed higher cellular and humoral of outer membrane protein enriched extracts from Pasteurella immune responses against pathogenic E. coli than the other test multocida isolated from turkeys. American Journal of birds that corroborates their resistance from experimental Veterinary Research 50: 676-683. infection. Continuation of the study may affirm the disease resistance potential of this native breed at genetic level.

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Chung S, Secombes C J (1988) Analysis of events occurring Mockett AP, Cook JK, Huggins MB (1987) Maternally- within teleost macrophages during the respiratory burst. derived antibody to infectious bronchitis virus: its detection in Comparative Physiology and Biochemistry 89: 539-544. chick trachea and serum and its role in protection. Avian Pathology 16: 407-416. DOI:10.1080/03079458708436391. Daly JG, Olivier G, Moore AR (1995) A calorimetric assay for the quantification of brook trout (Salvelinus fontinalis) Mukherjee R (2006) Isolation and immunobiochemical lymphocyte mitogenesis. Fish and Shellfish Immunology 5: characterization of sonicated extract, secretory and membrane 266-273. proteins of E. coli isolates of dog. M. V. Sc. Thesis submitted to West Bengal University of Animal and Fishery Sciences, Emam M, Mehrabani-Yeganeh H, Barjesteh N, Nikbakht G, Kolkata. Thompson-Crispi K, Charkhkar S, Mallard B (2014) The influence of genetic background versus commercial breeding Munir M, Abbas M, Khan MT, Zohari S, Berg M (2012) programs on chicken immunocompetence. Poultry Science Genomic and biological characterization of a velogenic 93: 77-84. doi: 10.3382/ps.2013-03475. Newcastle disease virus isolated from a healthy backyard poultry flock in 2010. Virology Journal 16: Feng ZQ, Lian T, Huang Y, Zhu Q, Liu YP (2013) Expression 9-46. DOI: 10.1186/1743-422X-9-46. pattern of genes of RLR-mediated antiviral pathway in different-breed chicken response to Marek’s Disease virus Olsen RH, Bisgaard M, Löhren U, Robineau B, Christensen H infection. BioMed Research International. doi: (2014) Extended-spectrum β-lactamase-producing Escherichia 10.1155/2013/419256 coli isolated from poultry: a review of current problems, illustrated with some laboratory findings. Avian Findlay VL, Munday BL (2000) The immunomodulatory Pathology 43:199-208. doi: 10.1080/03079457.2014.907866. effects of levamisole on the salmon, Salmo salar L. Journal of Fish Disease 23: 369-378. DOI: 10.1046/j.1365- Paul I, Isore DP, Joardar SN, Samanta I, Biswas U, Maiti TK, 2761.2000.00231.x. Gaguly S, Mukhopadhyay SK (2012) Orally administered β- glucan of edible mushroom (Pleuratus florida) origin up Girlich D, Poirel L, Carattoli A, Kempf I, Lartigue M, Bertini regulates innate immune response in broiler. Indian Journal of A, Nordmann P (2007) Extended-Spectrum β-Lactamase CTX- Animal Sciences 82: 745-748. M-1 in Escherichia coli isolates from healthy poultry in France. Applied and Environmental Microbiology 73: 4681- Qureshi MA (1998) Role of macrophages in avian health and 4685. doi: 10.1128/AEM.02491-06. disease. Poultry Science 77: 978-982.

He H, Genovese KJ, Swaggerty CL, Nisbet DJ, Kogut MH Roy B, Joardar SN, Samanta I, Das PK, Alam SS, Nandi S (2008) Differential induction of nitric oxide, degranulation, (2015) Detection of T- and B- cell antigens of fowl pox virus and oxidative burst activities in response to microbial agonist isolated from backyard chickens in India. Avian Diseases 59: stimulations in monocytes and heterophils from young 249-254. doi: 10.1637/11031-020415-Reg. commercial turkeys. Veterinary Immunology and Immunopathology 123: 177-185. doi: Samanta I, Joardar SN, Das PK, Sar T, Bandyaopadhyay S, 10.1016/j.vetimm.2008.01.033. Dutta TK, Sarkar U (2014a) Prevalence and antibiotics resistance profiles of Salmonella serotypes isolated from Lee SH, Lillehoj HS, Chun HK, Tuo W, Park HJ, Cho SM, backyard poultry flocks in West Bengal, India. Journal of Lee YM, Lillehoj EP (2007) In vitro treatment of chicken Applied Poultry Research 23: 1-10. doi: 10.3382/japr.2013- peripheral blood lymphocytes, macrophages, and tumor cells 00929. with extracts of Korean medicinal plants. Nutrition Research 27: 362-366. doi:10.1016/j.nutres.2007.04.001. Samanta I, Joardar SN, Das PK, Das P, Sar, TK, Dutta TK, Bandyopadhyay S, Batabyal S, Isore D (2014b) Virulence Lowry VK, Farnell MB, Ferro PJ, Swaggerty CL, Bahl A, repertoire, characterization and antibiotic resistance pattern Kogut MH (2005) Purified beta-glucan as an abiotic feed analysis of Escherichia coli isolated from backyard layers and additive up-regulates the innate immune response in their environment in India. Avian Diseases 58: 39-45. immature chickens against Salmonella enterica serovar Enteritidis. International Journal of Food Microbiology 98: Samanta I, Joardar SN, Das PK, Sar TK (2015) Comparative 309-18. doi:10.1016/j.ijfoodmicro.2004.06.008. possession of Shiga toxin, intimin, enterohaemolysin and major extended spectrum beta lactamase (ESBL) genes in E. Mitra D (2007) Immunobiochemical characterization of coli isolated from backyard and farmed poultry. Iranian Journal Escherichia coli serotypes isolated from poultry. M. V. Sc. of Veterinary Research 16: 90-93. Thesis submitted to West Bengal University of Animal and Fishery Sciences, Kolkata. Siwicki AK, Klein P, Morand M, Kiczka W, Studnicka M (1998) Immunostimulatory effects of dimerized lysozyme

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

A WAY OF REPRODUCTIVE MANIPULATION AND BIOLOGY OF Wolbachia pipientis

Habib Ali1,2,Youming Hou1,2,*,Baozhen Tang1,2 ,ZhanghongShi1,2, Bin Huang1,2, Abrar Muhammad1,2 1,2 and N B Sanda

1Fujian Provincial Key Laboratory of Insect Ecology, Department of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002 Fujian, China 2Key Laboratory of Integrated Pest Management on Crops in Fujian-Taiwan, Ministry of Agriculture, P. R. China, Fuzhou, 350002 Fujian, China

Received – February 11, 2016; Revision – February 28, 2016; Accepted – April 11, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).156.168

KEYWORDS ABSTRACT Wolbachia The endo-symbiotic relationship between microorganism and eukaryotes are very common and has been Endosymbiotic bacteria broadly investigated from all insect species. The genus Wolbachia are obligatory intracellular bacteria that induce evolutionary alterations and have been frequently reported in egg cytoplasm’s of various Integrated pest management invertebrates including insects, spiders, mites, scorpions, crustaceans and nematodes, which exert a (IPM) profound impact on host biology and behavior through a number of phenotypic alternations. Due to its ubiquitous phenotypic behavior, Wolbachia becomes a novel and promising natural micro-biocontrol Coleoptera agent to induce cytoplasmic incompatibility, killing of male embryos, parthenogenesis, and feminization. Autonomous transgenic capability, survival on several host species and the ability to modify the host reproductive systems makes it the most prominent and focusing scientific genomic research from last few decades. The aim of this review is to summarize the significance and recent discoveries of Wolbachia related to various arthropods which lead positive directions to be exploited in future for integrated pest management (IPM) strategies. Further, this review also discussed the biology, phylogeny, distributions, classifications and types of Wolbachia infections on insect orders and coleopterans in particular.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Youming Hou) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

157 Habib et al

1 Introduction fascination of researchers and genomic scientists due to its multiple behaviors, diverse host range, potential for rapid host Wolbachia pipientis are maternally inherited gram negative speciation and have a great ability to alter host embryonic bacteria which becomes a potential endo-symbiotic microbial progress and mitotic developments (Lassy & Karr, 1996). It biocontrol agent (Jeong & Suh, 2008). It belong to order has also great potential to control the population of insect’s Rickettsia and are reported in the cytoplasm of various insects pests as a microbial natural enemy with multiple ways of (Jeong & Suh, 2008), terrestrial nematodes (Bourtzis & Miller, infectious phenotypes to manipulate host biology of targeted 2003), crustaceans (Cordaux et al., 2012) and Arachnidan populations. Due to its ubiquitous behaviors, evolutionary, (scorpions)(Baradaran et al., 2014; Bryson, 2014). From last biologically and ecologically it has become a key potential few years Wolbachia gained much attention and extensively intracellular biocontrol agent in pest biocontrol strategies. This used against various pest and vector management (Xi & review highlights and evaluates updated genetical, biological Dobson, 2005) through manipulating the targeted reproductive and ecological role of Wolbachia in various insect orders system, interference with nutritive and metabolic pathways, which can be used as the potential role in future integrated pest distortions the biology of host by various infectious management (IPM) strategies. phenotypes like incompatibility of cytoplasm (CI), killing of male embryos, parthenogenesis and feminization (developing 2 Taxonomic characteristics of the Wolbachia the female characters into male)(Anbutsu & Fukatsu, 2010). These autonomous infectious mechanisms support Wolbachia species are members of the obligate intracellular reproductive phenomenon of the infectious organism and allow Rickettsiales and forge as a dual competitive microbial agent the Wolbachia to increase their infection. (DCMA) like parasitic relationships with numbers of arthropods and mutualistic relationships with various insect Various theoretical and empirical studies suggest that among orders, particularly with nematodes. During their 100-million- available alpha-proteobacteria, W. pipientis is more prominent year interaction with their hosts, the cytoplasmic transmitted and effective transmitter of intracellular symbiotic infections bacteria have evolved as ‘‘reproductive parasites which which known to infect more than 2/3 of global insect belongs to Class: Alpha-proteobacteria, order Rickettsiales and communities (Hilgenboecker et al., 2008) ranging from 20 - Family Rickettsiaceae, firstly reported by entomologist, 76% (Tagami & Miura, 2004). Moreover, some species of Marshall Herting and Samuel Wolbach in 1924 from sex cells class insecta (lice, Anoplura: Mallophaga) also had 100% of Culex pipiens Linnaeus (mosquito)(Hertig & Wolbach, Wolbachia infections (KYEI‐POKU et al., 2005). 1924). However, comprehensive publication was done in 1936 (Hertig, 1936). Wolbachia, not only known as inhibitor of reproductive systems but also known to direct distinguish effect on 3 Phylogeny and Classification of Wolbachia reduction the diverseness of mtDNA (Galtier et al., 2009), Although, some contradictory views also existed about the Among the family Anaplasmataceae, Wolbachiais is well effects of Wolbachia and diversity in nuclear material known for its capacity to alter the reproductive developments (Telschow et al., 2002), this is because Wolbachia and mtDNA of host (ovaries and testes). It is considered as most common are both transmitted via mother offspring’s and hypothesized endosymbiotic bacteria that infect millions of insect species that reducing the diversity of mtDNA hitches beside as from all around the globe (Jeyaprakash & Hoy, 2000; increasing of Wolbachia infections (Armbruster et al., 2003). Hilgenboecker et al., 2008). Within the class of alpha- proteobacteria, Wolbachia also has members such as Most of the earlier researchers and biologists believed that Anaplasma, Ehrlichia, Neorickettsia which are genetically Wolbachia is maternally inheritable symbionts, i.e. only much-closed genera to Wolbachia (Figure 1 (a)). vertically transfer from egg cytoplasm to offsprings. However recent studies indicate that Wolbachia could also cause On the basis of sequence information obtained with targeted infection through horizontal transfer from infected uninfected bacterial genes includes16S rDNA (partial small subunit species (Kawai et al., 2009). It can also be cultured outside the ribosomal DNA, groEl (heat shock protein gene), wsp (outer cell (Rasgon et al., 2006) that have great potential to transfer surface coat protein gene) (Baldo et al., 2006; Paraskevopoulos infection and live outside the host cell (culture cell) over a et al., 2006) and ftsZ (cell division gene), Wolbachia infections reasonable period of time (Dobson et al., 2002b).This have been characterized and divided into eight super groups A- discovery of in vitro production of Wolbachia leads researcher H (Zhou et al.,1998; Bordenstein et al., 2009; Ros et al., 2009) towards positive direction and not only open the doors of IPM Moreover, some recent literature reported that infectious programs in agriculture sector but also help to control the strains of Wolbachia can further be classified into 3 more insect vector of various human disease i.e. Dengue, super groups, that's A-K super orders (Zhou et al.,1998; Chikungunya, and Plasmodium (Moreira et al., 2009) Bordenstein et al., 2009; Ros et al., 2009; Salunke et al., 2010) as shown in Figure b (1): Table. 1. Over the last few decades, Wolbachia-arthropods association becomes a focusing research point and attract intense

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A way of reproductive manipulation and biology of Wolbachia pipientis 158

Figure 1 (a) Division of Wolbachia with genetically closed genera within class alpha-proteobacteria: (b) Division of Wolbachia strain into 11 super groups within different host species.

4 Identification and detection of Wolbachia

Wolbachia is completely dependent on the cytoplasmic atmosphere of the host. The average size of these bacteria is ranging from 0.8 to 1.5 µm (Herting et al., 1936) and covers by at least 2 cell membrane. Although these bacteria have been identified as maternally inherited infection through infected mother to offspring’s but the infections of Wolbachia is not only limited to reproductive systems of the host but it also infect and colonized in various tissues such as muscle cell, digestive organs, brain, fat body and hemolymph of the host (Min & Benzer, 1997; Cheng & Aksoy, 1999; Dobson et al., 1999; Cheng et al., 2000; Serbus et al., 2008; Albertson et al., 2009). Infection of Wolbachia can be confirmed within the tissues of various invertebrates by using various identification techniques such as visualized Giemsa stain (Hering et al., 1936), fluorescent dye DAPI, fluorescent cells nucleic acids dye SYTO 11 techniques (Albertson et al., 2009). Furthermore, in situ hybridization with specific Wolbachia DNA probes and staining of immune through antibodies (Moreira et al., 2009) is also used for the diagnosing the Wolbachia infection inside the host tissues. A very sensitive PCR (Polymerase chain reaction) technique along with other tools like Hybridization of DNA and analysis of sequence are also extensively used for Wolbachia detection.

Table 1 Division of Wolbachia infections into 11 supergroups

Super-groups Host name References index A, B, E Arthropods (Vandekerckhove et al., 1999; Lo et al., 2002; Lo et al., 2007) C, D Filarial Nematodes (Lo et al., 2007) F Both Arthropods and Nematodes (Campbell et al., 1992; Rasgon & Scott, 2004) G Spiders (Goodacre et al., 2006) H Termites (Bordenstein et al., 2009) I Siphonaptera (Ros et al., 2009) J Spirurida, Nematoda (Ros et al., 2009) K Prostigmata, Acarina (Ros et al., 2009)

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159 Habib et al

Table 2 Killing of male embryo discovered into following host species.

Host name References Coleoptera (Majerus & Zakharov, 2000) Diptera (Dyer & Jaenike, 2004) Lepidoptera (Jiggins et al., 1998; Jiggins et al., 2000; Fujii et al., 2001; Jiggins et al., 2001) Class Arachnida (Zeh et al., 2005; Koop et al., 2009)

5 Wolbachia-host association 1992; Cordaux et al., 2012). Sex in these crustaceans is determined by the action of a male hormone that suppresses The endosymbiont Wolbachia manipulate and alter the host female development. Wolbachia is thought to inhibit reproductive systems (Werren et al., 2008) through various development of the androgenic gland that produces this phenotypic mechanisms such as the killing of male sperm, hormone and also may block receptor sites required for feminization, cytoplasmic incompatibility and parthenogenesis. hormone activity. It is one of the most advantageous All phenotypes are focus on the enhancing the progenies of mechanisms of Wolbachia in which double offspring of the infected female of associated host population and therefore, female which eventually increase the rate of infections. So far, may cause the reason to increase the transmission and the limits of Wolbachia distribution via feminization have been availability of bacteria within the hosts. These effects may be discovered only in three orders of class Insecta (Table 3) and beneficial or detrimental, and are confounded by genetic and yet needs to be determined more. environmental factors. Our focus is to enlighten the host- interactions with most recent investigation related to insect 5.3 Cytoplasmic incompatibility communities by following phenotypic behaviors. Cytoplasmic incompatibility (CI) is the most abundant 5.1Killing of male embryos Wolbachia induced phenomena that alter the host reproduction.The presence of CI in Wolbachia have been Wolbachia is one of several types of maternally-inherited frequently reported in wide range of insects includes flour bacteria that kill males during embryonic development beetles, alfalfa weevils, wasps, plant hoppers, fruit flies, flour (Stevens et al., 2001) and significantly reduced the populations moths, wood louse, mites and numerous mosquitoes species. of males. Hence, infected females may lay a mixed brood of male and female eggs, but only female eggs survive to become CI may occurred by matting’s between Wolbachia infected adults. A broad range of endosymbionts like Flavobacteria, males and uninfected females, or between any partners infected Rickettsia, Arsenophonus, Wolbachia, and Spiroplasma are with different strains of Wolbachia. However, CI occurs in two associated with the host and responsible for induction of male directions; when Wolbachia-infected male cross with killing (Anbutsu & Fukatsu, 2010)but cytological mechanisms uninfected female causing unidirectional CI. Secondly when causing male-killing are still unknown (Hornett et al., 2006; both male and female carry opposite strains which are Sheeley & McAllister, 2009). Killing of a male reported in contrastive to each other known as bidirectional CI (Telschow class Insecta including Lepidoptera, Diptera and Coleoptera et al., 2005; Merçot & Poinsot, 2009). These types of CI and class Arachnidan (Table 2). caused higher embryonic death and organism produced less number of progenies (Breeuwer & Werren, 1990; O'Neill & 5.2 Feminization Karr, 1990; Bourtzis & O'Neill, 1998; Breeuwer & Werren, 2007; Bourtzis & Miller, 2014). Recently Tram et al. (2003) Feminizing strains of Wolbachia make changes in genetic of reported the occurrence of Wolbachia in male sperm and found male hosts and force it to develop into functional female which that it introduces a factor here that prevents embryogenesis in is known as alteration of sex or feminization. Such infections the fertilized egg, unless the female partner is infected with the are common in terrestrial isopods especially in woodlouse, same Wolbachia strain to allow the sperm’s ‘rescue’. Armadillidium vulgare have been best studied (Rousset et al.,

Table3 Feminization identified into different hosts.

Host name References Lepidoptera (Hiroki et al., 2002; Kageyama et al., 2002; Narita et al., 2007) Hemiptera (Negri et al., 2006) Terrestrial Isopods (Verne et al., 2007; Bouchon et al., 2008)

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A way of reproductive manipulation and biology of Wolbachia pipientis 160 Table 4 Cytoplasmic incompatibility identified into different host.

Host name References Coleoptera (Stevens & Wade, 1990) Diptera (Yamada et al., 2011; Zheng et al., 2011) Isopods (Moret et al., 2001) Acari (Zhang et al., 2015) Lepidoptera (Brower, 1976; Kellen et al., 1981) Hymenoptera (Reed & Werren, 1995) Homoptera (Noda, 1987)

Table 5 Discoveries of induction of parthenogenesis into different host. Host name References Thysanoptera (Arakaki et al., 2001) Acari (Husseneder & Collier, 2009) Hymenoptera (Pannebakker et al., 2005)

Unidirectional CI is most frequent and usually occurs between inhabit wide range of arthropods and nematodes to manipulate males infected with a single strain or female without any strain their reproductive systems. Parasitic behavior of intercellular of Wolbachia. While the bidirectional CI have been reported bacteria with invertebrates are very common and play an only in the condition when both partners are infected with the important role to manipulate host biological interactions such same Wolbachia strains. The reason of wide spreading this as reproductions, developments, gene expression (Zhao et al., phenotype because of two elements responsible for two 2013) and immune systems (Ivanov & Littman, 2011). A bulk different Wolbachia antagonistic functions (Werren, 1997; of researches have been conducted to identified the insect- Poinsot et al., 2003) known as modification and rescue. Like bacteria association particularly Wolbachia exerts negative other phenomena, CI also have limited investigations in impacts to reduce host fitness, responsible for degradation and various Wolbachia associated hosts (Yamada et al., 2007). early death (Min & Benzer, 1997), responsible for pathogen Therefore it is necessary to investigate this phenotype in all dissemination (Cook et al., 2008), reduce host survival rate insect communities. The most recent discoveries of Cl induced (McMeniman et al., 2009), manipulate reproductive system by Wolbachia are given below in Table 4. (McGraw et al., 2001) and inhibit the pathogenicity of host (Walker et al., 2011).With these above reproductive 5.4 Induction of parthenogenesis manipulations, the bacterium (Wolbcahia) thrives in its various hosts at the expense of the hosts' reproduction and act like Wolbachia also associated with parthenogenesis induction (PI) reproductive parasite to reduced host populations successfully. in different host species. Offspring’s of these insects have three different types of sex determination i.e. diploid male and 7 Mutualistic role of Wolbachia female (diplodiploidy); haploid male and diploid female (haplodiploidy); diploid female without a male (thelytoky) Endo-symbiotic microorganism, particularly Wolbachia are not (Normark, 2003). Wolbachia increases the targeted infected only played an important role in manipulating of host female offspring by second type (haplodiploid) of sex reproduction but also provide protection against targeted host determination in various insect species. PI bacteria are found in pathogens (Moran et al., 2008; Gross et al., 2009; Cook & both A and B divisions of Wolbachia, and phylogenetic McGraw, 2010). It is beneficial for the host species (Taylor et evidence suggests that PI has evolved several times al., 2013) by increasing the survival rate of host or insect independently in these bacteria. It therefore appears that PI can species (Vala et al., 2003; Dobson et al., 2004; Foster et al., evolve easily in the Wolbachia. Still lot of work needs to be 2005; Weeks et al., 2007; Zhao et al., 2013) or enhancing the done to determine, how PI Wolbachia cause gamete fecundity rate (Girin & Bouletreau, 1995; Dobson et al., duplications, disruption of the centrosome or spindle 2002a; Fry et al., 2004; Xie et al., 2011). In Tribolium formation, attachment of the spindle to the chromosomes, or confusum (Coleoptera: Tenebrionidae) infected male sperm spindle kinetics. So far, reported discoveries of Wolbachia via with Wolbachia takes long reproductive time than compared to PI in various arthropods are discussed below in Table 5. uninfected ones. Infection may also result to protect the host by suppressing the harmful genes (Drosophila melanogaster) 6 Parasitic role of Wolbachia (Starr & Cline, 2002; Clark et al., 2005). Moreover in Tetranychus urticae, Wolbachia also causes interference in the Wolbachia (Rickettsiales: Rickettsiaceae) are one of most metabolism of iron which decreases oxidative stress and active maternally inherited endosymbiotic proteobacteria that reduces the death of cells, eventually, enhance the chance of

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161 Habib et al reproduction within the host (Kremer et al., 2009). Another Order Coleoptera economically and ecologically recognized mutualistic example of Wolbachia is in wasp, Asobara tabida has a big and successful order cover almost 1/4 of all known (Dedeine et al., 2005) here it is very essential for animals species (Hunt et al., 2007). The intercellular embryogenesis, it also play a hypothesised role in ferritin relationships with microorganism are very common and expression (Kremer et al., 2009; Kremer et al., 2012), in fruits frequently investigated in many species of arthropods. Some flies, D. melanogaster, D. simulans it caused resistance against endosymbionts are very important and play a significant role in viral RNA infection (Hedges et al., 2008; Teixeira et al., 2008; development and survival of the various hosts. Last few Osborne et al., 2009), in mosquitoes, Aedes aegpti it activate decades members of class alpha-proteobacteria particularly the immune systems against numerous pathogens (Moreira et Wolbachia are widely identified in numerous species of order al., 2009; Bian et al., 2010; Van den Hurk et al., 2012) and are Coleoptera and more than thirty (30) beetle species have been also responsible for the microRNAs expressions (Hussain et discovered in this intracellular bacteria so far (Lachowska et al., 2011). al., 2010) and all belong to supergroups A and B, except Rhinocyllus conicus (Froehlich) which belongs to supergroup 8 Wolbachia discoveries in Order Coleoptera F (Lo et al., 2002) (Table 6).

Table 6 Wolbachia discoveries in coleopteran species.

Common name of weevil Scientific name Family References Azuki bean beetle Callosobruchus Chinensis Bruchidae (Kondo et al., 1999) Vine weevil Otiorhynchus sulcatus Curculionidae (Son et al., 2008) Ladybird beetle Adalia bipunctata L. Coccinellidae (Sokolova et al., 2002; Elnagdy et al., 2013) Two spotted Ladybug Adalia bipunctata Coccinellidae (Hurst et al., 1992) Flour beetle Tribolium confusum Tenebrionidae (Wade & Stevens, 1985) Black flour beetle Tribolium madens Tenebrionidae (Fialho & Stevens, 2000) Bark beetle Pityogenes chalcographus Scolytinae (Wolfgang et al., 2009) Rice water weevil Lissorhoptrus oryzophilus Kuschel Curculionidae (Chen et al., 2012) Raspberry beetle Byturus tomentosus Byturidae (Malloch et al., 2000) Tribe naupactini Curculionidae (Rodriguero et al., 2010) Alnus ambrosia beetle Xylosandrus germanus Curculionidae (Kawasaki et al., 2010)

Northern Corn rootworm Diabrotica barberi Chrysomelidae (Roehrdanz & Levine, 2007) Bean beetles Callosobruchus Chinensis Chrysomelidae/ Bruchinae (Kondo et al., 2011)

Cabbage Seedpod weevil Ceutorhynchus obstrictus Curculionidae (Floate et al., 2011) Coffee berry borer Hypothenemus hampei Scolytidae (Vega et al., 2002) Alfalfa weevil Hypera postica Curculionidae (Klostermeyer, 1978; Hsiao & Hsiao, 1984) Western corn rootworm Diabrotica virgifera virgifera Chrysomelidae (Giordano et al., 1997; Barr et al., 2010) Mexican corn rootworm Diabrotica virgiferazeae Chrysomelidae (Giordano et al., 1997) Northern Diabrotica barberi Chrysomelidae (Roehrdanz & Levine, 2007) corn rootworm Rice weevil Sitophilus oryzae Curculionidae (Giordano et al., 1997) Korean endemic firefly Luciola unmunsana Lampyridae (Jeong et al., 2009) Rice water weevil Lissorhoptrus oryzophilus Curculionidae (Saito et al., 2005; Chen et al., 2012; Lu et al., 2013) Flea beetle Aphthona nigriscutis Chrysomelidae (Roehrdanz et al., 2006) Tobacco beetle Lasioderma serricorne (Fabricius) Anobiidae (Kageyama et al., 2010) Bread beetle or biscuit beetle Stegobium paniceum (Linnaeus) Anobiidae (Kageyama et al., 2010) Grain beetle Oryzaephilus surinamensis (Linnaeus Silvanidae (Kageyama et al., 2010) Cowpea weevil Callosobruchus analis (Fabricius) Bruchidae (Kageyama et al., 2010) Cabinet beetle Anthrenus verbasci (Linnaeus Dermestidae (Kageyama et al., 2010) Confused flour beetle Tribolium confusum Tenebrionidae (Ming et al., 2015)

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A way of reproductive manipulation and biology of Wolbachia pipientis 162 Conclusions intracellular parasite Wolbachia: implications for host mitochondrial DNA evolution. Journal of Medical Entomology Wolbachia belongs to a diverse group of endosymbiotic 40:356-360. bacteria which can manipulate the reproductive development of numerous arthropods. Tremendous achievements have been Baldo L, Hotopp JC, Jolley KA, Bordenstein SR, Biber SA, made from last few decades to understand the biology and Choudhury RR, Hayashi C, Maiden MC, Tettelin H, Werren mechanism of Wolbachia, specifically in the field of genetics JH (2006) Multilocus sequence typing system for the and molecular biology through a numbers of advanced endosymbiont Wolbachia pipientis. Applied and Wolbachia research tools and technologies. Now a days Environmental Microbiology 72:7098-110. Wolbachia has become a potential biocontrol agent due to its various infectious phenotypic behaviors including Baradaran M, Jalali A, Jolodar A (2014) Molecular diagnosis incompatibility of cytoplasm, killing of the male embryo and of Wolbachia endosymbiont from Iranian scorpion induction of parthenogenesis and feminization which can Hemiscorpius lepturus using polymerase chain reaction (PCR) change the biology of the host. Theoretical and experimental amplification of 16S rDNA gene. African Journal of analysis show that Wolbachia may greatly affect the Biotechnology 10:19802-19206. population of host species and efficiently used as a biocontrol agent in pest management strategies. Main purpose of this Barr KL, Hearne LB, Briesacher S, Clark TL, Davis GE (2010) review is to highlight the significances and current discoveries Microbial symbionts in insects influence down-regulation of of Wolbachia in various arthropods that lead a possible defense genes in maize. PloS one 5:e11339. positive role in the field of IPM programs but still needs lot of key questions to be addressed like” can Wolbachia broadly Bian G, Xu Y, Lu P, Xie Y, Xi Z (2010) The endosymbiotic used as biocontrol agent? On what conditions it behave like bacterium Wolbachia induces resistance to dengue virus in parasitic or mutualistic? Exact phenomena of this intracellular Aedes aegypti. PLoS pathogens 6:e1000833. bacterium to manipulate host reproductions? Great attention is likely to be made to answering of these questions and next few Bordenstein SR, Werren JH (2007) Bidirectional years of Wolbachia studies therefore promises to be an exciting incompatibility among divergent Wolbachia and one. incompatibility level differences among closely related Wolbachia in Nasonia. Heredity 99:278-287. Acknowledgement Bordenstein SR, Paraskevopoulos C, Hotopp JC, Sapountzis P, We are very grateful for the grant from the National Natural Lo N, Bandi C, Tettelin H, Werren JH, Bourtzis K (2009) Science Foundation of China (31470656). Parasitism and mutualism in Wolbachia: what the phylogenomic trees can and cannot say. Molecular Biology Conflict of interest and Evolution 26:231-41.

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Tram U, Ferree PM, Sullivan W (2003) Identification of Xi Z, Dobson SL (2005) Characterization of Wolbachia Wolbachia–host interacting factors through cytological transfection efficiency by using microinjection of embryonic analysis. Microbes and Infection 5:999-1011. cytoplasm and embryo homogenate. Applied and Environmental Microbiology 71:3199-3204. Vala F, Breeuwer J, Sabelis M (2003) Sorting out the effects of Wolbachia, genotype and inbreeding on life-history traits of a Xie RR, Chen XL, Hong XY (2011) Variable fitness and spider mite. Experimental & Applied Acarology 29:253-264. reproductive effects of Wolbachia infection in populations of the two-spotted spider mite Tetranychus urticae Koch in China. Van den Hurk AF, Hall-Mendelin S, Pyke AT, Frentiu FD, Applied Entomology and Zoology 46:95-102. McElroy K, Day A, Higgs S, O'Neill SL (2012) Impact of Wolbachia on infection with chikungunya and yellow fever Yamada R, Floate KD, Riegler M, O'Neill SL (2007) Male viruses in the mosquito vector Aedes aegypti. PLoS neglected development time influences the strength of Wolbachia- tropical diseases 6:e1892. induced cytoplasmic incompatibility expression in Drosophila melanogaster. Genetics 177:801-808. Vandekerckhove TT, Watteyne S, Willems A, Swings JG, Mertens J, Gillis M (1999) Phylogenetic analysis of the 16S Yamada R, Iturbe‐Ormaetxe I, Brownlie J, O'Neill S (2011) rDNA of the cytoplasmic bacterium Wolbachia from the novel Functional test of the influence of Wolbachia genes on host Folsomia candida (Hexapoda, Collembola) and its cytoplasmic incompatibility expression in Drosophila implications for Wolbachial taxonomy. FEMS Microbiology melanogaster. Insect Molecular Biology 20:75-85. Letters 180:279-286. Zeh D, Zeh J, Bonilla M (2005) Wolbachia, sex ratio bias and Vega FE, Benavides P, Stuart JA, O'Neill SL (2002)Wolbachia apparent male killing in the harlequin beetle riding infection in the coffee berry borer (Coleoptera: Scolytidae). pseudoscorpion. Heredity 95:41-49. Annals of the Entomological Society of America 95:374-378. Zhang YK, Ding XL, Rong X, Hong XY (2015) How do hosts Verne S, Johnson M, Bouchon D, Grandjean F (2007) react to endosymbionts? A new insight into the molecular Evidence for recombination between feminizing Wolbachia in mechanisms underlying the Wolbachia–host association. Insect the isopod genus Armadillidium. Gene 397:58-66. Molecular Biology 24:1-12.

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A way of reproductive manipulation and biology of Wolbachia pipientis 168 Zhou W, Rousset F, O'Neill S (1998) Phylogeny and PCR– sequences. Proceedings of the Royal Society of London B: based classification of Wolbachia strains using wsp gene Biological Sciences 265:509-515

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

MODELING THE EFFECT OF MECHANIZATION LEVEL INDEX ON CROP YIELD APPROACHING SYSTEM DYNAMICS METHODOLOGY

Behnam Jalalzadeh, Ali Mohammad Borghei* and Morteza Almassi

Department of Agricultural Mechanization, Science and Research Branch, Islamic Azad University, Tehran, IRAN

Received – February 22, 2016; Revision – March 17, 2016; Accepted – April 13, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).169.179

KEYWORDS ABSTRACT

Mechanization Level A system dynamics methodology used for estimating effects of mechanization level index on the mean yield of farm crop products, considering the main input and output of farming crops' production system Modeling of Iran. A collection of constant parameters, logical relations and statistically estimated functions which were effective in mechanized farm crops' production of Iran, defined as an autonomous system and time System Dynamics span for modeling is defined a period 70-year viz. 1981 up to 2051. Running the established model by Farm Crops simulation software, resulted in key parameters, needed for creating "The production function" as mean yield of farm crop products depended to mechanization level index. After testing validity of the created Simulation model, analyzing estimated production function resulted in recognizing three economic production regions. The first economic production region continued from 1.342 Kw/ha to 2.013 Kw/ha; The second production region started at 2.013 Kw/ha and ended at 2.386 Kw/ha, and finally the third production region started from 2.386 Kw/ha for mechanization level Index, while the maximum profitable point for farm crops production appeared at 2.218 Kw/ha in simulation procedure. All the analysis based on the available statistics for agricultural sector of Iran, since 1981 up to the recent time.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Ali Mohammad Borghei) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. ______Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

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1 Introduction and development program for food production. Farm crops have the main role in supplement of inputs for livestock and A major advantage of using dynamic simulation approaches is poultry feeding, dairy industries, edible oil productions and all their ability to represent actual processes rather than observed other the food-processing industries. Use of machinery in behavior (Neuwirth & Peck, 2013). The necessity of system farming crop's production, considered as the most expensive dynamics approach for investment analysis and system input in farming crop's production after land costs (Edwards, dynamics application, such as to evaluate different types of 2015). investment decisions for developing have been showed previously (Luban, 2009). Due to the nature of machinery So this research conducted to focus on defining mechanized inputs, estimation of machinery input based farm crop farm production collective agents of Iran as a system and a production dependent on common agricultural economics system dynamic modeling used for anticipating real situation methods is restricted. The operational field of such by virtual simulating; due to the complexity of changing experiments for applying mechanization as test inputs should conditions and environment of farm crop production during be covered by large-scale regions like a province, state or time (Fisher et al., 2000). Estimating production function for country. farm crops of Iran based on machinery inputs, and determining the optimum production point were the main purposes of this Furthermore, due to the diversity of planted farm crops' species research. So that the maximum profitable production point, the within a region, using the mechanization level indicators, maximum capacity of system for physical production and the measured by Kw/ha, to large scaled areas for observing the optimum proposed range for economic production, based on effect of input on farm production as output would be so the rate of machinery inputs could be forecasted and complicated. Thus a system dynamic based method designed determinable. for simulating the real situation of the mechanized farming production system of Iran. In a research under the title of "A 2 Materials and Methods System Dynamics Model for Evaluating Investment Strategies for Agriculture Development", some strategies planned and The main objective of this study was the collection of tested to optimize the profits from the investment in agriculture information involved in mechanized farm crop production in system of India (Gupta & Kortzfleisch, 1987). In a case study, Iran and to establish relationships between them which could Singh (2006) proposed a linear model about the relationship form a dynamic autonomous system. between the yield and mechanization level index estimated in Such a conceptually defined system should imitate the real India for estimation of a mechanization index and its impact on situation regarding the role of machinery in farming procedure, production factors. Additionally, another study for estimating a for predicting future conditions acceptably. Cash's liquidity yield model for irrigated and non- irrigated wheat and barley current set as the basic element for designing a dynamic based on mechanization level indicator in Iran was conducted simulating model and a conceptual diagram of cash flow in a by Abbasi et al. (2013), result of study revealed an additive system designed so that could illustrate how inputted capital to relationship for the effect of farm mechanization level, on system converts to mechanization input and then how it mean yield of wheat and barley in Iran. Strategic importance of converts to farm products and finally to money in an enclosed food security issues for each community, forces the relevant loop (Figure 1). sector managers to have a sustaining, Improvement, optimizing

Figure 1 Conceptualization of Cash flow diagram and conversion of money to assets in a mechanized farming production system designed for present research.

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Modeling the Effect of Mechanization Level Index on Crop Yield Approaching System Dynamics Methodology 171 Main structure of this modeling built for predicting future Category 2: Constant system inputs are fixed numerical agents, trajectory of involving parameters based on their past which assumed to be fixed during simulating time. trajectory for this all the analysis based on the available Considering the generally unchanging nature of the inputs or statistics for agricultural sector of Iran, since 1981 up to the lack of sufficient statistics about them, was the reason to recent time used in this study. At first step for building assuming this category of inputs as the fixed parameters and to simulation model, basic concepts for system creation defined be set independent of the time (Table 2). as the diversity of all types of farm crops and farm machinery, a conceptual homologous average value defined and calculated Category 3: Cause and effect relationship (Sterman, 2000) for all various parameters for the machinery function in farm between each pair of involved parameters, defined as crop production system of Iran. arithmetical functions; so that the cause factor set as independent variable and the effect factor set as the dependent A collection of 48 commercial mechanized farm crop products variable of function (Table 3). Intending to create a of Iran (FAOSTAT, 2015), involving cereals, pulses, oilseeds, mathematical relationship between an effect factor and its vegetables, kitchen garden crops, forage and industrial farm corresponding cause factor, at first step annually statistics for crops defined as material output from the system. Mean yield each variable from related references since 1981 to current of farm crop's products through the system, estimated by time for years 2012, 2013, 2014 or 2015 gathered depending calculating the ratio of total farm crop's production to the entire on availability of statistics. cultivated area of farm crops (Eq. 1). For predicting forthcoming behavior of a time-dependent Mean yield of farm crop products through the system (kg/ha) = parameter up to year 2051 based on the previous behavior of (Total farm crops production (kg)/ (Total cultivated area of that parameter, the Time-Series Analysis (Shumway & Stoffer, farm crops (ha) ….(1) 2011) have been used.

The portion of total available draft power in system involving Nine predefined time series models (Embedded in SPSS®), draft power of any type of all working 2WD and 4WD farm tested for finding the best fitting model for predicting the tractors, combine harvesters and pedestrian controlled (single future behavior of each time dependent parameter. Captions of axle) tractors, to total mechanically cultivated area of farm the nine tested models were Auto-Regressive Integrated crops resulted in "The mechanization level index" of the Moving Average (ARIMA) models, Exponential Smoothing system (Almassi et al., 2008). A time period since 1981 to Models, Simple, Holt's Linear Trend, Brown's Linear Trend, 2051 defined as a 70-years simulation time span; so that future Damped Trend, Simple Seasonal, Winters' Additive and trajectory of system outputs could be predictable virtually Winters' Multiplicative. All statistical calculations performed based on the previous trajectory of variable inputs of the by using SPSS® 22 software. system since 1981 to 2014. Then a collection of involved components in mechanized farm crop production of Iran and To find out the best-fitting function for relationship between the relationship between them, gathered as endogenous each pair of a cause parameter and its related effect parameter, parameters of the system, and classified in four categories of the Regression Analysis method has been used. Eleven system inputs: Predefined regression analysis models in analyzing software were Linear, Logarithmic, Inverse, Quadratic, Cubic, Power, Category 1: Initial values for system inputs, are values of time Compound, S-Curve, Logistic, Growth model and Exponential independent parameters at the start time of simulation in 1981 models. (Table 1).

Table 1Initial values as the system fixed inputs at the starting time of simulation.

“Type of Input as the Initial Value” “Value” (Unit) “Reference of Data” “Initial Total Compiled Available Motor 5,412,410 (Kw) Mechanization Development Center of Power in System” Ministry of Agriculture of Iran (2015) “Initial Mean Farm Crop Production Unit 1.573 ($/ha) The World Bank, 2015 Cost” “Initial Unit Value of Available 0.675 ($/Kw) The World Bank, 2015 Mechanization Capital” “Initial Sales Price Unit Value of Farm 0.001 ($/kg) FAOSTAT, 2015 Crop Products” “Initial Life Unit Cost of a Rural and Urban 13.682 ($/Family) The World Bank, 2015 Family”

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Table 2 Inputs of farm crop production system of Iran with fixed values.

“Constant Value Input Type” “Value” (Unit) “Reference of Data” “Draft Power Coefficient (Ratio of available draft power 75 (%) Almassi et al., 2008 to Indicated motor power of a tractor)” “Mean Annual General Inflation Rate of Iran” 20.56 (%) The World Bank, 2015 “Total Farm Land to Total Arable Land Ratio” 81.58 (%) FAOSTAT, 2015 “Mean Power of a Motor Driven Ambulant Vehicle (4 61.512 (Kw) The World Bank, 2015 wheeled Farm Tractors, Combine harvesters and 2 wheeled Pedestrian tractors)” “Machine Mean Work Time per Area Unit” 51.897 (Kw/ha) Rajabi et al., 2012 “Mean Total Technical Worktime of a Motor Equipped 11,253 (Hours) American Society of Machine” Agricultural Engineers, 2000 & FAOSTAT, 2015 “Mean Number of Family Members” 3.55 (Person) FAOSTAT, 2015 “Number of Farmer Families to Number of all Families 18.88 (%) FAOSTAT, 2015 Ratio” “Mechanization Rate of Farm Crops” 71.73 (%) Mechanization Development Center of Ministry of Agriculture of Iran (2015) “Total Machinery Costs as Percent of Total farm crops 21.1 (%) Abbasi et al., 2011 Production Costs” “Number of Loan Repayments” 5 (1/Year) Agriculture Bank of Iran, 2015 “Total Loan Repayment Time” 5 (Dimensionless) Agriculture Bank of Iran, 2015 “Banks Loan Yearly Mean Interest Rate” 15 (%) Agriculture Bank of Iran, 2015 “Fixed Costs of a Machine as Percent of Total Machine 64.89 (%) Ashtiani et al., 2006 Costs” “Mean Yearly Paid Loans by Banks for Farm 0.0311 (%) Mechanization Development Machinery Supplement as a Percent of GDP” Center of Ministry of Agriculture of Iran (2015) “Portion of Farmers Revenue Spend for Mechanization 0 (%) Information and Development” Communication Technology Center for Ministry of Agriculture of Iran (2012)

Table 3 The cause and effect relation formulation between the system internal parameters.

“Cause Factor (t)- Independent” “Effect Factor (Y)- Dependent” “Effect as a Function of Cause”

“Total Available Capital ($)” “GDP (Current US$)” Y = (2.366E+7) + (1.458E-7) t - (8.538E-23) t2

“GDP (Current US$)” “Total Population (Person)” Y = (1.97E+7) + (0.002E) t - (1.021E-14) t2

“Total Population (Person)” “Total Arable Area (ha)” Y = (1.59E+7) - (2.052E - 9) t2 + (3.262E - 17) t3

“Mechanization Level Index “Mean Yield of Farm Crop Y = (3538.588) + (1683.504) t + (45.481) t2 (kw/ha)” Production (kg/ha)”

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Modeling the Effect of Mechanization Level Index on Crop Yield Approaching System Dynamics Methodology 173

Category 4: A collection of 33 logical relationships between 22. Mechanization Development Capital ($) = Mechanization input and outputs of the designed system defined as below: Development Costs as Percent of Total Net Revenue of Farm Production (%) × Net Revenue of Farm Production ($) /100 + 1. Mechanization level (Kw/ha) = Total Available Draft Power Banks Finances Input ($) (Kw) / Total Farm Area (ha) 23. The inputted capital into Mechanization System ($) = 2. Total Available Draft Power (Kw) = Total Available Mechanization Development Capital ($) + Total System Fixed Mechanical Power (Kw)/ Draft Power Coefficient (%) ×100 Costs ($) 3. Total Mass of Farm Products (kg) = Mean Yield of Farm 24. Total System Fixed Costs ($) = Farm Production Total Products (Kg/ha) ×Total Farm Area (ha) Mechanization Costs ($) ×Fixed Costs as Percent of Total 4. Farm Production Total Costs ($) = Total Farm Area (ha) × Costs (%)/ 100 Mean Farm Production Unit Cost ($/ha) 25. Total Banks Finances Input ($) = GDP (Current US$) × 5. Fully Mechanized Production Costs ($) = Farm Production Total Bank Finances as Percent of GDP (Current US$) (%) / Total Costs ($) × Mechanization Rate (%) /100 100 6. Farm Production Total Mechanization Costs ($) = Fully 26. Total Required Work Time of System (hour/Year) = Total Mechanized Production Costs ($) × Total Mechanization Cost Farm Area (ha) × Power Work Time per Area Unit Percent of Total Production Cost (%)/100 (hour/ha/Year) 7. Total System Fixed Costs ($) = Farm Production Total 27. Expected Lifetime of a Machine (Year) = Total Technical Mechanization Costs ($) × Fixed Costs as Percent of Total Work time of a Machine (hour) / Yearly Work time of a Costs (%) /100 Machine (hour/Year) 8. Total Farming System Costs ($) = Farm Production Total 28. Total Farm Area (ha) = Farm Land to Arable Land Ratio Costs ($) +Total Loan Repayment ($) + Total Life Costs ($) (%) × Total Arable Area (ha) / 100 9. Initial Net Revenue of Farm Production ($) = Initial Gross 29. Total Farming System Costs ($) = Total Loan Repayment Revenue ($) - Initial Total Costs ($) ($) + Farm Production Total Costs ($) + Total Life Costs ($) 10. Number of Total Families (Family) = Total Population 30. Each Loan Installment Value ($) = "Banks Finances Input (Person)/ Mean Number of Family Members (Person/Family) ($)" × ("Total Repayment Time"+1) ×"Loan Interest Rate (%) 11. Net Revenue of Farm Production ($) = Total Revenue Rate /200) +"Banks Finances Input") / "Number of total ($) - Total Costs Rate ($) + Initial Net Revenue of Farm Repayments" Production ($) 31. Repayment Step = Total Repayment Time / Number of 12. Total Gross Revenue of Farming ($) = Unit Sales Price of Repayments Farm Products ($/kg) × Total Production of Farm Products 32. Total Population (Person) = Effect of GDP on Population (kg) (Person/$) × GDP (Current US$) 13. Inputted power to Mechanization System (Kw) = Inputted 33. Mean Yield of Farm Products (kg/ha) = Effect of Capital into Mechanization System ($) / Unit Value of Mechanization Index on Yield (kg/Kw) × Mechanization level Mechanization Capital ($/Kw) (Kw/ha) 14. Value of Total Available Capital in Mechanization System ($) = Total Available Mechanical Power (Kw) × Unit Value of All defined fixed and variable parameters and relations in Mechanization Capital ($/Kw) categories 1, 2, 3 and 4 assembled as a dynamic system for 15. Number of Farmer Families = Farmer Families Ratio (%) × simulation and predicting future values of both dependent and Number of Total Families /100 independent parameters of production function of Iran's 16. Total Available Mechanical Power (Kw) = Total Power farming production system. Input Rate (Kw) - Total Power Exit Rate (Kw) + Initial Compiled Power (Kw) The conceptually designed model by using the simulation 17. Initial Power Exit (Kw) = Initial Compiled Power (Kw) / software ”Vensim ® PLE 6.3” were converted to an applicable Expected Lifetime of a Machine (Year) × Time Step (Year) software (Figure 2). 18. Current Power Exit (Kw) = Delaying Input Power to Mechanization System (Kw) equal to Expected Lifetime of a 3 Results and Discussion Machine 19. Total Power Exit (Kw) = Initial Power Exit (Kw), up to Overall structure of the system consisted of 95 input and Expected Lifetime of a Machine + Current Power Exit (Kw), output parameters among these 5 are initial value type inputs, after Expected Lifetime of a Machine. 14 constant value type inputs, 6 Level type parameters and 70 20. Number of all Tractors, Combine harvesters and Pedestrian are Auxiliary type parameters (Ventana Systems, Inc. Controlled Tractors = Total Available Mechanical Power (Kw) 2007). Furthermore, four causes and effect type functions, and / Mean Power of a Machine (Kw). 33 logically defined relations, finally assembled as basic 21. Yearly Work time of a Machine (hour/Year) = Total structure of agricultural machinery utilization for the Required Work Time of System (hour/Year) / Number of mechanized production of farm crops in Iran. Tractors, Combine harvesters and Pedestrian Controlled Tractors

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Figure 2 Operational Schematic of the main simulation model built by Vensim 6.3 PLE simulation software.

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Modeling the Effect of Mechanization Level Index on Crop Yield Approaching System Dynamics Methodology 175

Table 4 Comparative results for outputs of the modeled system vs real situation for point wise validation test of system outputs.

“Test Parameter” According to the Vensim Output for Model Output and “Percent of Measured Simulation Model Measured Records Difference Records at 2011 at 2011 Difference (%) Value of Total Available Machines ($) 4,962,974,123 5,599,450,215 636,476,091 12.82 Total Available Motor Power (Kw) 35,126,904 30,348,299 -4,778,605 -13.6 “Total Available Draft Power (Kw) 26,345,178 22,761,225 -3,583,953 -13.6 Total Arable Land Area (ha) 17,541,000 18,564,676 1,023,676 5.84 Mechanization Level of System (Kw/ha) 1.502 1.503 0.001 0.064 Unit Value of Mechanization Capital 186.175 184.506 -1.669 -0.9 ($/Kw) GDP (Current US$) 18,838,322,969 17,926,061,905 -912,261,064 -4.84 Total Farm Crop Yearly Production (Kg) 72,007,472,000 76,165,242,880 4,157,770,880 5.77 Mean Yield of Farm Crop Production 5,152 5,029 -123 -2.39 (kg/ha) System Population (Person) 74,799,000 76,624,552 1,825,552 2.44 Mean Life Costs of Rural & Urban 3,705.99 3,734.69 28.696 0.77 Families ($) Total Number of all Tractors, Combine 553,098 493,375 -59,723 -10.8 Harvesters and other Power driven vehicles (Set)

Mean Farm Crop Production Costs ($/ha) 429.365 429.489 0.123 0.03 Power Inputting to System (Kw) 916,416 779,851 -136,565 -14.9 Mean difference of 14 forecasted parameters and related really records in 2011 (%) 6.34

For checking the reality of system behavior and validating (The equation “2” assumed as production function formulae functionality of the built model response, four static and three and some testes implemented to show the reality of this dynamic tests have been done. As a static test, the built system assumption in continue). at first step should be capable of predicting the values of system output parameters in a specified previous time section In comparison the results of this research with the results of which statistical data for the same outputs had been recorded at other correspondent studies, the effect of mechanization level that time previously. Collating system output for a number of on mean yield of irrigated and non-irrigated wheat and barley 14 parameters in the year 2011 through the running in this study, compared with results of same previous studies. software with the registered statistics for the same items So the mean yield of both irrigated and non- irrigated wheat resulted that the built system could predict the value of 14 and barley were as the ratio of 41% of The mean farm crops outputs of the system by mean error of ±6.34% different from yield, so the effect of mechanization level index on the mean the mean of really recorded values in 2011 (Table 4). yield of food grain crops estimated as a triple degree nonlinear function by multiplying equation “2” by 0.41 (Eq. 3) Examining of the ten predefined regression models for relationships between crop yield and mechanization level index Y = 1872.755 − 970.555t + 1806.699t2 − 448.092t3 (Embedded in SPSS® 22) based on simulation model outputs … (3) for these parameters, resulted in the effect of “The In a case study in India for estimation of the mechanization mechanization level Index” on “The mean yield of 48 farm level index and its impact on farm production factors, a linear crop products." model has been proposed for establishing the relationship (Eq. 4) between the yield of food grains and the mechanization The arithmetic relation determined as a third-degree level index (Singh, 2006). polynomial formula in this study (Eq. 2) Y = 708.97 + 911.65t … (4) Y = 3406.087 − 1316.370t + 1827.328t2 − 337.958t3 Where; 0.40 Kw/ha ≤ t ≤ 2.80 Kw/ha … (2)

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Furthermore, another study for estimating the yield model of 2) as a production function of the system, some criteria which st both, irrigated and non-irrigated wheat and barley based on should be fulfilled. (Debertin, 1985) are 1 should have a third- mechanization level indicator in Iran, resulted a linear degree polynomial formula for the production function; 2nd it relationship (Eq. 5) for the effect of farm mechanization level, should be consistent with the principle of diminishing returns' on mean yield of wheat and barley in Iran (Abbasi et al., 2013). law in agricultural production economics for both physical production and net profit; 3rd it should involved in three Y = 625.7 + 798.65t … (5) standard production regions and 4th, The maximum profitable Where; 0.477 Kw/ha ≤t ≤ 2.073 Kw/ha production point should fall in the second region of production function. Consistency with the basic reference patterns of agricultural production (Sterman, 2000) is another proof for validation of The maximum value for mean yield of 48 types of farm crops the established model. To test this capability of the model, by was 9,161 kg/ha, while the mechanization level index reached continuous increasing of the system leading input, the amount to 2.386 Kw/ha. These results fulfilled the principle of of main system output should be initially increased and after diminishing returns in agricultural production economics reaching the maximum point, it should be starting to decrease. (Colman & Young, 1989). Ratio of net revenue of total farm crop production to total available capital in system as Comparison results showed that against the current research, mechanization inputs (A dimensionless parameter for omitting both previously proposed linear models (Singh, 2006) and yearly inflation effects of prices) defined as “Economic (Abbasi et al., 2013) didn't fulfill the principle of diminishing Effectiveness of the Mechanized Farming Production System returns in agricultural production economics (Figure 3). of Iran”.

For establishing relationship between the mechanization level for farm crop production and the mean yield of farm crops (Eq.

Figure 3: Graphical comparative results of this study and previous studies on the response of mean yield of food grain crops vs changes in the mechanization level index.

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Modeling the Effect of Mechanization Level Index on Crop Yield Approaching System Dynamics Methodology 177

Figure 4 The maximum economic efficiency of system while overloaded by increase in banks loans to find the optimum value of mechanization level Index (2.218 Kw/ha).

Running the established model based on current values for all The Marginal physical product (M.P.P), calculated by first inputs, didn’t result in maximum point of economic efficiency. derivation of total physical product (Eq. 7). Then to find the maximum value for economic efficiency, system overloaded by banks’ loans increase up to 150% of M.P.P=−1316.370+3654.656t − 1013.874t2 … (7) current value, as input from start time to end time of simulation. Results of this experiment showed that the Also the average physical product (A.P.P) calculated as ratio of maximum reachable value for defined economic efficiency of “The total physical product” to “t” (The Mechanization level the system will appear on year 2040 as 7.0 (dimensionless), index), (Eq. 8). while at this time mechanization level index will reach to 2.218 Kw/ha as the maximum point for the net profit of production A. P. P = −1316.370 + 1827.328t − 337.958t2 …(8) (Figure 4). According to the equations (6), (7) and (8) the first economic For diagnosis the three economic production regions (Colman production region begins from 0 up to 2.013 Kw/ha for & Young, 1989), the mean yield of farm crop products (Eq. 2) mechanization level Index, which consisted of two regions, set as equal to the total physical product (T.P.P) of the system from 0 up to 1.342 Kw/ha (Maximum value of M.P.P) and (Eq. 6). from 1.342 up to 2.013 Kw/ha (Maximum value of A.P.P). The second economic production region begins from 2.013 up to T. P. P = 3406.087 − 1316.370t + 1827.328t2 − 2.386 Kw/ha (Maximum value of T.P.P). Finally, the third 337.958t3 … (6) production region begins from 2.386 Kw/ha (Table 5).

Table 5 The boundary values of Production Function and distinguishing three economic production regions.

Maximum Value T.P.P. M.P.P. A.P.P. Profit of Production The Boundary Values Start of 3rd Economic Start of 2nd Part of Start of 2nd Maximum Economic Efficiency of Production Production Region 1st Economic Economic of Production Function Production Region Production Region Mechanization Level 2.386 1.342 2.013 2.218 Index (Kw/ha)

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178 Jalalzadeh et al

Figure 5 Triple production regions for the production function of farm crops as depended to machinery Input

Because of estimating the maximum profitable production main input could increase up to 2.386 (±6.34%) as Kw/ha with point of system equal to 2.218 Kw/ha by dynamically the probability level of 100%. overloading the system cash input in simulation procedure (Figure 4), and calculating the maximum value of T.P.P and Recommended region for farm crops production in Iran, A.P.P as 2.386 and 2.013 Kw/ha respectively, thus the defined as “Second Economic Production Region” from maximum profitable production point of system fell at second 2.013(Kw/ha) ± (6.34%) up to 2.386(Kw/ha) ± (6.34%) production function for farm crops of Iran. This run of model considering the machinery input. fulfilled the 4th condition as mentioned in “Developing Realistic Agricultural Production Functions” (Debertin, 1985). Comparing the results of this study with the current value of And due to estimating accuracy rate of model outputs for the mechanization level index (0.895 Kw/ha) showed that, at least system mean total outputs as 93.66% in comparison to the there is a need to increase mechanization level index of the recorded actual values in 2011 (Table 4), the total estimation system by 1.323 (±6.34%) as Kw/ha to reach the maximum error of production function of Iran's farm production system profitable point for farming production system of Iran. estimated as 6.34% in comparison to the recorded values in real situation (Eq. 9). Conflict of interest

“2.013(Kw/ha) ± (6.34%)” ≤ “2.218(Kw/ha) ± (6.34%)” ≤ Authors would hereby like to declare that there is no conflict of “2.386(Kw/ha) ± (6.34%)” … (Eq. 9) interests that could possibly arise.

Conclusion References

Results showed that due to the restrictions of field Abbasi K, Almassi M, Borghai AM, Minaii S (2013) experiments, estimating a valid production function of farm Estimating yield model of main farm crops based on crop products as the yield index depended to mechanization mechanization level indicator in Iran, Journal of Agricultural level index, could be accomplished by using a virtual modeling Machinery 4: 344-351 Retrieved from based on system dynamics approach. The estimated function http://jame.um.ac.ir/index.php/jame/article/view/34826 on for Iran's farm crops production (Eq.2 same as Eq. 6) was January 2016. consistent with results of previous studies and fulfilled the basic principles of agricultural production economics. Abbasi S, Gholami M, Rashidi M (2011) Comparison of methods for semi-mechanized and mechanized economic Finally analyzing the created production function resulted that, production of irrigated wheat in the city Kaboudarahang, First the maximum profitable point for farming production system National Conference on new issues in agriculture, Islamic of Iran based on machinery input, will occur by 2.218 Azad University, Saveh Branch, Iran, October 2011. (±6.34%) as Kw/ha for the value of mechanization level index. And the physical production of the yield index will exceed to its maximum value, while the mechanization level index as the

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Modeling the Effect of Mechanization Level Index on Crop Yield Approaching System Dynamics Methodology 179 Agriculture Bank of Iran (2015) Retrieved from https://sc.lib.miamioh.edu/bitstream/handle/2374.MIA/192/full http://www.bki.ir/Portals/0/SBank/AgriBankActivities.pdf on text.pdf?sequence=2&isAllowed=y on 14 December, 2015 15 August 2015 Information and Communication Technology Center for Almassi M, Kiani SH, Loveimi N (2008) Principles of Ministry of Agriculture of Iran (2012) Agricultural Statistics, 2 Agricultural Mechanization, Tehran: Jungle Javedaneh : 32-68, Retrieved from http://www.maj.ir/Portal/File/ShowFile.aspx?ID=6f66d3e3- American Society of Agricultural Engineers (2000) ASAE 0884-4823-b12d-6319a2edad84 on 15 August 2015 D497.4 MAR99, Agricultural Machinery Management Data, 354, Retrieved from Luban F (2009) Using simulation to evaluate Investment http://www.nrcs.usda.gov/Internet/FSE_DOCUMENTS/16/nrc projects: Proceeding of the Ninth International Conference on s143_009503.pdf on 15 August, 2015 Investments and Economic Recovery, Bucharest, Romania, May 22-23, 2009, 139-144 Ashtiani AR, Ranjbar I, Toorchi M (2006) Determining the economic life of the three models of farm tractors in Iran. Mechanization Development Center of Ministry of Agriculture Journal of Agricultural Sciences, 12: 221-230. of Iran (2015) Retrieved from http://www.agmdc.ir/HomePage.aspx?TabID=4843&Site=Ajm Colman D, Young T (1989) Principles of Agricultural dcPortal&Lang=en-US on 15 August 2015 Economics, Cambridge: Cambridge University Press 2: 5-29. Neuwirth C, Peck A (2013) A conceptual spatial system Debertin LD (1985) Developing Realistic Agricultural dynamics (SSD) model for structural changes in grassland Production Functions for Use in Undergraduate Classes, farming. 20th International Congress on Modelling and Southern Journal of Agricultural Economics, 17: 207-213, Simulation, Adelaide, Australia, 1–6 December 2013, 628-634. Retrieved from http://ageconsearch.umn.edu/bitstream/29983/1/17020207.pdf Rajabi MH, Soltani A, Zeinali E, Soltani E (2012) Assessment on 22 August, 2015. of energy consumption in the production of wheat in Gorgan. Journal of Plant Production Research 19 : 143-171. Edwards W (2015) PM-710 estimating Farm Machinery Costs, Iowa State University Extension, Retrieved from Shumway RH, Stoffer DS (2011) Time Series Analysis and Its http://www.econ.iastate.edu/research/extension/p7155 on 15 Applications, New York, NY, Springer August, 2015 Singh G (2006) Estimation of a Mechanization Index and Its FAOSTAT, (2015) Food and Agriculture Organization of the Impact on Production and Economic Factors (A Case Study in United Nations, Retrieved from India), Bio-systems Engineering 93: 99–106. http://faostat3.fao.org/download/Q/QC/E on 15 August, 2015 doi:10.1016/j.biosystemseng.2005.08.003

Fisher DK, Norvell J, Sonka S, Nelson MJ (2000) Sterman JD (2000) Business Dynamics, Systems Thinking and Understanding technology adoption through system dynamics Modeling for a Complex World, Boston, Irwin McGraw Hill. modeling: implications for agribusiness management. International Food and Agribusiness Management Review 3: The World Bank (2015) Download Data, Retrieved from 281–296. http://data.worldbank.org/country/iran-islamic-republic on 15 August 2015 Gupta G, Kortzfleisch G (1987) a System Dynamics Model For Evaluating Investment Strategies for Agriculture Ventana Systems Inc. (2007) Vensim®, User’s Guide Version Development, Computer Science and Systems Analysis: 5, 7, 78-80, Retrieved from http://is.bwl.uni- Technical Report, Miami University, Retrieved from mannheim.de/index.php?option=com_docman&task=doc_vie w&gid=10 on 15 August, 2015.

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

REPRODUCTIVE BIOLOGY OF Goodea atripinnis (JORDAN, 1880) (CYPRINODONTIFORMES: GOODEIDAE) UNDER CONTROLLED CONDITIONS

Joao Roberto Silva-Santos1, Ma. Consolación Martínez-Saldaña2, Roberto Rico-Martínez3, José Luis Gómez-Márquez4 and José Luis Arredondo-Figueroa5,*

1 Doctoral Program in Biological Science, Basic Sciences Center, Autonomous University of Aguascalientes, Aguascalientes, Mexico 2 Department of Morphology, Basic Sciences Center, Autonomous University of Aguascalientes, Aguascalientes, Mexico 3 Department of Chemistry, Basic Sciences Center, Autonomous University of Aguascalientes, Aguascalientes, Mexico 4 Department of Biology, Faculty of Higher Education, Zaragoza, National Autonomous University of México, Federal District, Mexico 5 Department of Zootecnia, Agronomy Sciences Center, Autonomous University of Aguascalientes, Aguascalientes, Mexico

Received – February 25, 2016; Revision – March 09, 2016; Accepted – April 15, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).180.193

KEYWORDS ABSTRACT

Reproduction The study was conducted to investigate the reproductive biology of Goodea atripinnis under controlled conditions in the Aguascalientes state. Fifty wild brooders were collected, acclimatized and reared to Goodea atripinnis obtain two F1 that were cultured until reaching sexual maturation. Then, two males and one female were placed in 40-L aquariums maintained at an average water temperature of 24.4 ± 0.37°C under a 14 h Goodeidae light: 10 h dark photoperiod. The courtships description was obtained by observations and digital Culture photographs. Furthermore, the gestation period, fertility, total weight (TW, g) and total length (TL, mm) of the both F1 offspring were recorded. Additionally, the TL and TW distributions, TL-TW relationship, Photoperiod sex ratio and size at first maturity were evaluated in each F1. During courtship, both brooders performed a quiver display before copulating. The females gave birth at eight and nine months of age. Females Temperature compared with males, reached a higher TW and TL. The TW-TL relationship differed significantly between the F1 sexes. The male: female sex ratio was 2.7:1.0 in the first F1 and 0.96:1.0 in the second one. The mature females of both F1 displayed an average of 24 offspring, with a mean TL of 15.4 ± 1.3 mm and TW of 0.040 ± 0.013 g. During their first year of life, the females gave birth twice with a 45-

day interval. The sexual maturity size (L50) of brooders averaged 42.0 mm for males and 47.9 mm for females. The results demonstrated a high reproductive potential in G. atripinnis.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (José Luis Arredondo-Figueroa) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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181 Joao et al

1 Introduction 2 Materials and methods

The family Goodeidae includes 16 genera and 41 viviparous 2.1 Collection and quarantine of wild brooders species distributed over the central Mexico plateau (Domínguez-Domínguez et al., 2010). The pioneer studies on Wild brooders of G. atripinnis were collected on May 5, 2013, these species were focused on their general biology (Mendoza, in Arroyo Viejo Agua Zarca in San José de Gracia 1962; Fitzsimons, 1972; Kingston, 1979). In Mexico, most Municipality (22°07’13.6’’N, 102°30’19.6’’W) in research has focused on various viviparous fish such as Aguascalientes State. No specific permissions were required Girardinichthtys spp. (Díaz-Pardo & Ortiz-Jiménez, 1986; for collection at this location, because this species it is Macías-García & Saborío 2004; Navarrete-Salgado et al., considered as a least concern (LC) in The International Union 2007; Cruz–Gómez et al., 2010; Cruz-Gómez et al., 2011; for Conservation of Nature (IUCN) Red List of Threatened Gómez–Márquez et al., 2013; Cruz-Gómez et al 2013), Species (Snoeks et al., 2009) and this species is not listed in Hubbsina turneri (Moncayo-Estrada, 2012), and Zoogoneticus The Norm Official Mexicana number 059 of 2010, from the quitzeoensis (Ramírez-Herrejón et al., 2007). Secretaria del Medio Ambiente and Recursos Naturales (SEMARNAT) (D.O.D.L.F., 2010), and their populations are Studies on Xenotoca variata (García-Ulloa et al., 2011) and abundant in Aguascalientes State. The fish were captured using Skiffia multipunctata (Kelley et al., 2005; Kelley et al., 2006) a fishing basket and net with a mesh size of 0.5 and 1.0 cm, under culture conditions were performed to observe the respectively and these methods are not invasive and not reproductive behavior and recognize the effects of captivity involved endangered on native fish species. Fifty brooders conditions on this behavior. In Europe, viviparous fishes, such exhibiting sexual dimorphism were selected and introduced as Ataeniobius toweri, Ameca splendens and Chapalichthys into 40-L plastic bags containing water from the collection site pardalis, have been maintained under culture for study and and 100 g of iodine-free marine salt and 1 mL of anti-stress preservation (Koldewey et al., 2013). The above-mentioned solution (Neutra Stress, Grupo Acuario, Mexico) per 10 L of works, combined with histological descriptions, have improved water. Oxygen was injected as described by García-Ulloa the comprehension of reproductive events (Koya et al., 2003; (2011). The captured brooders were transported to the Ortiz-Ordóñez et al., 2007; Uribe et al., 2005; Uribe et al., Aquaculture Unit (AU) in the Agricultural Science Center 2006; Uribe et al., 2010a; Uribe et al., 2010b; Uribe et al., (ASC) at the Autonomous University of Aguascalientes in 2011; Uribe et al., 2012; Uribe et al., 2014). Jesus María Municipality, Mexico.

In Aguascalientes State, the previous studies have focused on The G. atripinnis brooders were placed under quarantine the classification and distribution of native freshwater fishes conditions in two 60-L plastic containers with chloride-free (Fitzsimons, 1972). In 1981, 23 species belonging to 19 genera water and constant aeration at room temperature. During the and eight families were reported (Rojas-Pinedo,1981), but in quarantine period, the fish were treated with antibacterial and 1996, only 18 species belonging to 16 genera and eight antifungal substances to avoid infectious diseases. The fish families were reported (Martínez-Martínez & Rojas-Pinedo, were fed commercial food (Wardley Tropical Fish Flake Food, 2008). Recently, a study on the identification, distribution and Wardley-Hartz Company, USA) containing 44% crude protein, genotypes of native ichthyofauna of Aguascalientes was 10% crude lipids and 2% crude fiber) daily at 9:00 and 14:00 conducted, and six native species were described, including G. h. The quarantine time ended when no infectious diseases were atripinnis (Arroyo-Zúñiga, 2015). registered, and the brooders accepted balanced food.

Currently, there are few studies regarding G. atripinnis. This 2.2 Experimental conditions species is not included in the red list of threatened Mexican species but is one of the two species (together with After the quarantine period, 25 wild brooders were placed in Poeciliopsis infans) that has experienced drastic changes and each of two 200-L semicircular plastic containers. Each environment modifications in recent years. Other viviparous container was constantly aerated using a ¼-hp Sweetwater fishes, such as Allotoca dugesii and Xenotoca variata, have blower (Aquatic Ecosystem, FL, USA). The fish were fed to probably been eliminated from the state (Martínez-Martínez & satiety daily at 9:00 and 14:00 h with the Wardley Tropical Rojas-Pinedo, 2008). The anthropogenic impacts on Flake Food (Wardley-Hartz Company, USA). Under these continental aquatic systems can be considered permanent in conditions, two F1 broods were obtained in July of 2013 from many cases and will soon be problematic from the standpoint two different females; the first one included 26 and the second of restoration. Thus, the performance of studies focused on the 56 offspring. The offspring composing each F1 brood were maintenance, preservation and rearing of native fish species for placed in 60-L plastic receptacles with continuous aeration at propagation and repopulation purposes is very important room temperature and fed twice daily; 50% of the water of (García-Ulloa et al., 2011). The goal of the present research each receptacle was changed every week with chloride-free was to describe the reproductive biology of the viviparous fish water. Each fish was weighted monthly for ten months using a G. atripinnis in captivity under controlled conditions of digital balance Precisa XT 220A, (Precisa Gravimetrics, USA) photoperiod and water temperature. with a precision of 0.0001 g to obtain the total weight (TW in

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Reproductive biology of Goodea atripinnis (Jordan, 1880) (Cyprinodontiformes: Goodeidae) under controlled conditions 182 g). At each weighting, the total length (TL in mm) of each fish by paired-curve analysis to test for significant differences was measured using a 6” Petrul vernier caliper with a precision between males and females in each F1 brood. For discerning of 1 mm. Furthermore, the water temperature and dissolved the growth of fish during the experiment, the following oxygen (DO) level were monitored daily with a digital formula was employed (Ricker, 1975; Salgado-Ugarte et al., dissolved oxygen meter (model YSI 550A, Yellow Springs 2005): Instruments, USA). During the experimental period, the DO averaged 6.86 ± 0.98 mg/L, and the water temperature TW = a TLb. fluctuated from 18 to 21°C. Both F1 broods exhibited sexual morphometric characteristics at six months of age, when virgin Where TW and TL are the total weight and total length of the males and females were selected, separated and placed in fish, respectively, and a and b are constants estimated by linear seventeen 40-L glass aquarium in groups of two males and one regression analysis. female per aquaria. Every aquarium was equipped with a ground filter and a 100-watt heater to maintain high DO levels The sex ratio was calculating using the total number of fish of and a water temperature of 24.0°C. A photoperiod of 14 h of each sex and dividing each of these two values by the total light and 10 h of darkness was maintained during the number of females or males, whichever was smaller. The value experimental period throughout twelve months. The water obtained was analyzed under the null hypothesis of a 1:1 ratio temperature and DO in each aquarium were monitored daily. using a ² test and 95% confidence level (Daniel, 2002). The Every two weeks, the pH was registered with a digital pH batch fecundity was calculated as BF= number of young * meter (Waterproof pHTestr 20, Oakton Instrument, USA), and 100/TL (Grier et al., 2005). The sexual maturity size (L50) for total alkalinity and hardness were checked with Aquacheck test males and females was estimated as the size at which 50% of strips (Hach Company, USA). The following conditions were the fish were sexually mature in every class frequency (Pratt maintained throughout the experiment: the water temperature & Otake, 1990). The graphs were elaborated with the software at 24.4 ± 0.37°C; DO at 6.5 ± 0.8 mg/L, pH at 8.6 ± 0.18, total GraphPad Prism version 5.0 (GraphPad software Inc., USA). alkalinity at 192 ± 24 mg CaCO3/L and total hardness at 88.5 ± 35 mg CaCO3/L. 3 Results

Photographs taken with a digital camera (Nikon Coolpix P600, 3.1 Courtship Japan) were used for all the aquarium observations regarding the sexual courtship behavior of males and females as well as Courtship behavior began one month after virgin brooders of to describe the fin pigmentation changes in both sexes and the G. atripinnis were introduced into the aquarium. In both sexes, abdominal modifications that occurred in females after a black coloration developed in the anal, dorsal and caudal copulation. Moreover, the following data were recorded: the fins. The male placed his head in front of a female and total number of offspring per female; the survival rate, TW and quivered his body awaiting a response. When the female TL of the offspring; the TW and TL of each female before and quivered too, both brooders performed side-by-side after birth; the number of brooders produced by each female; synchronized swimming along the aquarium. Copulation most and the number of days between birth events. likely occurred at this time but was not clearly observed due to its short duration. After copulation, the black color was 2.3 Biological parameters missing from the fins (Figure. 1).

Diagrams of the TL distribution were elaborated (Salgado- Ugarte et al., 2005). The TW-TL relationship was calculated

Figure 1 Fin pigmentation in brooders of G. atripinnis during courtship and aggression behaviors between congeners. (a) Female with pigmented fins before courtship, (b) female with unpigmented fins after courtship, (c) male with pigmented fins before courtship and (d) male with unpigmented fin after courtship. ______Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

183 Joao et al

Figure 2 Characterization of abdominal morphology during gestational periods in a (a) female at the beginning of gestation, (b) female during the middle of gestation and (c) female at the end of gestation.

The males demonstrated territorial and aggressive behavior At eight months, males and females of the first F1 brood when two of them were introduced in the same aquaria. showed a TL of 49.9 ± 2.1 mm and 56.6 ± 6.4 mm, Commonly, the larger male attacked the smaller fish, causing respectively, and respective TWs of 2.11 ± 0.66 g and 3.01 ± lesions and provoking death (n = 5). In February and early 0.87 g. The males of the second F1 brood presented a TL of March of 2014, the first gravid females were detected. Three 48.5 ± 3.5 mm and a TW of 1.79 ± 0.33 g. The females of the states could be differentiated in the females: a) the beginning second F1 brood exhibited a TL of 57.5 ± 3.8 mm and a TW of of gestation, when the females, some which conserved the 3.27 g ± 0.56 g. The Mann-Whitney U test indicated TL and black fins, lacked an obviously enlarged belly but evidenced TW significantly differences between the sexes in both the first abdominal morphological changes; b) the middle of gestation, (P< 0.03) and second (P< 0.001) F1 broods. when females did not exhibit black fins but displayed a large belly as a result of an increased egg size and the presence of 3.3 TW-TL relationship embryos; and c) the end of gestation, when abdominal enlargement was advanced, and embryos could sometimes be For the first F1 brood, a significant difference was evident in observed moving inside the abdominal cavity (Figure. 2). the TW-TL relationship of males and females (F=4.7; P< 0.05); the growth equation was TW = 0.00173TL2.9454 for Gravid females initiated the liberation of offspring in the males and TW = 0.0175TL2.9443 for females. For the second F1 middle of March of 2014 at an age of eight months for the first brood, the TW-TL relationship was TW = 0.0095TL3.2958 for F1 brood and nine months for the second F1 brood. The males and TW = 0.0071TL3.4697 for females, with a significant liberation was influenced by factors such as the maturation of difference between the sexes (F= 32.11, P<0.01) (Figure. 3). embryos and the stress caused by the presence of males or a transfer from one aquarium to other. 3.4 Sex ratio

3.2 TL and TW distributions Males of G. atripinnis exhibited a species-distinctive andropodium, which could be identified during the first two Three-month-old males and females of the first F1 brood and half months of life. Of the 26 fish in the first F1 brood, 19 reached an average TL of 37.2 ± 8.2 and 37.2 ± 4.9 mm, (73%) were males and seven (26%) were females, with a sex respectively. Two-month-old males and females of the second ratio of 2.7 males: 1 female; a ² test indicated significant F1 brood reached a TL of 30.0 ± 2.3 mm and 32.2 ± 1.8 mm, deviation from a 1:1 sex ratio (² =2.7, P< 0.05). The fish of respectively. The TW was 0.82 g ± 0.26 g for males and 0.84 ± the second F1 brood comprised 53 organisms: 26 (49%) were 0.30 g for females. For the second F1 brood, the TW reached males, 27 (51%) were females and the resulting sex ratio was 0.35 ± 0.08 for males and 0.41 ± 0.08 g for females. 0.96:1, respectively, which was not significantly different from a 1:1 ratio (²=0.009, P> 0.05).

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Reproductive biology of Goodea atripinnis (Jordan, 1880) (Cyprinodontiformes: Goodeidae) under controlled conditions 184

Figure 3 Total weight-total length relationship for male and female G. atripinnis of both F1 broods. Negative allometric development shown by (A) males and (B) females of the first F1 brood and positive allometric development shown by (C) males and (D) females of the second F1 brood.

Table 1 Fertility and size characteristics of G. atripinnis females.

Female Aquarium Number of offspring Offspring TW of offspring TL of offspring TL of females TW TW number number survival (%) (g) (mm) (mm) *(g) ** (g) 1 2 25 100 0.044 15.9 56 3.4 2.4 2 2 18 100 0.039 15.6 61 4.3 2.8 3 3 35 100 0.046 16.4 62 5.3 4.0 4 3 15 87 0.033 17.7 70 6 4.9 5 4 38 100 0.037 14.9 61 4.45 3.3 6 5 28 96 0.030 14.7 63 4.9 3.4 7 6 26 100 0.058 14.9 61 4.3 3.2 8 6 33 100 0.027 14.1 62 5.2 4.2 9 6 45 100 0.033 14.4 72 7.7 5.7 10 9 11 100 0.064 16.9 60 3.7 3.1 11 10 14 93 0.062 18.1 58 3.5 2.7 12 11 33 97 0.025 12.9 56 3.4 2.5 13 11 29 97 0.036 15.2 64 4.4 3.8 14 12 18 100 0.061 17.5 60 4.9 3.0 15 15 15 100 0.053 16.5 56 3.4 2.6 16 15 18 28 0.020 13.1 60 3.6 3.0 17 16 25 100 0.032 13.6 58 3.7 2.8 18 16 11 55 0.033 14.4 60 5.2 3.3 19 17 25 100 0.040 15.9 61 4.6 3.4 Total 462 Average 24.31 92.26 0.040 15.40 61.10 4.52 3.37 SD 9.64 18.74 0.013 1.51 4.17 1.08 0.84 SD = Standard deviation, * before giving birth, ** after giving birth.

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185 Joao et al

3.5 Fecundity first maturation size of 42 mm for males and 47.9 mm for females (Figure. 4). The first birth in the second F1 brood was recorded at a parental age of eight months. The average number of offspring 4 Discussions per female was of 24.32 ± 9.64. At birth, the offspring measured 15.4 ± 1.3 mm for TL and 0.040 ± 0.013 g for TW. 4.1 Courtship During the birth, the offspring survival rate was 92% ± 19%. The TL of the pregnant females ranged from 46 to 72 mm. The The presence of two males in each aquarium provoked an females in gestation had an average TL of 61.11 ± 4.18 mm. increase in aggression and competence by the female. The An average weight loss of 1.15 ± 0.25 g occurred each time a males situated their heads on the caudal fin of their competitor, female gave birth (Table 1). shifted in circles and nibbled. This behavior has been described in some fishes of the family Goodeidae, with large males 3.6 Gestational period displacing small ones through a brief persecution, after which the fighting stops (Kingston,1979). The females of G. atripinnis exhibited an average of two births during their first year, with a range of one to four births. The The introduction of two males for each female is recommended period between the first and second births averaged 45 days (± in G. multiradiatus because the presence of one male is 11.2) (Table 2). insufficient to guarantee female fecundation (Macías-García, 1994). In G. atripinnis, suggestions have been made that 3.7 First maturation size pairing only one male and female of equal size is adequate to assure courtship and fecundation. The selection of males by

The L50 for the TL of males and females of the first F1 brood females has been observed in G. multiradiatus; the presence of was 42.8 and 48.9 mm, respectively, whereas in the second F1 males 75% of the female size inhibits courtship. The use of brood, this value was 41.3 mm in males and 47.4 mm in two males and one female is also recommended to stimulate females. The average TLs obtained for each sex indicated a courtship because in some viviparous species, females select the male for copulation (Kingston, 1979; Kelley et al, 2005).

Figure 4 The size of the first state of maturation in males and females of G. atripinnis for the first and second F1 broods. A - Males of the first F1 brood matured at a TL of 42. 8 mm, whereas. B - females of the first F1 brood matured at a TL of 48.9 mm. In the second F1 brood, (C) males matured at a TL of 41.3 mm, whereas (D) females attained maturity at a TL of 47 mm.

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Reproductive biology of Goodea atripinnis (Jordan, 1880) (Cyprinodontiformes: Goodeidae) under controlled conditions 186 Table 2 Birth numbers, parturition dates and inter-birth periods for G. atripinnis females.

Aquarium Number of births 1st birth 2nd birth Days between births 2 2 04/28/2014 06/05/2014 38 3 3 03/30/2014 05/17/2014 48 4 2 03/13/2014 05/14/2014 52 5 2 05/04/2014 07/15/2014 72 6 4 03/11/2014 04/19/2014 39 9 2 04/14/2014 05/15/2014 31 11 2 03/21/2014 05/06/2014 46 12 2 03/30/2014 05/22/2014 53 15 2 03/17/2014 04/21/2014 35 16 3 03/14/2014 05/04/2014 51 17 4 03/20/2014 05/02/2014 43 Mean 46.1 SD 11.2 SD = Standard deviation. The table only includes the females with two or more parturitions.

In G. atripinnis, three reproductive phases viz a) orientation, (Mendoza, 1962). This condition may occur in G. atripinnis b) display and c) copulation have been reported. The first because the Goodeidae family does not present superfetation, phase is subdivided into watching and following. The sexual which is exclusive to the Poeciliidae family (Turner, 1933; behavior perceived in the present study was similar to that Burns, 1985; Macías-García, 1994; Contreras Mac Beath & observed previously (Nelson,1975); the male positioned Ramírez-Espinoza, 1996; Gómez-Márquez et al., 2008; Uribe himself in front of the female displaying his rigid head toward et al., 2010a) and has only been observed as an occasional the aquaria wall and tilting his tail toward the female. Once the event in Girardinichthtys viviparus (Díaz-Pardo & Ortiz- female retained a static position, the male entered a display Jiménez, 1986). phase and initiated body quivering in an S form, keeping the same position as that assumed in the observation phase. Females of G. atripinnis maintained under laboratory Sometimes, the female displayed quiver movements, indicating conditions are not carnivorous (Kingston, 1979). Some her receptivity. Such movements are similar to those reported viviparous fish, such as Ameca splendens and Atenobious in other viviparous fishes and are named conduct quiver toweri, can live in colonies and are not predators. In contrast, behavior because the fishes vibrate their entire body rapidly in Chapalichtis pardalis show cannibalistic behavior, and for this small amplitudes in an S or C shape (Kingston, 1979). Of the reason, the separation of gravid females before birth is five displays mentioned, the most commonly observed in the recommended (Koldewey et al., 2013). present study was a sigmoid display with head tilting. However, the body of males was generally positioned with the Females of G. atripinnis exhibited two types of breeding head pointed toward the tail of the female or close to the stimulation in the present experiment. First, the females gently aquaria wall, or vice versa. After displaying, the male bit offspring, provoking them to swim to the surface of the approached the female keeping a head-to-head position to water column. Large females suctioned offspring with their realize a synchronic swing, finally moving his body toward the mouth and then expulsed them with force. In the second caudal fin of the female. In the present study, it was not behavior, females used their caudal fin to move the water possible to observe copulation. However, this event is column and stimulate the offspring. In both behaviors, the reportedly of short duration in certain species, lasting two to females incited fingerlings to swing reducing the mortality rate five seconds in G. atripinnis and approximately 0.6 to 1.7 with this action. seconds in Xenotoca eiseni (Greven & Brenner, 2010). The synchronization of the male and female is necessary for a 4.2 Distribution of TW-TL successful copulation because the andropodium is not an intromittent organ and male must concealment female for The TW-TL relationship results obtained in the present copulation (Nelson, 1975). experiment suggest that G. atripinnis may attain a TL of 56 mm by nine months of age, with a tendency to reach 70 mm Females of G. atripinnis can be classified as receptive and non within one year. G. luitpodi (G. atripinnis) reached a TL of 78 receptive (Nelson, 1975; Greven & Brenner, 2010). When to 85 mm by one year of age (Mendoza,1962), whereas females were receptive during the present study, it was Allophorus robustus presented a TL ranging between 60 and necessary to reintroduce a male after each birth. This condition 90 mm (Casebolt et al., 1998; De Lapeyre et al., 2010). Under has also been reported in Allophorus robustus and Neophorus natural conditions in the epicontinental waters of diazi in that each cohort requires a separate insemination Aguascalientes State, the TL of wild G. atripinnis males and

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187 Joao et al females measured from 100 to 110 mm, and similar results 4.3 TW-TL relationship were reported for this species in Lake Patzcuaro in Michoacan State, where mature females reached a TL generally ranging During the experiment, G. atripinnis showed a coefficient of from 90 to 110 mm but exceeding this size in exceptional cases “b” significantly different from three other species displaying (Mendoza, 1962). allometric growth (Ricker, 1975; Salgado-Ugarte et al., 2005). The organisms of the first F1 brood presented a “b” value Fishes of the family Goodeidae show sexual dimorphism below three, revealing negative allometric growth, which (Díaz-Pardo & Ortiz-Jiménez, 1986). G. atripinnis exhibit two indicates a higher than proportionate increase in TL with features that allow the recognition of sexual dimorphism: a) increasing TW. The second F1 brood showed a value one is the modification of the anal fin in males to generating an exceeding three and indicating positive allometric growth and andropodium that can be recognized as an anal fin divided in a proportionately greater increase in TW with increases in TL two portions (Uribe et al., 2010b), and b) the other feature is (Salgado-Ugarte et al., 2005). Although both F1 broods were the difference in TL between the sexes, with adult females cultured under the same conditions, other factors might have 20% longer compared with males (Kobelkowsky, 2005). influenced the growth rate. Some factors affecting growth rate in fishes have been divided into the following categories: a) The fingerlings obtained in the present study showed similar intrinsic factors such as genetics, physiology, maturation stage, morphological characteristics between the sexes during the health state and behavior and b) extrinsic factors such as water first months of life. During the second and third months, sexual temperature, accessibility of food, dissolved oxygen dimorphism characterized by andropodium formation and the concentration and the presence of toxic metabolites (Hepher & manifestation of fin coloration changes began. These Pruginin, 1985). characters facilitated sex separation. The TL differences between the sexes began in the first F1 brood at six months of High variability has been observed for “b” estimation among age and in the second one after five months of age. A rapid different populations of the same species, probably due to TL increase in female size provided greater longevity and better variations, the procedure used to measure this variable, and the resistance to reproductive stress, assuring good development nutritional condition of the population (Ricker, 1975; Frota et and health for future cohorts (Gómez-Márquez et al., 2013). al., 2004). In Hubbsina turneri, the TW-TL relationships demonstrated a wide dispersion of data for large fish, which The Goodeidae family exhibits great size diversity. In small was most likely associated with the reproductive season fish such as Skiffia multipunctata, the TL averaged 25 mm in (Moncayo-Estrada, 2012). A similar case was reported in males and 28 mm in females; in Mexico, the genus Allotoca Ameca splendens, for which differences in the TW-TL includes seven species usually showing a TL from 34 to 54 relationship in diverse locations were most likely due to the mm. Allotoca catarinae reach a TL of 47 mm in males and 63 environmental conditions (Ortiz-Ordóñez et al., 2007). mm in females (Domínguez-Domínguez et al., 2005). Among the medium-sized fishes, Girardinichthtys viviparus reach an 4.4 Sex ratio average TL of 49 mm in males and 61 mm in females (Gómez- Márquez et al., 2013). In G. multiradiatus, males manifest a The sex ratio variability detected for G. atripinnis was TL of 42 mm, and females reach 48 mm (Domínguez- similarly reported for G. multiradiatus in San Miguel Arco Domínguez et al., 2005), with a maximum of 49 mm recorded Reservoir in Mexico State, where a male: female ratio of 3:1 in wild populations (Cruz-Gómez et al., 2011). Among the was reported (Navarrete-Salgado et al., 2007), and in Villa largest fishes, Chapalichthys encaustus can attain an average Victoria Reservoir, also in Mexico State, where a male: female TL of 63 mm in males and 61 mm in females, whereas in ratio of 1:2.77 was found (Cruz-Gómez et al., 2011). In San Allophorus robustus, the males measure 102 mm, compared Martin Village Reservoir in Querétaro State, Mexico, a male: with 93 mm in females (Domínguez-Domínguez et al., 2005). female ratio of 1:1.7 was registered (Cruz-Gómez et al., 2005). G. atripinnis is considered a large species in the Lerma- A proportionately higher number of females are commonly Chapala-Santiago basin, with a TL ranging between 66 and 83 found in natural habitat, such as the male: female ratio of 1:2.4 mm in males and females, which makes this species relevant reported for G. viviparous, a species living in urban lakes in for consumption. This species is consumed by the rural Mexico City, Mexico (Gómez-Márquez et al., 2013). In Lake population in some Mexican states, such as Michoacan (Kelley Cuitzeo in Michoacan State, Mexico, females of Hubbsina et al., 2005; Colon et al., 2009). turneri were recorded in high abundance, with a male: female ratio of 1:41M (Moncayo-Estrada, 2012). Out of captivity, A search of the mainstream scientific literature resulted in no females of several species of viviparous fish are the more data regarding the TW of this species; for this reason, a abundant sex (Macías-García et al., 1998). comparison with other species of the family Goodeidae was not possible. However, it is important and necessary to register The 1:1M G. atripinnis sex ratio recorded in the second F1 these data to increase the understanding of this group of brood in the present experiment has also been documented for viviparous fishes. H. turneri in Lake Zacapu, Michoacán, Mexico, where a ratio close to 1:1M was registered (Moncayo-Estrada, 2012).

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Reproductive biology of Goodea atripinnis (Jordan, 1880) (Cyprinodontiformes: Goodeidae) under controlled conditions 188 Similar ratios have been reported for Zoogoneticus Gómez et al., 2011). In G. atripinnis, an average of 30 quitzeoensis in the reservoir of Mintzita in Morelia, offspring per brood has been reported, but a high number were Michoacan, Mexico (Ramírez-Herrejón et al., 2007). frequently found when the maternal TL was high (White & Turner, 1984). In a wild female with a TL of 111 mm, 110 In natural populations, selection processes influence the sex fingerlings were reported in one brood. In the current study, ratio toward 1:1 (50% males and 50% females), preserving a this relationship was not observed, perhaps because all the steady evolutive strategy (Maynard, 1978). In Aguascalientes females presented a similar TL. State in the natural distribution area of G. atripinnis, the predators do not have a specific affinity for one particular Schoenherr (1977) determined that fertility in viviparous fish is gender, nor is the competence for niche and food important the total number of embryos present within the female at the because G. atripinnis share the habitat with other species, such time of his capture and preservation, therefore in this study the as Yuriria alta, Algansea tincella and Scartomyzon austrinus. fertility was determined as the number of embryos obtained for However, upholding low fecundity but a high survival rate of each birth. The partial fecundity in viviparous fishes is defined fingerlings can be a strategy for tolerating adverse conditions. as the number of young counted in the ovary during dissection A great variety of mechanisms for sexual determination have or in a brood at birth, suggesting that this term may be been mentioned in relation to the differential distribution of expressed as the number of fingerlings in relation to the female sex, including genetic causes and environmental factors, such standard length (SL), TL (in mm or cm) or TW (in g) x 100 as water temperature, pH and social behavior (Devlin & to evaluate and estimate intraspecific fecundity (Grier et Nagahama, 2002; Van Aerle et al., 2004; Guerrero-Estévez & al., 2005). In G. atripinnis, reports of an average fecundity Moreno-Mendoza, 2012). In the present experiment, water of 55 to 60 embryos exist (Uribe et al., 2005), whereas the temperature most likely played a role in male production; average in the present study was 40 embryos despite a similar however, the sex ratio approaches 1:1 because multiple factors TL for females. are in equilibrium (Valenzuela et al., 2003). The offspring born of G. atripinnis in the present study The sex ratio is influenced by genetic, environmental and registered an average TL of 15.4 ± 1.3 mm, with a TW of social factors, although the effects of these factors could differ 0.040 ± 0.013 g, respectively. These data were obtained from between species (Rosenfeld & Roberts, 2004; Guerrero- 19 births and differed from those reported in G. luitpoldii (G. Estévez & Moreno-Mendoza, 2012). In the case of genetic atripinnis) in Lake Patzcuaro, where the high TL measured factors, there is no simple model that is useful for all fishes. In was of 23.7 mm, and some of the fish reached a maximum of Gambusia affinis, reports of heterogametic females exist. In 31.2 mm before giving birth (Mendoza, 1962). In other studies, Xiphophorus spp., both heterogametic females and males exist, juveniles of the same species showed a TL of 20 mm, a supporting the idea of a recent sex chromosome divergence longitudinal arrangement in the ovary, and a distribution (Barollier et al., 1999). The available sexual studies are aligned in parallel along the main ovarian axis in two ovarian specific for each species and sometimes for a particular chambers (Uribe et al., 2005). This change in TL was observed population of fishes. The influence of water temperature on the in offspring born with different sizes and pigmentation in sex ratio has been confirmed in 59 different species belonging aquariums and occurred because of matrotrophic development to 13 families of fishes (Ospina-Álvarez & Piferrer, 2008). in this species, allowing accessibility to nutrients during the However, insufficient studies exist for viviparous fishes, and embryonic developmental phase while the embryos remain in the mainstream has been focused on members of the the reproductive tract (Lombardi & Wourms, 1988; Hollenberg Poeciliidae family, in which males are not abundant when the & Wourms, 1995). In G. atripinnis, the increase in embryonic fish are maintained in high-temperature water (Sullivan & mass results from the maternal transfer of nutrients during the Schultz, 1986; Römer & Beisenherz, 1996). gestational period.

4.5 Fertility 4.6 Pregnancy period

This is the first report of G. atripinnis fertility in Females in this experiment presented sequential births with a Aguascalientes State. The average offspring number was of gestation period of 45 days (± 11 days). This result is 24.32 ± 9.64 per birth. This result was similar to that reported consistent with the gestation period reported in Goodeidae fish for G. luitpoldii (G. atripinnis) studied in Lake Patzcuaro, under laboratory conditions, where a gestation period of 30 to namely, 19.1 offspring in the 44 ovaries examined (Mendoza, 90 days occurred and was influenced by the number of 1962), and differed from the maximum of 60 embryos reported daylight hours, the water temperature and the nutritional status for G. atripinnis (Uribe et al., 2005). The small differences of females. Furthermore, not all females in one population are mentioned could be the result of the rearing conditions in pregnant simultaneously because eggs are not synchronically captivity compared with those under natural conditions produced (Kingston, 1979). For G. atripinnis, the gestation (Navarrete-Salgado et al., 2007). Differences in the fertility of period has been determined to last 60 to 75 days (Mendoza, viviparous fish have been attributed to a relationship between 1962) or two months (Kingston, 1979). In comparison, the TL and age, which was demonstrated in G. viviparous, as was gestation period was shorter in the present study because the a relationship between the number of embryos and TL (Cruz- water temperature influenced embryonic development. In

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189 Joao et al

Xenotoca variata, a small variation in temperature between females occurred at a TL of 32 mm (Cruz-Gómez et al., 2011), two and three °C generated important changes in breeding and 30 mm at Villa Victoria Dam and Lake Texcoco, performance (García-Ulloa et al., 2011). respectively, both in the State of México (Díaz-Pardo & Ortiz- Jiménez, 1986). Another example involves H. turneri females, G. luitpoldii (G. atripinnis) can produce one birth annually at which reached sexual maturity at an SL of 30 mm in Lake the end of the dry season between April and June (Mendoza, Zacapu, Michoacan State, Mexico; however, the minimum SL 1962; Orbe-Mendoza, 2002). This timing coincides with that for reproduction was 25.9 ±0.21 mm in Lake Cuitzeo, found in the present study; however, an average of two births Michoacan, Mexico (Moncayo-Estrada, 2012). per female and as many as four births were observed in the present study. A similar pattern was reported in H. turneri and Conclusions was attributed to individual condition, habitat characteristics and the length of the gestational period (Moncayo-Estrada, Under the experimental conditions of the present study, G. 2012). atripinnis showed a sex ratio near 1:1M. The average TL of nine-month-old fish was 49.2 mm in males and 57.05 mm in The reproductive period of G. atripinnis has been reported to females; the average TW was 1.95 ± 0.22 and 3.14 ± 0.18g, last three months from April to June (Mendoza, 1962), but respectively. lasted seven months from March to October in the present study, in similarity with a third study reporting an April to Under a 14 h light: 10 h dark photoperiod and an average September reproductive period (Bárragan & Magallón,1994). temperature of 24 °C, sexual maturity occurred at a TL of 42 This species could most likely reproduce all year as long as the mm in males and 48.3 mm in females. The females first gave water temperature remained optimal. Such a result has been birth between eight and nine months of age, with the reported in A. splendens in the Ameca River, México, where possibility of year-long reproduction under stable the water temperature varied from 25.0 to 28.2 °C, allowing photoperiodic and thermal conditions. multiple reproductive cycles (Ortiz-Ordóñez et al., 2007). However, only one reproductive event was recorded annually G. atripinnis females can copulate as many as four times in cold waters (Díaz-Pardo & Ortiz-Jiménez, 1986). Other during the reproductive season, with an average of two times. species, such as G. multiradiatus inhabiting San Miguel Arco The gestational period lasted ninety days, with an average Reservoir in México State and San Martín Reservoir, production of 24.32 ± 9.64 fingerlings per female in the first Querétaro, México, also presented one reproductive season year of life. The fingerlings reached an average TL of 15.4 ± (Navarrete-Salgado et al., 2007; Cruz-Gómez et al., 2010; 1.3 mm and TW of 0.040 ± 0.013 g. Cruz-Gómez et al., 2011). The variation in the reproductive cycle of G. atripinnis and other Goodeids may be considered The information generated in the present study establishes a evidence for the plasticity of populations acclimated to foundation upon which to initiate the rearing of G. atripinnis different geographic conditions and the particular under controlled conditions. This study has shown the characteristics of new habitats (Del Mar Torralva et al. 1997). relevance of promoting reproductive studies on native fishes, especially those with scientific and ecological value that has 4.7 Size at first reproduction been underestimated.

In Goodeidae fish, scientific reports on sexual maturity are Acknowledgments scarce. In particular, the G. atripinnis maturation period was described as two years (Mendoza, 1962); however, under the We want to express our acknowledgment to CONACyT rearing conditions of the present study, maturation occurred at (Mexico National Council for Science and Technology) for the only seven to eight months of age. For males of this species, Scholarship Number 376450. Special thanks to all authors for early sexual maturation can be attributed to photoperiodic and the collaboration in this journal for their useful comments and water temperature conditions that induce gametogenic data analysis. Also, thanks to Angela Araujo García, Albert del development. The reproductive season can vary with Refujio Moreno Mena, Jorge Ramón Rocha Ruiz and Sonia environmental conditions, such as water temperature and Cruz for the excellent technical support. feeding strategies, but not all the organisms reproduce simultaneously, even those of the same size or age (Salgado Conflict of Interest Ugarte et al., 2005; Uribe et al., 2010b; Cruz-Gómez et al., 2011). Authors would hereby like to declare that there is no conflict of interests that could possibly arise In the present study, the males of G. atripinnis developed sexually at a lesser TL and age compared with females; males References and females in both the F1 brood attained sexual maturity at an average TL of 42 and 48.3 mm, respectively. The size at the Arroyo-Zúñiga KI (2015) Caracterización taxonómica y first reproduction can vary among populations of the same genotipificación de especies nativas del estado de species; for example, the first reproduction of G. multiradiatus

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Reproductive biology of Goodea atripinnis (Jordan, 1880) (Cyprinodontiformes: Goodeidae) under controlled conditions 190 Aguascalientes. M. Sc. Thesis submitted to The Universidad Martín, Querétaro, México. BIOCYT: Biología, Ciencia y Autónoma de Aguascalientes. México, Pp: 97. Tecnología 3: 94-106.

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Hepher B, Pruginin Y (1985) Cultivo de peces comerciales, Martínez-Martínez J, Rojas Pinedo A (2008) Peces. In: Avila Basado en las experiencias de las granjas piscícolas en Villegas H, Melgarejo ED, Cruz Angón A (Eds), La Israel.1st (Ed), Limusa, Mexico. biodiversidad en Aguascalientes, estudio de estado, Comisión Nacional para el Conocimiento y Uso de la Biodiversidad Hollenberg F, Wourms JP (1995) Embryonic growth and (CONABIO), Instituto del Medio Ambiente del Estado de maternal nutrient sources in Goodeid fishes (Teleostei: Aguascalientes (IMAE), Universidad Autónoma de Cyprinodontiformes). Journal of Experimental Zoology 271: Aguascalientes (UAA), México, Pp: 32-35. 379-394. DOI: 10.1002/jez.1402710508. Maynard SJ (1978) The Evolution of Sex.1st (Ed), Cambridge Kelley JL, Magurran AE, Macías García C (2006) Captive University Press, New York. breeding promotes aggression in an endangered Mexican fish. Biological Conservation 133:169-177. Mendoza G (1962) The reproductive cycles of three viviparous DOI:10.1016/j.biocon.2006.06.002 teleosts, Alloophorus robustus, Goodea luitpoldii and Neoophorus diazi. The Biological Bulletin 123 : 351-365. Kelley JL, Magurran AE, Macías-García C (2005) The DOI:10.2307/1539280 influence of rearing experience on the behaviour of an endangered Mexican fish, Skiffia multipunctata. Biological Moncayo-Estrada R (2012) Análisis historico de la biología de Conservation 122: 223-230. DOI: la Cherehuita (Hubbsina turneri) (Pisces: Goodeidae), especie 10.1016/j.biocon.2004.07.011 endémica y en peligro de extinción de México. Revista Chapingo. Serie ciencias forestales y del ambiente 18:101-110. Kingston DIL (1979) Behavioral and morphological studies of DOI: 10.5154/r.rchscfa.2011.02.020 the Goodeid genus Ilyodon, and comparative behavior of the fishes of the family Goodeidae. Unpubl., PhD. Thesis submitted to The University of Michigan.

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Reproductive biology of Goodea atripinnis (Jordan, 1880) (Cyprinodontiformes: Goodeidae) under controlled conditions 192 Navarrete-Salgado NA, Cedillo-Díaz BE, Contreras-Rivero G, Salgado-Ugarte IH, Gómez-Márquez JL, Peña-Mendoza B Elías -Fernández G (2007) Crecimiento, reproducción y (2005) Métodos actualizados para análisis de datos biológicos- supervivencia de Girardinichthys multiradiatus (Pisces, pesqueros. 1st (Ed), FES Zaragoza UNAM, México. Goodeidae) en el embalse San Miguel Arco, Estado de México. Revista Chapingo 13:15-21. Schoenherr AA (1977) Density dependent and density independent regulation of reproduction in the gila topminnow Nelson GG (1975) Anatomy of the male urogenital organs of Poeciliopsis occidentalis (Baird and Girard). Ecology 58:438- Goodea atripinnis and Characodon lateralis (Atheriniformes: 444. DOI: 10.2307/1935619 Cyprinodontoidei), and G. atripinnis courtship. Copeia 3:475- 482. DOI: 10.2307/1443645 Snoeks J, Laleye P, Contreras-MacBeath T (2009).Goodea atripinnis. The IUCN Red List of Threatened Species 2009 Orbe-Mendoza AA, Acevedo-García J, Lyons J (2002) Lake available on http://www.iucnredlist.org/details/169399/0, Patzcuaro fishery management plan. Reviews in Fish Biology accessed on 15 November 2014. and Fisheries 12:207-217. DOI:10.1023/A:1025087705940 Sullivan JA, Schultz RJ (1986) Genetic and environmental Ortiz‐Ordóñez E, Uría Galicia E, López‐López E, Maya JP, basis of variable sex ratios in laboratory strains of Poeciliopsis Carvajal Hernández AL (2007) Reproductive cycle by lucida. Evolution 40:152-158. DOI:10.2307/2408612. histological characterization of the ovary in the butterfly goodeid Ameca splendens from the upper Río Ameca Basin, Turner CL (1933) Viviparity superimposed upon ovo- Mexico. Journal of Applied Ichthyology 23: 40-45. viviparity in the Goodeidae, a family of Cyprinodont teleost DOI:10.1111/j.1439-0426.2006.00790.x. fishes of the Mexican Plateau. Journal of Morphology 55:207- 251. DOI: 10.1002/jmor.1050550202 Ospina-Álvarez N, Piferrer F (2008) Temperature-dependent sex determination in fish revisited: prevalence, a single sex Uribe CM, Grier HJ, Parenti LR (2012) Ovarian structure and ratio response pattern, and possible effects of climate change. oogenesis of the oviparous goodeids Crenichthys baileyi PLoS One 3:e2837-e2837. DOI:10.1371/journal.pone.0002837 (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (teleostei, Cyprinodontiformes). Journal of Morphology 273:371-387. Pratt HL, Otake T (1990) Recommendations for work needed DOI:10.1002/jmor.11028. to increase our knowledge of reproduction relative to fishery management. NOAA Technical Report NMFS 90:509-510. Uribe MC, De la Rosa-Cruz D, Grier HJ (2014) Proliferation of oogonia and folliculogenesis in the viviparous teleost Ramírez-Herrejón JP, Medina-Nava M, Salazar-Tinoco CI, Ilyodon whitei (Goodeidae). Journal of Morphology 275:1004- Zubieta TLE (2007) Algunos aspectos reproductivos de 1015. DOI: 10.1002/jmor.20277 Zoogoneticus quitzeoensis Hubbs y Turner (1939) (Osteichtyes-Goodeidae) en la represa La Mintzita Morelia, Uribe MC, De la Rosa-Cruz G, García-Alarcón A (2005) The Michoacán, México. Biológicas Revista de la DES Ciencias ovary of viviparous teleost. Morphological differences between Biológico Agropecuarias Universidad Michoacána de San the ovaries of Goodea atripinnis and Ilyodon whitei Nicolás de Hidalgo 9:63-71. (Goodeidae). In: Uribe MC, Grier HJ (Eds), Viviparous Fishes, New Life Publication, Florida, Pp:217-236. Ricker WE (1975) Computation and interpretation of biological statistics of fish populations. Bulletin of the Uribe MC, Grier HJ (2011) Oogenesis of microlecithal oocytes Fisheries Research Board of Canada 191:145-157. DOI: in the viviparous teleost Heterandria formosa. Journal of 10.2307/3800109 Morphology 272:241-257. DIO:10.1002/jmor.10912

Rojas-Pinedo A (1981) Distribución de la ictiofauna del estado Uribe MCA, Aguilar-Morales M, De las Rosa-Cruz G, García- de Aguascalientes. Profesional Thesis. Universidad Autónoma Alarcón A, Campuzano-Caballero JC, Guerrero-Estévez SM de Aguascalientes. México Pp: 72. (2010a) Ovarian structure and embryonic traits associated with viviparity in Poeciliids and Goodeids. In: Uribe MC, Grier HJ Römer U, Beisenherz W (1996) Environmental determination (Eds), Viviparous Fishes II, New Life Publication, Florida, Pp: of sex in Apistogramma (Cichlidae) and two other freshwater 211-230. fishes (Teleostei). Journal of Fish Biology 48:714 – 725. DOI: 10.1111/j.1095-8649.1996.tb01467.x Uribe MCA, De la Rosa Cruz G, Alarcón AG, Guerrero- Estévez SM, Morales MA (2006) Histological features of Rosenfeld CS, Roberts RM (2004) Maternal diet and other atretic stages of the ovarian follicles of two viviparous teleost factors affecting offspring sex ratio: a review. Biology of species: Ilyodon whitei (Meek, 1904) and Goodea atripinnis Reproduction 71:1063-1070. (Jordan, 1880)(Goodeidae). Hidrobiológica 16:67-73. DOI:10.1095/biolreprod.104.030890

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Uribe MCA, Grier HJ, Mejía-Roa V, Yáñez-García N, García- Van Aerle R, Runnalls TJ, Tyler CR (2004) Ontogeny of Alarcón A, De la Rosa-Cruz G, Aguilar-Morales M (2010b) gonadal sex development relative to growth in fathead Functional structure of the testis and spermatogenesis of minnow. Journal of Fish Biology 64:355-369. Goodeids and Poeciliids. In: Uribe MC, Grier HJ (Eds), DOI:10.1111/j.0022-1112.2004.00296.x Viviparous Fishes II, New Life Publication, Florida, Pp: 151- 172. White MM, Turner BJ (1984) Microgeographic differentiation in a stream population of Goodea atripinnis (Goodeidae) from Valenzuela N, Adams DC, Janzen FJ (2003) Pattern does not the Mexican Plateau. Environmental biology of fishes 10:123- equal process: exactly when is sex environmentally 127. DOI: 10.1007/BF00001669 determined? The American Naturalist 161:676-683. DOI: 10.1086/368292

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EFFECT OF PAPAYA AND STORAGE TIME ON THE QUALITY OF THE NEWLY DEVELOPED PAPAYA CRACKERS

Habiba Khatun1,*, Nusrat Jahan1, Mojaffor Hosain2, Trisna Mardy1 and Shahinur Rahman1

1Departmentof Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200 2Departmentof Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200

Received – March 03, 2016; Revision – March 17, 2016; Accepted – April 20, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).194.200

KEYWORDS ABSTRACT Crackers Crispy nutritious papaya crackers were developed from locally available papaya and whole wheat flour o Physicochemical by baking in oven at 130 C for 35 min. Crackers were packed in high density polyethylene pouches and characteristics stored under ambient condition for 90 days. The influence of papaya and storage condition on physicochemical compositions was evaluated in present study. Physicochemical analyses of fresh Papaya crackers revealed the presence of moisture (3.3-3.6%), ash (4.07-4.12%), protein (6.35-8.13%), fat (1.75-1.94%), fibers (11.50-13.75%), minerals like sodium (6.1-6.61 mg/100g), magnesium (122.44- Whole wheat flour 115.28 mg/100g), potassium (469.62-443.34 mg/100g), calcium (24.99-21.97 mg/100g) and phosphorus (307.66-229.13 mg/100g). Among various studied physicochemical parameters only slight increases Storage time was reported in the moisture content while in case of other parameters like fat, ash, fiber, protein and mineral content some reduction was reported at the 90 days of storage. This study introduced the formulation of crackers by using whole wheat flour and papaya which can be a good source of fiber and minerals.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Habiba Khatun) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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1 Introduction exporting papaya crackers. Based on the above discussion, the present work was aimed to formulate and analyze the Papaya (Carica papaya L.) is one of the important and nutritional composition of papaya crackers and also, to assess versatile fruits of the family Caricaceae and grown worldwide the storage stability of prepared papaya crackers. in the tropics and subtropics including India, Bangladesh, Malaysia, Australia, Hawaii, Philippines, Sri Lanka, South 2 Materials and methods Africa and other countries in tropical America (Anuara, 2008). In Bangladesh, papaya is available all around the year, 2.1 Sample collection therefore ripe papaya is consumed as fruit and green papaya as vegetable. According to BBS (2011), papaya production in Fresh, fully mature and sound papaya and whole wheat flour Bangladesh was 1.25 lac metric tons from 1.24 thousand were purchased from local market at Basherhat for preparation hectares of land with an average yield of 7 ton/ha. However, it of crackers. was also reported that papaya production in Bangladesh is far below compared to other neighboring countries (Chowdhury, 2.2 Formulation of papaya crackers 2008). Furthermore, post-harvest losses of papaya is recorded almost 39.9% in Bangladesh (Hasan, 2010) and it is a major Three formulations of papaya crackers viz F1, F2 and F3 were cause of unavailability of papaya (Mondal et al., 1995). developed using papaya and whole grain wheat flour with the Unavailability of effective preservation and processing addition of seasoning (baking powder, corn flour, sugar, garlic, techniques are major reasons of post-harvest losses of papaya. ginger and salt) as given in Table 1.

Nowadays, fast food snacks especially crackers have emerged Table 1 Different formulations of papaya crackers. as popular items because of unique flavor and texture to the modern consumers of all age groups. Excess use of these fast Formulation Ingredient foods can be harmful for human beings and according to Papaya Whole Seasoning Thunstrom (2011), higher consumption of these fast food items (%) wheat (%) (%) can cause several diseases like diabetes, cardiovascular F1 40 50 10 disease, cancer, dental caries and osteoporosis, overweight as well as obesity because of less fiber and excess fat content. F2 30 60 10 F3 20 70 10 On the other hand, dietary fiber is tremendous attention in food processing where bakery products could be modified by 2.3 Preparation of papaya crackers addition of value added ingredients (Vergara-Valencia et al., 2006; Sudha et al., 2007). Consumption of food made of whole Clean and disease free whole wheat was turned into flour by grain is associated with the lowering of glucose level in blood, hammer mill. Simultaneously, papaya was washed with clean cardiovascular disease, cancers and other diseases because of water and peeled and sliced longitudinally and boiled for 5 min its dietary fiber and micronutrients (McKeown et al., 2002). at 180oC. After boiling, papaya and whole wheat flour were High amount of dietary fiber, vitamin E, vitamin B complex, taken in different combination in a bowl as described in Table minerals, antioxidants and phytoestrogens are found in the bran 1. Boiled papaya was mashed and all seasoning ingredients and germ of whole grains (Jacobs et al., 1998). In Bangladesh, (garlic, ginger) were added properly for making dough. Dough there are less whole wheat products whereas in developed was transformed into sheet of 2mm thick and cut into desirable countries, whole wheat products have been gained much shapes. After that, the cut pieces were baked at 130oC for popularity. 35min in oven. After baking, crackers were cooled and packaged (50g) in high density polyethylene pouches. Fresh papaya could be explored for the production of papaya crackers. It can be used incorporation with wheat flour for Fresh crackers were subjected to initial physicochemical better quality crackers. According to Tietze (2002), mature analysis before packaging. The sealed packages were stored at papaya is a rich source of vitamin A, E, B complex, ascorbic ambient condition (temperature 25±2oC and relative humidity acid compared to carrots and oranges. Furthermore, a recent 68±3%) for storage studies at 30 days intervals for 90 days. study also shows that papaya contains arginine amino acid, which is essential for male fertility (Rovira, 2009). 2.4 Physico-chemical analysis

Therefore, papaya crackers with whole grain flour may be one Physico-chemical analyses namely, moisture, ash, protein and of potential processed products in Bangladesh. In some areas fat content were carried out according to standard methods of of Bangladesh, papaya is very cheap due to lack of storage, AOAC (1997). The crude fiber was determined by the method transportation and processing facility. Moreover, substantial of Ayesha et al. (2002) with slight modification. amount of foreign exchange could be earned by processing and

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Effect of papaya and storage time on the quality of the newly developed papaya crackers 196 The available minerals (calcium, magnesium, potassium, It was observed that different ratio of papaya and whole grain phosphorous and sodium) were determined by atomic flour significantly affected the moisture content of crackers. absorption methods described by Wahab et al. (2010) with Figure 1A shows that moisture content of papaya crackers some modifications. For this, one gram of the crackers sample increased throughout the storage period. Change in moisture was taken in 50 ml flask. Then, 10 ml of concentrated nitric content was comparatively less for first 30 days of storage but acid and 5 ml of concentrate edperchloric acid were added to later on the level of moisture content increased with the the flask and digested using a hot plate until complete removal increasing duration of storage. Maximum increase (101.52%) of white smoke and solution become clear. 20-30ml of distilled was reported in F1 sample, whereas minimum increase water was then added and filtered, and final volume was made (72.22%) was observed in F3 sample at the end of storage up to100 ml with distilled water. Absorbance was measured by duration. The increase in moisture of crackers may be due to using atomic absorption spectrophotometer. high water vapor transmission rate through packaging material. Finding of present study are in agreement with the findings of 2.5 Statistical Analysis Jagdeesh et al. (2007) and Purohit & Rajyalakshmi (2011). Furthermore, Manikantan et al. (2014) also observed an All measurements were carried out in triplicate. The data were increase in moisture of banana chips stored in polypropylene analyzed using statistical program SPSS (version 20.0) and based nanocomposite packaging films. significant differences were compared by analysis of variance test (ANOVA) following Duncan’s Multiple Range Tests at 3.2.2 Ash content the significance level of 5%. Results of ash analysis were presented in Figure 1B. Ash 3 Results and Discussion content of fresh crackers of F1, F2 and F3 were reported 4.12, 4.09 and 4.07% respectively (Figure 1B). Significant 3.1 Physico-chemical characteristics of papaya and whole difference in ash content of fresh crackers was not observed wheat flour due to variation in papaya and whole wheat grain flour ratio. Ash content of crackers gradually decreased with the Data regarding physico-chemical characteristics of green increasing of storage time (Figure 1B). Highest ash content papaya and whole wheat flour have been presented in Table 2. (3.63%) was observed in F2 sample, whereas F1 and F3 It was observed that green papaya showed high moisture showed same ash content (3.55%) at the end of storage period. (88.2%), ash (0.63%), very low protein (0.57%), fat (0.63%) Moisture absorption may be possible reason behind the and crude fiber content (0.75%). The results of present study decrease in ash content of crackers. However, no significant are in agreement with the findings of Krishna et al. (2008). On effect of storage time was reported on the ash content of the other hand, wheat flour showed moisture (12.5%), ash crackers. This result was in line with the finding of Akubor & (1.7%), high protein (12.3%), fat content (1.8%) and fiber Adejo (2000) who observed similar trend in decreasing of ash content (1.9%). The results are comparable with the findings of content in stored plantain chips. Belderok et al. (2000) who observed slight higher protein and less fiber content (13% and 1.5% respectively) in whole wheat 3.2.3 Protein content endosperm. Figure 1C showed ratio of papaya and whole wheat flour and it Table 2 Physico-chemical characteristics of green papaya and was reported that this combination significantly affected the whole wheat flour. protein content of fresh crackers. Protein content in crackers increased with the increase of whole wheat flour content. Composition Green papaya Whole wheat flour However, maximum protein (8.14%) was observed in fresh F3 sample and followed by F2 (7.48%) and F1 (6.36%) sample. Moisture (%) 88.2±0.23 12.5±0.19 Data regarding change in protein content of crackers revealed Ash (%) 0.63±0.08 1.7±0.10 that there was a gradual decrease in protein content throughout Protein (%) 0.57±0.11 12.3±0.34 the storage period (Figure 1C). Fat (%) 0.63±0.14 1.8±0.19 Crude fiber (%) 0.75±0.12 1.9±0.23 Maximum decrease (28.75%) was found in F3 sample while All values are means ±SD F2 sample had minimum decrease (16.04%). One of possible reason of decreasing in protein content of crackers during 3.2 Storage studies of papaya crackers storage is moisture absorption from the atmosphere. Similarly, Akubor & Adejo (2000) reported a gradual but insignificant 3.2.1 Moisture content decrease in protein content in plantain chips stored in 0.7 mm thick polyethylene bags (26.6 × 18.8 cm). The moisture content of freshly prepared cracker samples F1, F2 and F3 was 3.3, 3.55 and 3.6% respectively (Figure 1A).

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Figure 1 Change in physico-chemical compositions (A-Moisture, B-Ash, C-Protein, D-Fat and E-Fiber content) of crackers during storage period

3.2.4 Fat content showed maximum decreasing trend from 1.94% to 0.98% and minimum trend 1.84% to 1.22% was observed in F2 sample. Fat content of fresh crackers was significantly affected by ratio Decreasing trend in fat content of stored crackers may be of papaya and whole grain flour. Fat content of fresh cracker resulted due to hydrolysis and oxidation of oil component. increased with the decreasing of papaya paste (Figure 1D). These results are in good agreement with Manikantan et al. Moreover, Figure 1D clearly indicates that fat content (2014) who noted increased free fatty acid in stored banana decreased with the storage period. During storage period, F3 chips.

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Effect of papaya and storage time on the quality of the newly developed papaya crackers 198 3.2.5 Fiber content in the fiber content was reported with the passage of storage time, whereas maximum decrease (39.54%) was resulted in F2 Fiber content in F1, F2 and F3 combinations of freshly sample and minimum (24.17%) in F1 sample. Moisture prepared papaya crackers of were 11.5, 12.14 and 13.75% absorption during storage time may be possible reason of respectively (Figure 1E). The storage period showed decreasing the amount of fiber content in different significant effect on fiber content of crackers and a reduction combinations of papaya crackers.

Figure 2 Change in mineral contents (A-Calcium, B-Magnesium, C-Sodium, D-Potassium and E-Phosphorous) of crackers during storage period

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3.2.6 Mineral content Association of Official Analytical Chemists (1997) Official methods of analysis. 16th edition. Washington, D.C. 2. Results of study revealed that incorporation of whole wheat flour had significant effect on mineral contents of papaya Ayesha S, Abid N, Anjum FM (2002) Chemical Composition crackers (Figure 2). Maximum calcium, magnesium, sodium, of Three Wheat (Triticumaestivum L.) Varieties as Affected by potassium and phosphorous value were found in F3 sample NPK Doses. International Journal of Agricultureand Biology and it was 27.24%, 123.85%, 6.78%, 503.72% and 327.02% 4:537-539. respectively. On the other hand, value of mineral contents decreased in all cracker samples with longer storage period. BBS (2011) Year Book of Agricultural Statistics of However, maximum mineral contents were found in F3 sample Bangladesh. Bangladesh Bureau of Statistics. Statistics at 90 days of storage period, whereas minimum minerals in F1 Division. Ministry of Planning, Government of the Republic of sample (Figure 2). Increasing of moisture might have Bangladesh. influenced the mineral contents of crackers during storage period. It is interesting to note that there was no published Belderok B, Mesdag H, Donner DA (2000) Bread-Making paper regarding change in mineral content of crackers during Quality of Wheat - A century of breeding in Europe. Springer storage. Netherlands. Chowdhury MGF, Islam MN, Islam MS, Islam AFMT, Conclusion Hossain MS (2008) Study on preparation and shelf-life of mixed juice based on wood apple and papaya. Journal of soil This study revealed that the ratio of wheat flour and papaya and nature 2:50-60. significantly affected the nutrient compositions of crackers. Additionally fiber content was increased and baking process Hassan MK (2010) A guide to postharvest handling of fruits instead of frying resulted in less fat content of the crackers. and vegetables, Department of Horticulture. Bangladesh Storage study also revealed that storage time significantly Agricultural University Mymensingh, 2202. affected the quality of crackers. Furthermore, it was also reported that high density polyethylene was not sufficient Jacobs DR Jr, Marquart L, Slavin J, Kushi LH (1998) Whole- enough to keep the quality of crackers during storage. We also grain intake and cancer: an expanded review and meta- propose to pack crackers in laminated films containing analysis. Nutrition and Cancer 30:85–96. nitrogen flush for long term storage stability. In addition, the effect of storage conditions on oxidative stability of newly Jagdeesh SL, Hegde L, Kotimani S, Gorbal K, Reddy BS, formulated crackers could be assessed before the production at Swamy GSK, Basavaraj N, Raghvan GSV (2007) Influence of industrial scale. packaging on storage behavior of jackfruit papad. Journal of Food Science Technology 34:15–19. Acknowledgement Krishna KL, Paridhavi M, Patel JA (2008) Review on The research work was funded by University Grant nutritional, medicinal and pharmacological properties of Commission through Institute of Research and Training, Hajee papaya (Carica papaya Linn.). Indian Journal of Natural Mohammad Danesh Science and Technology University, Products and Resources 7:364-373. Dinajpur, Bangladesh. Manikantan MR, Shrama R., Kasturi R., Varadharaju N (2014) Conflict of interest Storage stability of banana chips in polypropylene based nanocomposite packaging films. Journal of Food Science and Authors would hereby like to declare that there is no conflict of Technology 51:2990–3001.DOI 10.1007/s13197-012-0839-0 interests that could possibly arise. McKeown NM, Meigs JB, Liu S, Wilson PWF, Jacques PF References (2002) Whole-grain intake is favourably associated with metabolic risk factors for type 2 diabetes and cardiovascular Akubor PI, Adejo EE (2000) Physicochemical, microbiological disease in the Framingham Offspring Study. American Journal and sensory changes in stored plantain chips. Plant Foods for of Clinical Nutrition 76:390-398. Human Nutrition 55:139–146. DOI: 10.1023/A:1008186224120. Mondal MF, Rahman MA, Pramanik MAJ (1995) Effects of different post harvest treatments on physio-chemical changes Anuara NS, Zaharia SS, Taiba IA, Rahman MT (2008) Effect and shelf life of mango. Bangladesh horticulture 23:1-5. of green and ripe Carica papaya epicarp extracts on wound healing and during pregnancy. Food and Purohit C, Rajyalakshmi P (2011) Quality of products containing defatted groundnut cake flour. Journal of Food Chemical Toxicology 46: 5. DOI: 10.1016/j.fct.2008.03.025. Science and Technology 48:26–35.doi: 10.1007/s13197-010- 0125-y.

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Effect of papaya and storage time on the quality of the newly developed papaya crackers 200 Rovira DD (2009) Technology and Engineering. Accessed European Journal of Health Economics 12:127–140.doi: online on 23rd March, 2010 from http://books.google.ca/ 10.1007/s10198-010-0234-6. books?isbn=0813821355. Tietze HW (2002) Health and Fitness. Harald W. Tietze Sudha ML, Vetrimani R, Leelavathi K (2007) Influence of Publishing Property Limited, Bermagui. fibre from different cereals on the rheological characteristics of wheat flour dough and on biscuit quality. Food Chemistry Vergara-Valencia N, Granados-Pérez E, Agama-Acevedo E, 100:1365-1370.doi:10.1016/j.foodchem.2005.12.013. Tovar J, Ruales J, Bello-Pérez LA (2006). Fibre concentrate from mango fruit: Characterization, associated antioxidant Thunstrom JN (2011) Economic policies for healthier food capacity and application as a bakery product ingredient. intake: the impact on different household categories. The Lebensmittel-Wissenchaft und-Technologie 40:722- 729.doi:10.1016/j.lwt.2006.02.028.

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

SOMATIC EMBRYOGENESIS AND CALLUS PROLIFERATION IN Picrorhiza kurroa ROYLE ex. BENTH

Ritu Mahajan*, Nisha Kapoor and Iqbal Singh

School of Biotechnology, University of Jammu, Jammu (J&K) India

Received – February 19, 2016; Revision – March 17, 2016; Accepted – April 21, 2016 Available Online – April 25, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).201.209

KEYWORDS ABSTRACT

Picrorhiza kurroa Picrorhiza kurroa Royle ex. Benth is an endangered and important medicinal plant of alpine Himalayas. Developing an efficient protocol for its mass multiplication is essential to meet the requirements of In vitro pharmaceutical industries and also in conservation of this plant under its natural habitat. Present study was undertaken to develop a protocol for in vitro mass multiplication of P. kurroa. Result of study Endangered revealed that highest frequency of shoot regeneration was achieved on Murashige and Skoog’s basal Callus medium supplemented with 1.0mg/l BAP, 0.5 mg/l Kn and 1.0mg/l GA3, while the best rooting was observed in MS medium supplemented with 2.5 mg/l IBA. MS medium supplemented with 3.0 mg/l Somatic embryos 2,4-D resulted in highest frequency of embryogenic callus. Callus inoculated on MS media supplemented with BAP and IAA resulted in both shoot and root formation while the callus on MS media supplemented with NAA and IBA resulted only root formation. The somatic embryos were established from callus on MS medium supplemented 2.5 mg/l 2,4- D after four weeks. MS medium containing 1.0 mg/l BAP and 1.0 mg/l GA3 resulted into shoots from well developed somatic embryos. This protocol will provide a system for the germplasm conservation in P. kurroa by multiplication and regeneration of true to type plants.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Ritu Mahajan) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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1 Introduction The explants were then surface sterilized with 70% ethanol,

mercuric chloride (HgCl2), sodium hypochlorite and 1ppm

The increasing demand of medicinal plants for the Potassium permanganate (KMnO4) in laminar air flow. A development of several drugs and chemotherapeutics has single or combinations of two or more sterilants were used for threatened their natural habitats and now they are in brink of the surface sterilization of the explants. extinction (Mugula et al., 2012; Bodeker et al., 2013). These plants are demanding to be conserved for their potential utility. 2.2 Culture medium and conditions One of such plant is Picrorhiza kurroa Royle ex. Benth belonging to family Scrophulariaceae which is endemic to MS (Murashige & Skoog 1962) basal medium (Himedia) was Himalayan region (Patial et al., 2012). The plant grows from used for the shoot establishment. The medium was Kashmir to Sikkim at an altitude of 3,000-5,000 meters. The supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. The rhizomes of the plant are the principle source of iridoid pH of the medium was adjusted to 5.8 before autoclaving. MS glycosides out of which the major is Picrosides and kutkosides medium with varying concentrations of growth hormones was (Patil et al., 2013). These iridoids have numerous therapeutic prepared for shoot induction, multiplication and callus uses like antihepatotoxic, choleretic, hypolipidemic, proliferation. Cultures were maintained at 25 ±2º C in 16/8 h antiinflammatory, antispasmodic (Tiwari et al., 2012; Sultan et light/dark cycle in tissue culture room. Subculturing was al., 2016). The dried extracts from the plant has a cure for liver carried after 4–6 weeks interval. Care was taken to prevent any disorders, fever, jaundice and allergies (Baruah et al., 1998). further contamination of subcultures from the microbes. The herbal preparation of the plant plays a major role in antidiabetic and anticancerous activity (Pradhan 2011; Kumar 2.3 Shoot proliferation and rooting & Ramesh 2014; Mallick et al., 2015). Two weeks after the initial establishment of aseptic cultures, The over exploitation of the rhizomes of P. kurroa for the the shoot cultures thus obtained were further transferred for extraction of bioactive constituents along with the poor multiplication on to MS medium containing different cultivation and small population size has depleted this species combination and concentrations of BAP (Benzyl Amino from its natural habitat (Verma et al., 2012). As a result, the Purine), Kn (Kinetin) and GA3 (Gibberellic Acid). Ten plant is now listed as an endangered plant species in red data different MS medium combinations with growth regulators book by International Union for Conservation of Nature were tried for shoot multiplication (Table 1). For root (IUCN). The conventional method of propagation using induction, after 6 weeks the well developed in vitro grown rhizomes and seeds restricts multiplication as seed germination shoots were separated singly and implanted on MS medium is very poor in P. kurroa, so conservation using in vitro supplemented with different concentrations of IBA (Indole approaches is essential (Rawat et al., 2013). Butyric Acid) (Table 2).

It is necessary to develop a protocol for mass multiplication of 2.4 Callus induction and organogenesis this endangered high value medicinal plant so as to assure its conservation and the pharmaceutical demand. The present Fully expanded young leaves of in vitro grown plants of P. study was undertaken with the objective to improve the kurroa were cut into small pieces using sterilized blade and protocol for high frequency shoot multiplication and callus then inoculated on MS medium containing 2,4 D (2, 4- mediated somatic embryogenesis in P. kurroa under in vitro Dichlorophenoxy Acetic Acid) at different concentrations conditions. (1.0mg/l to 4.0mg/l) for callus induction and proliferation. The proliferated callus after six weeks was transferred to 2 Materials and Methods regeneration media containing BAP, IAA and NAA (Napthalene Acetic Acid). 2.1 Plant material and surface sterilization 2.5 Somatic embryogenesis and plant regeneration The explants were excised from young and healthy growing parts of P. kurroa collected from high altitudes of Kishtwar After eight weeks of culturing, the small pieces of friable and area of Jammu and Kashmir. The plant parts were washed embryogenic callus were transferred to MS medium containing under running tap water so as to remove all the soil 2,4-D (2.0, 2.5, 3.0 mg/l) for induction of somatic embryos. completely. Different explants like auxillary buds and After 4 weeks of subculturing, greenish yellow embryoids internodal segments were excised from plant parts and were were observed. These pro embryoids were subcultured on same then collected in a beaker containing 2-3 drops of Tween 20 medium for maturation for next two weeks. Mature somatic (Himedia) in distilled water. After 10 min the explants were embryos were transferred to regeneration MS medium washed thrice with distilled water. containing BAP (0.5, 1.0, 1.5, 2.0 mg/l) and GA3 (1.0mg/l).

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Somatic embryogenesis and callus proliferation in Picrorhiza kurroa Royle ex. Benth 203

Table 1 Different concentrations and combinations of growth hormones used for shoot and root multiplication in P. kurroa.

Growth regulators Concentration of growth regulators (mg/l) MS + BAP 0.5, 1.0 MS + Kinetin 0.5, 1.0

I. 1.0mg/l BAP+ 0.5.0mg/l Kn Shoot multiplication medium MS + BAP + Kinetin II. 0.5 mg/l BAP+ 1.0mg/l Kn III. 1.0 mg/l BAP+ 1.0mg/l Kn I. 0.5 mg/l BAP+ 1.0 mg/l Kn + 1.0 mg/l GA3

MS +BAP+Kinetin+GA3 II. 1.0 mg/l BAP+ 0.5mg/l Kn + 1.0 mg/l GA3 III. 1.0 mg/l BAP+ 1.0 mg/l Kn + 1.0 mg/l GA3

Root multiplication medium MS + IBA 0.5, 1.0, 1.5, 2.0, 2.5, 3.0

3 Results and Discussion multiplication medium the plants regenerated into multiple shoots (Figure 1b). After six weeks the number of shoots per 3.1 Surface Sterilization explant was counted and also their length was recorded.

The explants used in the present study were taken from the P. The maximum percentage of shoot growth observed after six kurroa plant parts, collected from field. These were surface weeks was 85.63% with an average number of 8.7± 0.18 sterilized with sodium hypochlorite used at the concentration shoots per explant. The average shoot length was 5.92 ± 0.29 of 5.0% which resulted in 46.6% of uncontaminated explants cm. However, when the concentration of kinetin was increased Best sterilization treatment was 70% ethanol for 30 sec to 1.0mg/l, the percentage shoot growth decreased to 80.21% followed by HgCl2 (0.1%) for 2 min and then KMnO4 (1ppm) and the average number of shoots was 7.8± 0.15 (Table 3). for 3 min resulting in 80.0% of uncontaminated explants. Venkatachalam et al. (2012) reported a high frequency (87.3%) Similar results were also reported by Sen et al. (2014) where of shoot proliferation (6.2 shoots/explant) in MS medium they observed that more than 3 min of surface sterilization with supplemented with 2.0 mg/l BAP in combination with 0.5 mg/l mercuric chloride is lethal to all of his tested varieties of A. Kinetin in Gloriosa Superba while Patial et al. (2012) observed aspera. Thus the herbaceous explants are always treated with maximum number of shoots on MS medium supplemented low concentration of sterilants so that their soft tissues are not with kinetin in P. kurroa. affected or damaged. In the present study, it was also observed that the shoots 3.2 Shoot multiplication obtained on MS medium supplemented with BAP only, were healthy with long and thick leaves but the number of shoots The explants were established in MS basal medium after two was less whereas the number of shoots was more in MS media weeks of inoculation (Figure 1a). The established shoots were containing kinetin but the shoots were thin (Figure 2). transferred to shoot multiplication medium after four weeks. Reduction in the number of shoots regenerated per explant The best shoot multiplication was observed in MS medium with BAP as only growth regulator was also reported in A. supplemented with 1.0 mg/l BAP, 0.5 mg/l Kn and GA3 squamosa (Nagori & Purohit, 2004). (1.0mg/l). After six weeks of subculturing in shoot

Table 2 Effect of different sterilants on the surface sterilization of explants.

S. No. Treatments Time duration Percent survival of the explants 1 70% ethanol 30-60 sec. Nil

2 KMnO4 (1ppm) 3 min Nil

3 70% ethanol + HgCl2 (0.05%) 30 sec. + 2 min. 13.3

4 70% ethanol + HgCl2 (0.1%) 30 sec. + 2 min. 36.6

5 70% ethanol + HgCl2 (0.2%) 30 sec. + 4 min. 30.0

6 70% ethanol + HgCl2 (0.5%) 30 sec. + 2 min. 15.9 7 Sodium hypochlorite (5% v/v) 5 min. 46.6 8 Sodium hypochlorite (10% v/v) 5 min. 20.0

9 70% ethanol + HgCl2 (0.2%) + KMnO4 (1ppm) 30 sec. + 2 min + 3 min 66.6

10 70% ethanol + HgCl2 (0.1%) + KMnO4 (1ppm) 30sec. + 2 min + 3 min 80.0

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(A) (B) Figure 1 (A) Proliferation of leaves from auxiliary buds after two weeks MS medium (B) Shoot multiplication in MS medium with

BAP, Kn and GA3

Figure 2 Effect of BAP and Kinetin in P. kurroa after 4 weeks of subculturing

Figure 3 Root multiplication in MS medium with IBA after six weeks of sub culturing.

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Somatic embryogenesis and callus proliferation in Picrorhiza kurroa Royle ex. Benth 205

Table 3 Effect of different concentrations of plant growth regulators on in vitro shoot multiplication in P. kurroa.

S.N Concentration of growth Percentage Shoot growth Average no. of shoots per Average shoot length o regulators (mg/l) explant (cms) 1 MS + 0.5 BAP 52.80 2.2± 0.54 2.17 ± 0.58 2 MS + 1.0 BAP 66.20 2.7± 0.38 3.61 ± 0.22 3 MS + 0.5 Kn 50.80 1.1± 0.32 1.21± 0.37 4 MS + 1.0 Kn 59.06 1.9± 0.51 1.92± 0.52 5 MS + 1.0 BAP + 0.5 Kn 68.60 6.8± 0.21 4.22± 0.36 6 MS + 0.5 BAP + 1.0 Kn 63.41 5.7± 0.25 5.64± 0.80 7 MS + 1.0 BAP + 1.0 Kn 72.49 4.2± 0.51 5.21± 0.71 8 MS + 1.0 BAP + 0.5 Kn+ 1.0 85.63 8.7± 0.18 5.92± 0.29 GA3 9 MS + 0.5 BAP + 1.0 Kn+ 1.0 76.54 8.2± 0.22 5.53± 0.40 GA3 10 MS + 1.0 BAP + 1.0 Kn+ 1.0 80.21 7.8± 0.15 5.22± 0.69 GA3 *Data is from twenty shoots in three replicates and is represented as mean±SD

3.3 In vitro rooting and hardening plant species (Baul et al., 2011; Praveena & Veeresham 2014; Rahdari et al., 2014). The rooted plants were removed from The addition of auxin in the rooting medium results in long flasks, washed carefully to remove agar and transferred to a length and higher number of roots as compared to hormone potting mixture in plastic pots containing sand: soil (1:1). The free medium. Development of healthy and long rootlets is plants were initially covered with jars in glass house for two helpful for the establishment of plantlets in the field. Direct, weeks to maintain the humidity. About ninety percent rate of healthy root initiation and elongation was observed in P. survival was obtained upon hardening. kurroa after subculturing of elongated single shoots onto MS basal medium supplemented with different concentrations of 3.4 Callus induction IBA (Table 4; Figure 3). Different concentrations of 2,4-D in MS medium resulted in Best rooting medium was MS medium supplemented with 2.5 callus formation from the leaf explants (Table 5). The callus mg/l IBA resulting in 84.54% of root growth. The average was creamy white and friable in MS medium containing number of roots per explant was 10.35±0.18. The roots were 2.0mg/l and 3.0mg/l 2,4-D (Figure 4). It was observed that the long, healthy with numerous root hairs. As the concentration of high concentration of 2,4-D (4.0mg/l) resulted in browning of IBA was increased to 3.0mg/l, the percent root growth and the explants with no callus formation. Similarly, Zuraida et al., average number of roots was decreased to 79.22% and (2015) has also reported that higher concentration of 2,4-D 9.11±0.21 respectively (Table 4). Similar effects of IBA were resulted in browning of the callus in Pelargonium radula. also observed during in vitro rooting in several other medicinal

Table 4 Effect of IBA concentration on in vitro rooting.

S. No. MS + Growth regulators %age Average no. of roots per Types of roots (mg/l) rooting explant 1. MS +0.5 IBA 35.40 1.23±0.27 Small thread like roots no root hairs. 2. MS +1.0 IBA 55.80 4.97±0.62 Thin, fragile roots with less root hair. 3. MS +1.5 IBA 68.56 7.14±0.34 Thin, fragile roots with root hairs. 4. MS +2.0 IBA 74.58 8.44±0.23 Thin, fragile roots with few root hairs 5. MS +2.5 IBA 84.54 10.35±0.18 Long, well developed roots with numerous root hairs. 6 MS +3.0 IBA 79.22 9.11±0.21 Long, well developed roots with numerous root hairs. *Data is from fifteen plants in three replicates and is represented as mean±SD

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Figure 4 Callus proliferation in MS medium supplemented with 3.0mg/l 2,4-D.

(A) (B) Figure 5 (A) Shoot and root induction from callus in MS media supplemented with 2,4-D, BAP and IAA (B) Root induction from leaf explants in MS medium supplemented with 2,4-D, IAA and NAA

After six weeks, MS medium supplemented with 2.5mg/l 2,4- 2011; Kim et al., 2012; Giri et al., 2013; Barberini et al., 2016). D + 1.0mg/l BAP + 1.0mg/l IAA resulted in the formation of In the present study, cytokinins were used as promoters for the shoots along with the roots from the callus. When the induction of somatic embryos. The combination of both 2,4-D proliferated callus was inoculated on MS medium containing along with BAP and GA3 in MS medium significantly 2.5mg/l 2,4-D + 2.0mg/l NAA + 0.5mg/l IAA, then only root affected the number of embryo production. After four weeks, formation was observed (Figure 5a and 5b). The concentration 2,4-D at the concentration of 2.5mg/l resulted in the formation of plant growth regulators needed to induce callus, varies from of greenish yellow callus. species to species and also depends on the source of explant (Ngomuo et al., 2014). It has been reported in many plants that The callus when subcultured on the same medium resulted in 2,4-D is mostly used as auxin for callus induction (Junaid et the formation of many globular somatic embryos. Further al., 2007; Silveira et al., 2013) and addition of a low subculturing resulted in the formation of globular, torpedo and concentration of cytokinins in callus culture medium often heart shaped somatic embryos. Mature somatic embryos were enhances callus regeneration (Singh et al., 2009). transferred to regeneration medium containing BAP and GA3. The mature embryos turned green on MS medium 3.5 Somatic embryogenesis and shoot regeneration supplemented with 1.0mg/l BAP and 1.0mg/l GA3 after a week and then after two weeks the shoots differentiated with Somatic embryogenesis has already been reported in many one or two plumules was observed (Figure 6). endangered plants for their in vitro conservation (Lee et al.,

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Somatic embryogenesis and callus proliferation in Picrorhiza kurroa Royle ex. Benth 207

Figure 6 Shoot regeneration from somatic embryos in MS medium supplemented with 2,4-D, BAP and GA3

Table 5 Effect of plant growth regulators on callus induction and regeneration after 6 weeks.

S. No MS+Growth hormones (mg/l) Type of Response in P. kurroa Rate of callus induction 1 MS+1.0 2,4-D No callus --- 2 MS+2.0 2,4-D Creamy white ,friable, small in size + 3 MS+3.0 2,4-D Creamy white, friable, large in size ++ 4 MS+4.0 2,4-D Browning of the explant --- 5 MS+2.5 2,4-D+ 1.0 BAP Creamy white , friable callus, with shoots +++ 6 MS+2.5 2,4-D+ 1.0 BAP + 1.0 IAA Creamy white , callus, with small shoots and roots +++ 7 MS+2.5 2,4-D+2.0 NAA +0.5 IAA Creamy white callus ,with large roots +++ ---- Absent, + Less callus, ++ moderate growth, +++ Good growth rate

Conclusions Acknowledgements

The in vitro techniques help in conservation and multiplication The authors are thankful to National Medicinal Plant Board of medicinally important plants that are overexploited for their (NMPB), New Delhi for their financial support. The authors bioactive constituents. The present study has developed a are also thankful to School of Biotechnology, University of protocol for mass multiplication and somatic embryogenesis in Jammu, Jammu for providing the basic facilities to carry out P. kurroa which will facilitate its rapid and large scale this research work. propagation. This study can facilitate the in vitro conservation of elite genotypes of this medicinally important endangered References plant. Further the somatic embryogenesis opens a chance for the improvement of the plant through transgenic approach. Barberini S, Danti R, Lambard M (2016) Somatic plant Though further studies needs to be done for the improving the regeneration from selected common cypress (Cupressus germination of somatic embryos and their further sempervirens L.) clones resistant to the bark canker disease. establishment in the green house. Plant Cell, Tissue and Organ Culture 124: 393-403. DOI 10.1007/s11240-015-0902-4 Conflicts of interest Baruah CC, Gupta PP, Nath A, Patnaik LG, Dhawan BN Authors would hereby like to declare that there is no conflict of (1998) Anti-allergic and anti-anaphylactic activity of picroliv-- interests that could possibly arise. a standardised iridoid glycoside fraction of Picrorhiza kurroa. Pharmacological Research 36: 487-92. DOI 10.1006/phrs.1998.0397

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Baul TK, Hossain MM, Mezbahuddin M, Mohiuddin M hypocotyls segments. Scientia Horticultarae 99: 89–98. DOI (2011) Vegetative propagation of Litsea monopetala , a wild 10.1079/IVP2005682 tropical medicinal plant: Effects of indole-3-butyric acid (IBA) on stem cuttings. Journal of Forestry Research 22: 409-416. Ngomuo M, Emerald Mneney E, Ndakidemi PA (2014) The in DOI 10.1007/s11676-011-0111-x. vitro propagation techniques for producing banana using shoot tip cultures. American Journal of Plant Sciences 5: 1614-1622. Bodeker G, Klooster VT, Weisbord E (2014) Prunus africana DOI 10.4236/ajps.2014.511175 (Hook.f.) Kalkman: The Overexploitation of a Medicinal Plant Species and Its Legal Context. Journal of Alternative and Patil AA, Sachin BS, Shinde DB, Wakte PS (2013) Complementary Medicine 20: 810-822. DOI Supercritical CO2 assisted extraction and LC-MS 10.1089/acm.2013.0459 identification of picroside I and picroside II from Picrorhiza kurroa. Phytochemical Analysis 24: 97-104. DOI Giri D, Tamta S (2013) Induction of Somatic Embryogenesis 10.1002/pca.2383 in Vulnerable Medicinal Plant Hedychium spicatum Buch-Ham ex Smith. Plant Tissue Culture and Biotechnology 23: 147- Patial V, Devi K, Sharma M, Bhattacharya A, Ahuja PS (2012) 155. DOI10.3329/ptcb.v23i2.17506 Propagation of Picrorhiza kurroa Royle ex Benth: An important medicinal plant of Western Himalaya. Journal of Junaid A, Mujib A, Bhat MA, Sharma MP, Šamaj J (2007) Medicinal Plants Research 6: 4848-4860. DOI Somatic embryogenesis and plant regeneration in 10.5897/JMPR12.115 Catharanthus roseus. Biologia Plantarum 51: 641-646. DOI 10.1007/s10535-007-0136-3 Pradhan S (2011) Antihyperglycemic effect of various medicinal plants of Sikkim Himalayas - A review. Kim YJ, Lee OR, Kim KT, Yang DC (2012) High Frequency International Journal of Research in Phytochemistry & of Plant Regeneration through Cyclic Secondary Somatic Pharmacology 1: 124-130. Embryogenesis in Panax ginseng. Journal of Ginseng Research 36: 442–448. DOI: 10.5142/jgr.2012.36.4.442 Praveena C, Veeresham C (2014) Multiple shoot regeneration and effect of sugars on growth and nitidine accumulation in Kumar HM, Ramesh C (2014) Anticancer activity of nano shoot cultures of Toddalia asiatica. Pharmacognosy Magazine encapsulated formulation from the extracts of Picrorhiza 10: S480-S486. DOI 10.4103/0973-1296.139777 kurroa against human cancer cell lines. Journal of Pharmacognosy and Phytochemistry 2: 182-185. DOI Rahdari P, Khosroabadi M, Delfani K, Hoseini SM (2014) 10.1016/0006-2952(93)90364-3 Effect of Different Concentration of Plant Hormones (IBA and NAA) on Rooting and Growth Factors in Root and Stem Lee OR, Sathiyaraj G, Kim YJ, In JG, Kwon WS, Kim JH, Cuttings of Cordyline Terminalis. Journal of Medical and Yang DC (2011) Defense genes induced by pathogens and Bioengineering 33: 190-194. DOI 10.12720/jomb.3.3.190-194 abiotic stresses in Panax ginseng C.A. Meyer. Journal of Ginseng Research 35: 1-11. DOI 10.5142/jgr.2011.35.1.001 Rawat B, Rawat JM, Mishra S, Mishra NS (2013) Picrorhiza kurroa: current status and tissue culture based biotechnological Mallick MN, Singh M, Parveen R, Khan W, Ahmad S, interventions. Acta Physiologiae Plantarum 35: 1-12. DOI Najm MZ, Husain SA (2015) HPTLC Analysis of Bioactivity 10.1007/s11738-012-1069-9 Guided Anticancer Enriched Fraction of Hydroalcoholic Extract of Picrorhiza kurroa. BioMed Research International Sen MK, Nasrin S, Rahman S, Jamal AHM (2014) In vitro 2015: 513875. DOI 10.1155/2015/513875 callus induction and plantlet regeneration of Achyranthes aspera L, a high value medicinal plant. Asian Pacific Journal Mugula BB, Vries BJ, Bingi SW (2010) Mechanisms for of Tropical Biomedicine 4: 40-46. DOI 0.1016/S2221- sustainable use of biodiversity in and beyond natural 1691(14)60206-9. ecosystems: a study on conservation and commercial production of Prunus africana in Uganda. International Journal Silveira DG, Lino LSM, Souza ADS, Souza FVD (2013) of Biodiversity and Conservation 2: 180-185. Somatic embryogenesis of Neoglaziovia variegata (Arruda) Mez, an important source of fiber from native Brazilian Murashige T, Skoog F (1962) A revised medium for rapid bromeliads. Brazilian Archives of Biology and Technology 56: growth and bio-assays with tobacco tissue cultures. 547-555. DOI 10.1590/S1516-89132013000400004. Physiologia Plantarum 15: 473-497. DOI 10.1111/j.1399- 3054.1962.tb08052 Singh P, Singh A, Shukla KA, Singh L, Pande V, Nailwal TK (2009) Somatic embryogenesis and in vitro regeneration of an Nagori R, Purohit SD (2004) Plantlet regeneration in Annona endangered medicinal plant sarpgandha (Rauvolfia serpentina squamosa through direct shoot bud differentiation on L.). Life Science Journal 6: 57-62.

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Somatic embryogenesis and callus proliferation in Picrorhiza kurroa Royle ex. Benth 209 Sultan P, Jan A, Pervaiz Q (2016) Phytochemical studies for Anticancer Medicinal Herb. In: International Conference on quantitative estimation of iridoid glycosides in Picrorhiza Biotechnology, Biological and Biosystems Engineering, kurroa Royle. Botanical Studies An International Journal 57:7. December 18-19, Phuket, pp. 246-249. DOI 10.1186/s40529-016-0121-2 Verma P, Mathur AK, Jain SP, Mathur A (2012) In vitro Tiwari SS, Pandey MM, Srivastava S, Rawat AK (2012) TLC Conservation of Twenty-Three Over exploited Medicinal densitometric quantification of picrosides (picroside-I and Plants Belonging to the Indian Sub Continent. The Scientific picroside-II) in Picrorhiza kurroa and its substitute Picrorhiza World Journal 2012: 929650. DOI 10.1100/2012/929650 scrophulariiflora and their antioxidant studies. Biomedical Chromatography 26: 61-68. DOI 10.1002/bmc.1626 Zuraida AR , Mohd Shukri MA , Erny Sabrina MN, Nazreena OA (2015) Improvement of Regeneration of Pelargonium Venkatachalam P, Ezhili N, Thiyagarajan M (2012) In vitro radula via Somatic Embryogenesis. British Biotechnology Shoot Multiplication of Gloriosa Superba L.– An Important Journal 5: 166-173. DOI 10.9734/ BBJ/2015/15337

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EFFECT OF TEMPERATURE AND LOADING DENSITY ON DRYING KINETICS OF WHEAT

Mojaffor Hosain1,*, Raihanul Haque2, Md. Nazrul Islam3, Habiba Khatun4 and Md. Shams-Ud-Din3

1Department of Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200 2Department of Food Engineering and Technology, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200 3Department of Food Technology and Rural Industries, Bangladesh Agricultural University, Mymensingh 4Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200

Received – February 19, 2016; Revision – March 17, 2016; Accepted – April 28, 2016 Available Online – April 28, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).210.217

KEYWORDS ABSTRACT Wheat

Drying kinetics The effect of temperature and loading density on drying kinetics of wheat were studied experimentally in a laboratory-scale cabinet dryer. The study was conducted at drying temperatures of 40, 45, and 50oC 2 Cabinet dryer with loading density of 2.12 and 4.24kg/m at constant air velocity of 0.6 m/sec. The Henderson and Pabis model was applied to fit the drying conditions. Results of study revealed that drying rate constant Diffusion Coefficient gradually decreased with the increasing of loading density of wheat. The effective moisture diffusivity for wheat was increased with the increasing of drying temperature, and it was in the range of 3.27×10-11 2 -11 2 Activation energy cm /sec to 7.95×10 cm /sec while the activation energy of 17.64 kcal/ g-mole was found from the Arrhenius equation.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Mojaffor Hosain) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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211 Mojaffor et al

1 Introduction density (layer) in a cabinet dryer. Apart from this, effective diffusivities and activation energy were calculated in selected Wheat (Triticum aestivum) belongs to the family Graminae, is drying conditions. one of the important cereals to make flour for diet and livestock feed. Wheat can be easily processed to flour and 2 Materials and Methods semolina. Further, wheat is also used as rich source of carbohydrate, fiber, vitamin B complex and minerals 2.1 Sample collection (Sramkova et al., 2009). It is believed that wheat is originated in the Nile Valley by 5000 BC and the Mediterranean region is The Prodip (BAW-1008) wheat was used in this study and the center of domestication (Zohary & Hopf, 2000). In collected from the central farm of Bangladesh Agricultural Bangladesh, wheat cultivation is started from the late 1980s. University during harvesting time. After collecting, wheat was Firstly, it is consumed in rural areas but now, it is second winnowed to get fresh and quality sample material. On the highest consumed cereal crop (Banglapedia, 2006). According same day, prepared sample was subjected to drying process. to BARI report 2010, about 1 million ton of wheat is produced against the annual demand of 3.0-3.5 million tons in 2.2 Drying process Bangladesh. Consequently, 2.0-2.5 million tons of wheat is imported every year in Bangladesh to meet the national Drying process of wheat was accomplished in cabinet dryer demand. On the other hand, postharvest loss of wheat is found (model OV-165, Gallenkamp Company), consists of several almost 3.62% in Bangladesh, among this storage loss is 1.54%. chambers in which samples were placed in trays. Usually, a fan However, improved drying system with proper drying is used for blowing air over a heater and above the samples management is important to minimize the storage loss of wheat placed in trays of cabinet dryer. The air velocity was measured (Bala et al., 2010). by Anemometer.

Drying is one of the oldest but important techniques to Fresh wheat with known loading density (2.12 kg/m2 and 4.24 preserve and improving the storage quality of agricultural kg/m2) was subjected to cabinet drying process at three produces. Drying technique involves the removal of moisture different dry bulb temperatures (40, 45 and 50°C) with content to safe level by means of heat and mass transfer constant air velocity (0.6 m/sec) to know the effect of loading (Ertekin & Yaldiz, 2004), as a result of this, it prevents the density and temperature on drying kinetics of wheat. enzymatic and microbial spoilage of food products. Furthermore, dried products need less space for storage and 2.3 Theoretical consideration drying technique makes agricultural products easier to handle without losing remarkable amount of vitamins (Izadifar & 2.3.1 Mechanical modeling of drying curves Mowla, 2003; Sagar & Kumar, 2010). In Bangladesh, drying technique varies from natural sun drying to mechanical drying Different drying models are used to fit the drying curves to depending on product and affordability in cost. Sun drying is experimental data for determining most efficient drying model. most common drying technique for wheat but it requires An effective drying behavior model is very important to know relatively long drying time and is associated with drying loss the drying characteristics of wheat for a cabinet dryer. In this of wheat. study, the experimental drying data of wheat at different conditions were fitted into Henderson and Pabis model given On the other hand, mechanical drying namely cabinet drying is below (Figiel, 2010): alternative option with several advantages including high thermal conductivity, energy saving, better space utilization MR = a exp (-kt)………. (1) and precise controlling system (Maskan, 2000). Several researchers investigated drying characteristics of fruits and The moisture ratio of fresh wheat was calculated by the vegetables in various drying system. Thin layer drying following equation (Figiel, 2010): characteristics of pomegranate arils with varying temperature o (50, 55 and 60 C) in cabinet drying system are studied by MR = (Mt - Me)/ (Mo – Me) ….(2) Kingsly & Singh (2007). The effects of different drying temperatures and air velocity levels on drying behavior of Where, MR is moisture ratio, M0,Mt and Me are initial carrot have been studied by Erenturk & Erenturk (2007). In moisture content, moisture content at specific time and addition, different drying models are used to design dryers for equilibrium moisture content (kg water/kg dm) respectively. efficient moisture transfer analysis along with to describe the drying behavior of agricultural produces (Midilli et al., 2002; For low equilibrium moisture content and moisture ratio (MR< Da Silva et al., 2009; Kumar et al., 2011). 0.6), equation reduces to

Hence, present study was aimed to investigate the drying MR = Mt/ Mo …. (3) behavior of wheat with varying drying temperature and loading

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Effect of Temperature and Loading Density on Drying Kinetics of Wheat 212 2.3.2 Effective moisture diffusivity The temperature dependence of the effective diffusivity may In drying process, moisture moves to surface by a physical be described by an Arrhenius-type relationship (Madamba et mechanism known as diffusivity, which can be explained by al., 1996; Ozdemir & Devres, 1999; Sanjuan et al., 2003; Fick’s diffusion equation (Abe & Afzal, 1997). On the other Akgun & Doymaz, 2005; Wang et al., 2006) and written as: hand, effective moisture diffusivity depends on chemical composition, temperature, density and porosity of substance (Perry et al., 1984). Several researchers have been stated that effective moisture diffusivity (Deff) of food substance can be calculated by ‘methods of slope’ technique (Karathanos et al., 1990; Zogzas et al., 1996; Sharma et al., 2005). It is assumed that food dehydration is occurred by diffusion process. Therefore,mass transfer during drying process can be described 2 by Fick’s second law of diffusion. The expression is: Where, D0 = frequency factor of Arrhenius equation (m /s), Ea = activation energy (J/mol), T = absolute temperature (K) and R = universal gas constant (J/mol K). The activation energy

… (4) (Ea) could be calculated from the slope of straight line of ln Deff versus reciprocal of T.

Where, M is moisture content (dry basis), De is effective diffusion co-efficient and T is time (hr) 3 Results and Discussion

The above diffusion equation is usually used for a sphere 3.1 Effect of temperature on drying kinetics shaped body such as grain with radius ‘r’ (Brooker et al., 1974; Crank, 1975; Islam, 1980) can be written as: Wheat was dried at 40, 45 and 50°C in thin layer to know the effects of temperature on drying kinetics of wheat, whereas experimental data were analyzed using equation (6). Moisture ratio was plotted against drying time on semi-log graph paper … (5) (Figure 1) and the regression equations were developed as below:

2 -0.226t Where, Deff is the effective diffusivity (m /s) and r is radius. MR= 0.9203e at 40°C ------(11) MR= 0.9193e-0.285t at 45°C------(12) The equation (5) can be simplified by taking the first term of MR= 0.9062e-0.353t at 50°C------(13) series (Tutuncu & Labuza, 1996) and written as From Figure (1) and above developed equations (11 to 13), it is clear that the moisture ratio (MR) decreased continuously with …. (6) the drying progresses. Results of the study also revealed that drying time to a specific moisture ratio decreased with higher drying temperature of wheat. Figure (1) also suggests that drying rate constant gradually increased with the increasing of drying temperature. Higher temperature with low humidity may increase the drying rate at initial stage. But longer drying process would result in case hardening; therefore, it would … (6) reduce the drying rate significantly and deteriorate the product quality due to cooking instead of drying. The slope and effective diffusivity can be calculated from the above equation and written as: Similar findings have been stated by Sarker et al. (2012) for potato; Kumar et al. (2011) for carrot pomace; Khaliduzzaman et al. (2009) for garlic; Kamruzzaman & Islam (2006) for . …(7) aroids. Thus optimum drying temperature is crucial parameter in mechanical drying process with counter current operation (Karel et al., 1975; Potter & Hotchkiss, 1978; Islam, 1980). 2.3.3 Calculation of activation energy … (8)

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Figure 1 Effect of temperature on drying kinetics for wheat

Figure 2 Effect of loading density on drying kinetics of wheat at 40°C

Figure 3 Effect of loading density on drying kinetics of wheat at 45°C.

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Effect of Temperature and Loading Density on Drying Kinetics of Wheat 214

Figure 4: Effect of loading density on drying kinetics of wheat at 50°C.

3.2 Effect of loading density on drying kinetics 3.3 Effect of temperature on diffusion co-efficient of wheat

To measure the effects of loading density on drying kinetics of Diffusion coefficient (De) and inverse absolute temperature -1 wheat, the experimental data were analyzed using equation (6). (Tabs ) were plotted on semi-log paper and regression lines Moisture ratio was plotted against drying time on semi-log were drawn (Figure 5). The Arrhenius equation was found as graph paper to draw regression lines (Figure 2, 3 and 4). below:

-8881.89Tabs-1 The following regression equations were developed for two De = 72.126 e …(20) loading densities and three dry bulb temperatures: 2 Where, De is Diffusion coefficient (cm /s) and Tabs is Absolute MR= 0.9371e-0.159t at 40°C------2.12 kg/m2 ------(14) temperature (°K). MR= 1.0055e-0.105t at 40°C------4.24 kg/m2 ------(15) MR= 0.9386e-0.186t at 45°C------2.12 kg/m2 ------(16) MR= 1.0038e-0.147t at 45°C------4.24 kg/m2 ------(17) It was observed that the effective moisture diffusivity MR= 0.9621e-0.280t at 50°C------2.12 kg/m2 ------(18) increased with the increasing of drying temperature. The MR= 0.9924e-0.192t at 50°C------4.24 kg/m2 ------(19) highest diffusivity (7.95×10-11 cm2/sec) was noted at 50oC, whereas the lowest values (3.27×10-11 cm2/sec) was noted at Figure 2, 3 and 4 show that drying rate of wheat decreased as 40oC. Similar trend in effective moisture diffusivity with loading density of wheat increases where drying rate constant increasing drying temperature was reported by Roberts et al. was not decreased proportionally. Fig. 2 shows that rate (2008). constant 0.159 hr -1 was reported for loading density 2.12 2 -1 kg/m , whereas rate constant 0.105 hr was resulted at 40°C In this reaesrch, calculated activation energy (Ea) for wheat for 4.24 kg/m2 loading density. was 17.64 kcal/ g-mole. The activation energy for wheat was higher than those reported by Kamruzzaman & Islam (2006) On the other hand, rate constant 0.186 hr -1 and 0.147 hr -1 were for aroids (5.12 kcal/ g-mole); Islam et al. (1997) for mango observed at 45°C for loading density 2.12 kg/m2 and 4.24 (4.4 Kcal/g-mole); Alam et al. (2014b) for blanched slices of kg/m2 respectively (Fig. 3). Again, at 50°C, loading density of onion (2.46 Kcal/g-mole); Alam et al. (2014a) for ginger 2.12 kg/m2 resulted in rate constant (0.315 hr -1), whereas rate (15.868 Kcal/g-mole); Sarker et al. (2012) for potato (5.06 constant (0.158 hr-1) was revealed for loading density of 4.24 Kcal/g-mole); Khaliduzzaman et al. (2009) for garlic (7.48 kg/m2 at same condition (Fig. 4). These results were supported Kcal/g-mole) and lower than those noted by Ginger & by the findings of Kamruzzaman & Islam (2006) who noted Mascheroni (2001) for whole wheat (27.00 Kcal/g-mole); that drying rate constant decreased with the increased loading Afzal Babu et al. (1997) for onion (26.83 Kcal/g-mole) and density of aroids. Similar behavior regarding the influence of Iqbal (2003) for cucumber (8.50 Kcal/g-mole) and for loading density on drying rate constant was observed by cauliflower (7.76 Kcal/g-mole). Kamrul (2006) for raw paddy.

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215 Mojaffor et al

Figure 5 Effect of temperature on diffusion coefficient

Conclusion Alam MM, Islam MN, Islam MN (2014b) Study on drying The Henderson and Pabis model was used to fit different kinetics of summer onion. Bangladesh Journal of Agricultural drying conditions for wheat in cabinet drying system. The Engineering 39 : 661-673. model revealed that drying of wheat could be accomplished commercially by cabinet dryer. It was observed that drying Bala BK, Haque MA, Hossain MA, Majumdar S (2010) Post time to a specific moisture ratio decreased with higher drying Harvest Loss and TechnicalEfficiency of Rice, Wheat and temperature. On the other hand, loading density of wheat Maize Production System: Assessment and Measures for showed negative effect on drying rate constant.The effective Strengthening Food Security. Bangladesh Agricultural moisture diffusivity varied from 3.27×10-11 cm2/sec to University. 7.95×10-11 cm2/sec with a temperature range of 40 to 50oC and was found to be increased with the increasing of Banglapedia (2006) BANGLAPEDIA—national encyclopedia temperature. Furthermore, activation energy (Ea) was found of Bangladesh. Available on 17.64 kcal/ g-mole. Drying temperature of 50oC with loading http://www.banglapedia.org/httpdocs/HT/W_0053.htm access density of 2.12 kg/m2 could be suggested for drying of wheat on 20 February, 2016. incabinet drying process. Brooker DB, Bakker FW, Hall CW (1974) Drying. The AVI Conflict of interest Pub. C. Ine. U.S.A.:185.

Authors would hereby like to declare that there is no conflict of Crank J (1975) The mathematics of diffusion. Clarendom interests that could possibly arise. Press, Oxford.

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Akgun NA, Doymaz I (2005) Modeling of olive cake thin- Ertekin C, Yaldiz O (2004) Drying of eggplant and selection of layer drying process. Journal of Food Engineering 68:455–461. a suitable thin layer drying model. Journal of Food DOI:10.1016/j.jfoodeng.2004.06.023 Engineering 63: 349–359. DOI:10.1016/j.jfoodeng.2003.08.007 Alam MM, Hossain MZ, Aktar M, Islam MS, Hasan Z (2014a) Drying and Dehydration Kinetics of Ginger. International Journal of Innovation and Applied Studies 9: 956-967.

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Ginger SA, Mascheroni RH (2001) Difusive Drying Kinetics Madamba PS, Driscoll RH, Buckle KA (1996) The thin layer in Wheat, Part 2: dryingcharacteristics of garlic slices. Journal of Food Engineering 29:75–97. DOI:10.1016/0260-8774(95)00062-3 applying the Simplified Analytical Solution to Experimental Data. Biosystems Engineering 81: 85-97. Maskan M (2000) Microwave/air and microwave finish drying DOI:10.1006/bioe.2001.0004 of banana. Journal of Food Engineering 44: 71–78. DOI:10.1016/S0260-8774(99)00167-3 Iqbal A (2003) Processing and preservation of cauliflower and cucumber by dehydrationand fermentation. M.S. thesis Midilli A, Kucuk H, Yapar Z (2002) A new model for single submitted to Department of Food Technology and Rural layer drying. Drying Technology 20 : 1503–1513. Industries, Bangladesh Agricultural University, Mymensingh, Bangladesh. Ozdemir M, Devres YO (1999) The thin layer drying characteristics of hazelnuts during roasting. Journal of Food Islam MN (1980) Use of solar energy for development of Engineering 42:225–233. DOI : 10.1016/S0260- shelf-stable potato product. Ph.D. Thesis submitted to Royal 8774(99)00126-0. Veterinary and Agriculture University, Copenhagen, Denmark. Perry RH, Green DW, Maloney JO (1984) Perry’s Chemical Islam MN, Uddin MB, Islam NM (1997) Development of self- Engineers’ Handbook. (6th Edn), McGraw-Hill, New York 13- stabledehydration mango products. Bangladesh Journal of 20. Agricultural Engineering 4: 65-73. Potter NN, Hotchkiss JH (1978) Food Science, CBS Publishers Izadifar M, Mowla D (2003) Simulation of Cross-Flow and Distributors, Shahdara, Delhi 78. Continuous Fluidized Bed Dryer for Paddy Rice. Journal of Food Engineering 58: 325-329. DOI:10.1016/S0260- Roberts JS, Kidd DR, Zakour OP (2008) Drying kinetics of 8774(02)00395-3 grape seeds. Journal of Food Engineering 89: 460-465. DOI:10.1016/j.jfoodeng.2008.05.030 Kamrul N (2006) Effect of Conditions on Milling Quality and Germination of Paddy. M.S. Thesis submitted to Department Sagar VR, Kumar SP (2010) Recent advances in drying and of Food Technology and Rural Industries, Bangladesh dehydration of fruits and vegetables: A review. Journal of Agricultural University, Mymensingh. Food Science and Technology 47: 15-26. DOI: 10.1007/s13197-010-0010-8 Kamruzzaman M, Islam MN (2006) Kinetics of dehydration of aroids and developed dehydrated aroids products. Journal of Sanjuan N, Lozano M, Garcia-Pascal P, Mulet A (2003) Chemical Engineering, IEB 24: 19-24. DOI: Dehydration kinetics of red pepper (Capsicum annuum L var http://dx.doi.org/10.3329/jce.v24i0.5579 Jaranda). Journal of the Science of Food and Agriculture 83:697–701. DOI: 10.1002/jsfa.1334 Karathanos VT, Villalobos G, Saravacos GD (1990) Comparison of two methods of effective moisture diffusivity Sarker A, Islam MN, Shaheb MR (2012) A study on the drying from drying data. Journal of Food Science 55: 218-231. DOI: behaviour of a local variety (lalpakri) of potato (Solanum 10.1111/j.1365-2621.1990.tb06056.x tuberosum L.). Bangladesh Journal of Agricultural Research 37: 505-514. Karel M, Fennema OR and Lund DB(1975) Principle of Food Science, Part II, Physical principles of Food Preservation. Sharma GP, Verma RC, Pankaj P (2005) Mathematical Marcel Deker, Inc. New York and Basel. modeling of infrared radiation thin layer drying of onion slices. Journal of Food Engineering 71: 282-286. Khaliduzzaman, Al-Emran M, Shams-Ud-Din M (2009) DOI:10.1016/j.jfoodeng.2005.02.010 Studies on the preparation of shelf-stable garlic powder. Bangladesh Research Publications Journal 2: 578-584. Sramkova Z, Gregova E, Sturdik E (2009) Chemical composition and nutritional quality of wheat grain. Acta Kingsly ARP, Singh DB (2007) Drying kinetics of Chimica Slovaca 2 : 115 – 138. pomegranate arils. Journal of Food Engineering 79:741-744. DOI:10.1016/j.jfoodeng.2006.02.033

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

PHYTOCHEMICAL ANALYSIS, ANTIMICROBIAL SCREENING AND HYPOGLYCEMIC EFFECT OF SOME SELECTED MEDICINAL PLANT EXTRACT FROM OMAN

S Hemadri Reddy*, Aisha Khalid AL-Hinai, Hajer Hafed AL-Yaqoobi and Fatima Jafar AL-Ajmi

Department of Applied Science, Higher College of Technology, Al Khuwair, Po box: 74, postal code: 117, Muscat, Sultanate of Oman

Received – December 30, 2015; Revision – January 15, 2016; Accepted – May 03, 2016 Available Online – May 03, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).218.224

KEYWORDS ABSTRACT

Medicinal plants In present study phytochemical screening and antimicrobial potential of three Omani traditional medicinal plants i.e. Ajuga iva, Pteropyrum scoparium and Rhazya stricta was carried out. The Phytochemical screening information generated from study will be helpful in drug administration and developing commercial drug form these studied traditional plants. All three selected plants were air dried under natural Plant extraction condition and phytochemical screenings were carried out under laboratory conditions. Antimicrobial Antimicrobial study properties of the selected plants were carried out against the selected microorganisms i.e. Bacteria in-vivo assay in mice (E.coli, Staphylococcus and Bacillus species) and fungi (Trichoderma and Aspergillus). Further, selected plants were also used to test their effect on reducing diabetic incidence. Results of study Blood sugar level revealed that selected plants have significant inhibitory effect on the multiplication of selected microorganisms. Similarly oral administration of plant extract also reduced the blood sugar level of the Body mass treated mice; it started reducing after 1 week of treatment and continued till 21 days of exposure as compared to control. Further, phytochemical screening of these plants indicated that these plants contain many of chemical substances which had important role in the reducing blood sugar level.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (S Hemadri Reddy) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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219 Reddy et al

1 Introduction filter paper and residues were also extracted twice by using 400ml of ethanol. The final extract is subjected for Rota Plants are important source of traditional medicine and are evaporation at 90°C under constant pressure with respect to useful in the treatment of various diseases with fewer side time duration. Different concentrations ranging from 0.1, 0.5% effects (Bako et al., 2005). Traditional medical practices are and 1.0% were made from extracted crude extracts with not only useful in disease healing but also have significant DMSO and distilled water. The concentrated crude extracts contribution in the discovery of pharmaceutically active obtained through Rota evaporator were weighed and dissolved substances from plants (Principe, 1991; Pearce & in DMSO initially and then final volume was made up to the Puroshothaman, 1992) which can contribute in the commercial required concentrations (1g / 100 ml, w/v). production of drugs. The role of plant biodiversity in health care has been well documented from ancient time (Posey, 2.2 Phytochemical screening 1999). Literature survey suggested that worldwide more than 50,000 plant species are successfully used for medicinal Phytochemical screening was carried out according to Reddy purposes and among these almost 13% are flowering plants et al. (2012). Different crude extracts collected from Rota (Schippmann et al., 2002). Various active ingredients like evaporator were qualitatively tested for the presence of various alkaloid, glycoside, saponin, essential oil, tannins and phytochemical constituents by using standard methods given mucilages have been isolated from various plant parts such as by Brain & Turner (1975) and the extractive values were root, stem, leaves, bark, fruit and seeds; these phytochemicals calculated by suggested protocols. determine a definite curing physiological response in the treatment of various humans and other animal ailments 2.3 Screening of Antimicrobial Activity (Adhikari et al., 2010). Because of the accelerated local, national and international interest in recent years, the demand In-vitro antimicrobial screening were carried out under for medicinal and aromatic plants has increased manifold and laboratory conditions, for this various microorganism were in pharmaceutical industry views plant wealth are a source of collected from microbiology laboratory, HCT, Muscat with income. Due to easy availability, less side-effects and stain numbers of E.coli (ATCC 25922), S aureus (ATCC sometimes only source of health care, the demand for 25932), B Subtilis (ATCC 6633), T. harzianum (ATCC 20847) medicinal plants is increasing in both developing and and A. brasiliensis (ATCC 16404). All the suggested developed countries. microorganisms were cultured on recommended cultural medium and finally transfer & maintained on agar broth for Ajuga iva is a small (5-10 cm) wooly, aromatic perennial herb O/N. with well documented anti-ulcerous, hypoglycemic and anti- inflammatory activities. The anti-ulcerous activity of Ajuga iva Antimicrobial activity of various plants extracts viz. A. iva, P. was confirmed experimentally. Similarly, medicinal properties scoparium and R. stricta have been carried out by using disc of P. scoparium and R. stricta were also reported by various diffusion method (Kavanagh, 1972). The inhibitory effect of researchers (Emad El-deen, 2005). The Hypoglycemic effect of each extracts was compared with the standard antibiotics the lyophilized aqueous extract of these plants was also shown penicillin and mycostatin against bacteria and fungi in normal and streptozotocin diabetic rats. Due to various respectively. medicinal values, present study was aimed to explore the antimicrobial and anti hypoglycemic properties of these 2.4 Effect of various extracts on blood sugar and body weight selected Omani plants. Furthermore, phytochemical analysis was also carried out for justifying the possible reason of Three concentrations viz 0.1%, 0.5%, and 1% of individual antimicrobial and anti hypoglycemic efficacy. extracts were prepared by dissolving one gram of each crude extract in 5 ml DMSO (Dimethylsulfoxide) to obtain 1% stock 2 Materials and methods solution from which experimental concentrations namely 0.1%, 0.5% and 1.0% were prepared in sterile distilled water at 2.1 Plant materials and extraction pH 9 (Ali et al., 2000). Thirty locally bred white male mice with initial weighing of 15 - 30 g were obtained from Animal The plant materials Ajuga iva, P. scoparium and R. stricta house, Higher College of Technology (HCT), Al-Khuwair, were collected from Rustaq during February 2014. Different Sultanate of Oman for testing effect of various extracts on plant parts like leaves, stem and root were air dried under blood glucose level. All the mice were divided into 10 groups; natural conditions for two days and dried plant materials were each group had 3 mice and these were acclimatized 4 days powdered by using home blender and 40g of plant extract were prior to experimentation at a temperature of 25˚C. Before extracted with 200ml of ethanol for 6 hours at room applying various treatments blood glucose level was measured temperature at orbital shaker through maceration method of with the help of Glucometer. extraction. The extracts were filtered through whatman No.1

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Phytochemical analysis, antimicrobial screening and Hypoglycemic effect of some selected medicinal plant extract from Oman 220

Table 1 Phytochemical screening of different plant extract.

Test A.iva P. scoparium R.stricta Alkaloids ( wagner`s test ) +++ +++ +++ Carboxylic acid - - - Coumarins +++ +++ +++ Fixed oil Test 1 +++ +++ - Test 2 +++ +++ - Flavonoids Ferric chloride test +++ +++ +++ Shimoda`s test +++ +++ +++ Zinc-HCl +++ +++ +++ Phenols reduction test +++ +++ +++ Quinones - - - Resins +++ +++ ++ Saponins - +++ +++ Steroids (Salkowski test ) +++ +++ +++ Tanmins (Ferric chloride test) +++ +++ +++ Glycosides (Borntragers test) +++ +++ + Here +++ represents the highest amount; ++ represents the moderate level presence; + represents the availability of less amount; - represents the absence of particular ingredients; all the values presented in the above table are the mean of three replicates.

They were fed pellets regularly with the same amount to each terpenoids, alkaloids, phenolics, flavonoids, saponins and cage, given tap water. Group 1 (control), was fed by simple tap cardiac glycosides are also responsible for exhibiting water and labelled as control while the Group 2, 3, 4 were hypoglycaemic potential. Medicinal plant products yield good treated with 0.1%, 0.5% and 1% of A. iva extract, Group 5, 6, results in the management of various forms of hyperglycaemia 7, were treated with 0.1%, 0.5% and 1% of P. scoparium as compared to synthetic drugs that usually contain single extract and Group 8, 9, 10 were treated with 0.1%, 0.5% and active principle, that on continuous administration could 1% of R. stricta extract respectively by oral gavage. 1ml of predispose various forms of idiosyncrasies in diabetic patients each extract was given regularly by syringe for duration of 21 (Ahmad et al., 1983; Anila et al., 2002; Jung et al., 2006; days. After 21 days, all the mice were weighed and blood Sanni, 2007). glucose level was measured by Glucometer (one touch select – AW06505403A). 3.2 Effect of various extracts on body weight and blood sugar levels 3 Results and Discussion 3.2.1. Body weight 3.1 Phytochemical screening of selected plant materials Result of study suggested that diversity of weight loss or gain Results of the phytochemical screening of A. iva and P. is depending on the concentration and type of extract. Among scoparium revealed the presence of various phyto-chemicals the various tested extracts, A. iva reported least effective in like alkaloids, coumarins, oils, flavonoids, phenols, resins, body weight gain while P. scoparium and R. stricta are at par steroids, tannins and glycosides. Furthermore, present study to each other and showed superiority over A. iva. Various also suggested that presence of carboxylic acids and quinines concentration of P. scoparium and R. stricta are significantly are limited and not reported from all the tested plant extracts. differ than the A. iva but these two are not significantly differ Table 1 revealed the presence of saponins in P. scoparium than the control. Mice treated with three different extract while it was not reported in the A. iva extract. Also the concentration of A. iva viz 0.1%,0.5% and 1.0% shows 0.92g presence of alkaloids, coumarins, flavonoids, phenols, resins, ,0.25 g and 0.1g weight gain respectively while this weight saponins, steroids, and tannins was reported from the R. stricta gain was 4.05g in control (Table 2), here it was also reported while the presence of quinones, oils, carboxylic and glycosides that increasing concentration of extract have negative impact was not reported from the same. According to Wadood et al. on the weight gain and least weight gain was reported in the (2013) medicinal plants possessing various bioactive mice treated with heighted concentration (1.0%) of A.iva. secondary metabolites which are responsible for various antimicrobial properties like antifungal, antibacterial and anti- Further, it was reported that mice treated with selected inflammation. Therefore the results obtained from present concentration (0.1%, 0.5% and 1.0%) of P. scoparium shows study indicated the presence of various bioactive substances 1.25g, 1.3g and 1.15g respectively, no significant difference which are responsible for various antimicrobial properties of was reported between various concentration and all the these extracts. Occurrence of phytochemicals such as treatments are almost at par to each other. Positive impact of

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221 Reddy et al

R. stricta treatment was observed on the treated mice and with formulations made using the identified effective extract and the increasing extract concentration rate of weight gain was fraction of this plant could serve the purpose better than the also increased from 0.48g to 1.85g respectively. The existing formulations with crude aqueous extract since the suppression of weight gain is a dose dependent and it may be experimental plants used in this study are used all over the because of the presence of an orexigenic compounds in the Sultanate of Oman as blood sugar control plant material extracts (Reddy et al., 2015). traditionally.

3.2.2. Hypoglycemic study 3.3. Antimicrobial activity

Blood sugar level of the experimental mice was analyzed after Antimicrobial activity of ethanolic extracts of selected plant 21 days of treatment application and reported that tested plant extracts was reported by disc diffusion method against the extract have hypoglycemic effect. Among various tested selected microorganisms while recommended antibiotic and extracts, R. stricta extract was found most suitable in reducing fungicide disc was used as control treatment. Results of the blood sugar level and all the tested concentration of this extract study revealed that all three plant extracts have antimicrobial effectively reduced the blood glucose level. This extract was properties against the selected bacteria and fungi. Among the followed by the A.iva extract and it also showed hypoglycemic three plant extracts, R. stricta extract showed highest zone of effect. P. scoparium reported as least effective treatment and inhibition against all tested microorganisms and it shows did not show any effect on reducing blood glucose level. superiority over the other two extracts of A. iva and P. Further, it was noticed that all the concentration of A.iva scoparium (Table 3, Figure 1). The antimicrobial property of extract (0.1, 0.5 & 1.0 %) did not show any significant glycosides ensures its use as an anti-infective agent (Rucker et differences and all were showing at par result (0.01, 0.02 & al., 1992; Murakami et al., 1993). Coumarins which was 0.05 mmol). Whereas mice treated by all the concentrations of reported in all the plant extracts, are well known for its R. stricta showed reduction in blood sugar level and the effect antitumor, antibacterial and anthelmintic properties (Chhabra was more profound with the increasing the doses of extract and et al., 1984). Highest zone of inhibition (21.6 mm) was it was reported at 0.1% (0.12 mmol), 0.5% (0.27mmol) and at reported in the well exposed by 1% R. stricta and it was also 1% (0.37mmol). While the mice treated with the different against the Staphylococcus. Further, it was reported that zone concentration of P. scoparium extract did not reduced blood of inhibition is directly proportional to the concentration and glucose level. Results of the study revealed that the extract of highest zone of inhibition was reported from the disc R. stricta and A. iva are more pronounced in reducing blood containing highest concentration of extract. The higher activity glucose level than P. scoparium. The hypoglycemic effect of ethanolic plant extracts against E. coli, S.aureus and B. produced by R. stricta extracts may be due to its positive effect subtilis indicates the presence of antimicrobial agents in the on the insulin releasing mechanism; this is showing resembling tested plants which are useful in curing various skin infections, with the mechanism of sulfonylureas (Miura et al., 2001; as these organisms often cause various skin infections in Okine et al., 2005). Findings of this study suggested that the R. human beings (Reddy et al., 2012). While antifungal assay also stricta extract have remarkable effect on the decreasing serum suggested the antifungal properties of these plant extracts and glucose level. This research supports the inclusion of this it caused reasonable zone of inhibition against the tested fungal plants in formulation of traditional antidiabetic, and the cultures (Table 3, Figure 1).

Table 2 Effect of ethanolic plant extracts on body weight and blood sugar.

Name of plant Concentration of Body Weight in Grams Blood sugar level m mol material plant extracts Before After 21 days Mean Before After 21 days Mean difference difference Control 22.1±2.00 26.15±1.38 4.05↑ 6.32±2.95 7.7±0.51 1.38↑ 0.1% 23.45±2.75 24.37±1.68 0.92↑ 5.85±0.91 5.84±1.52 0.01↓ A. iva 0.5% 25.77±1.33 26.02±1.66 0.25↑ 6.07±0.17 6.05±0.21 0.02↓ 1.0% 19.4±0.85 19.5±1.55 0.1↑ 4.85±0.26 4.80±0.35 0.05↓ 0.1% 25.82±0.86 24.57±2.12 1.25↓ 4.7±0.63 4.82±0.52 0.12↑ P. scoparium 0.5% 26.2±1.04 24.9±1.27 1.3↓ 5.85±0.49 6.12±0.33 0.27↑ 1.0% 24.35±1.04 23.2±1.30 1.15↓ 3.95±0.81 4.32±0.19 0.37↑ R. stricta 0.1% 22.3±1.95 22.82±1.82 0.48↓ 5.3±0.61 4.8±0.13 0.50↓ 0.5% 24.65±1.93 25.55±2.33 0.9↓ 6.3±0.63 4.9±0.0535 1.40↓ 1.0% 25.02±3.19 23.17±3.48 1.85↓ 7.6±1.57 5.25±0.09 2.35↓ Here all the values expressed are the SEM ± of 3 replicates calculated with descriptive statistics maintained for 21 days with plant extracts treatment.

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Phytochemical analysis, antimicrobial screening and Hypoglycemic effect of some selected medicinal plant extract from Oman 222

A.iva

P. scoparium

R. stricta

Figure 1 Antibacterial activity of three different plant extracts against E.coli, Staphylococcus and Bacillus (C: control; 1:0.1%, 2:0.5%,3: 1.0% )

Among various texted plant extracts, highest zone of inhibition as alkaloids, coumarins flavonoids, phenols, quinones etc., was noticed with R. stricta extract against T. harzzianum (10.6 possess significant bioactive properties. The study of mm) while the least antifungal activity was noticed in the A. antimicrobial efficacy against both bacteria and fungi revealed iva extracts which was against the A. brasiliensis (5mm). that these three plants have ability to kill or inhibit the growth of bacteria and fungi which experimentally support the use of Conclusion these plants in traditional medicine by traditional healers. The Hypoglycemic role of all the three plant extracts revealed a Results of this study experimentally established the medicinal great avenue for exploiting the herbal medicines to control one as well antimicrobial properties of the selected medicinal of the world’s most popular health concerns. The further plants. A significant effect of these plants was reported on the researches on the same plants are highly recommended to trace reducing blood sugar and in maintaining body weight, since bioactive principles with specific functional groups involved to these plant materials are used by Omanis around the Sultanate trace their medicinal importance. Therefore the present of Oman as traditional medicine against diabetes, reducing investigation helps to trace herbal medicine useful to treat weight and to control many more microbial diseases. The diabetes and also these traditional plants can be used as preliminary phytochemical screening indicates that ethanolic antimicrobials. plant extracts consist different types of phytoconstituents such

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223 Reddy et al

Table 3 Antimicrobial activity of three extracts against selected microorganisms.

Treatment Concentration of plant Zone of Inhibition (mm) extracts Bacteria Fungi E.coli S aureus B Subtilis T. harzianum A. brasiliensis A.iva Standard (Penicillin & 7.3 27 4.3 9 20.3 Mycostatin) 0.1% 10.6 11.3 12.3 2 1.6 0.5% 11.6 15.3 14.6 7.3 4 1.0% 13.3 18.3 18.3 9.3 5 P.scoparium Standard (Penicillin & 5.3 6.3 12.3 8.3 21.6 Mycostatin) 0.1% 12 11.3 14.3 4 4.6 0.5% 17 19.3 17.6 5.6 7.3 1.0% 18.6 21 18.6 7.3 7.3 R.stricta Standard (Penicillin & 5.3 3 8 7.6 24.6 Mycostatin) 0.1% 11.6 15.3 12 7.3 2.6 0.5% 12 18.6 14.3 10 9.6 1.0% 13.3 21.6 19.6 10.6 10.3 All the results are mean of three replicates

Acknowledgement Anila L, Vijayalakshmi NR, Tian C (2002) Beneficial effect of flavonoids from Sesamum indicum, Emblica officinalis and The authors are highly thankful to Higher College of Momordica charantia. Phytotherapy Research 14:592–595. Technology, Ministry of manpower, Alkhuwair, Muscat, Sultanate of Oman for providing necessary requirements. Bako SP, Bakfur MJ, John I, Bala EI (2005) Ethno-medicinal and phytochemical profile of some savanna plant species in Conflict of interest Nigeria. International journal of Botany 1: 147-150.

Authors would hereby like to declare that there is no conflict of Brain KR, Turner TD (1975) The practical evaluation of interests that could possibly arise. phytopharmaceuticals. 2nd Edn. Bristol: Wright Science technica Pp. 81-82. References Chhabra SC, Uiso FC, Mshiu EN (1984) Phytochemical screening of Tanzanian medicinal plants. Journal of Adhikari BS, Babu MM, Saklani PL, Rawat GS (2010) Ethnopharmacology 11: 157-179. doi:10.1016/0378- Medicinal Plants Diversity and their Conservation Status in 8741(84)90037-0. Wildlife Institute of Indi (WII) Campus, Dehradun. Ethno- botanical Leaflets 14: 46-56. Available at: Emad El-deen HM (2005) Population ecology of Rhazya http://opensiuc.lib.siu.edu/ebl/vol2010/iss1/6 access on 17th stricta Decne. in Western Saudi Arabia. International Journal January, 2016. of Agriculture and Biology 7: 932-938.

Ahmad Y, Fatima K, Le Quesne PPW, Atta-ur-Rahman (1983) Jung M, Park M, Lee HC, Kang YH, Kang ES, Kim SK (2006) Further alkaloidal constituents of the leaves of Rhazya stricta. Antidiabetic Agents from Medicinal Plants. Current Medicinal Phytochemistry 22: 1017– Chemistry 13:1203-1218. DOI: 1019.http://dx.doi.org/10.1590/S1415-47572013005000009 10.2174/092986706776360860.

Ali BH, Tanira MO, Bashir AK, Al-Qarawi AA (2000) Effect Kavanagh F (1972) Analytical microbiology Part II, New of Rhazya stricta Dence on Monoamine Oxidase and York, Academic Press, Pp .126. Cholinesterase Activity and Brain Biogenic Amine Levels in Rats. Journal of Pharmacy and Pharmacology 52: 1297-1300. Miura T, Itoh C, Iwamoto N, Kato M, Kawai M, Park SR, http://dx.doi.org/10.1211/0022357001777289 Suzuki I (2001) Hypoglycemic activity of the fruit of the Momordica charantia in Type 2 diabetic mice. Journal of Nutritional Science and Vitaminology (Tokyo) 47: 340–344.

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Phytochemical analysis, antimicrobial screening and Hypoglycemic effect of some selected medicinal plant extract from Oman 224 Murakami A, Ohigashi H, Tanaka S, Hirota M, Irie R, Takeda Leaf and Root Extracts of Asclepias curassavica (L). IOSR N, Tatematsu A, Koshimizu K (1993) Bitter cyanoglucosides Journal of Pharmacy and Biological Sciences 2:39-44. from Lophira alata. Phytochemistry 32: 1461 – 1466. DOI:10.1016/0031 -9422(93)85160-S. Reddy S, Al-NeeriI , Al-Issaei H, Al-Jabri S (2015) Effect of Selective Medicinal Plant Extract on Blood Glucose, Sperm Okine LKN, Nyarko AK, Osei-Kwabena N, Oppong IV, Shape and Various Physiological Parameters. American Barnes F, Ofosuhene M (2005) The antidiabetic activity of the Journal of Plant Sciences 6: 1109-1115. DOI: herbal preparation ADD-199 in mice: A comparative study 10.4236/ajps.2015.68115. with two oral hypoglycaemic drugs. Journal of Ethnopharmacology 97 : 31–38. Rücker G, Kehrbaum S, Sakulas H, Lawong B, Goeltenboth F DOI:10.1016/j.jep.2004.09.048. (1992) Acetylenic glucosides from Microglossa pyrifolia. Planta Medica 58: 266-269. DOI: 10.1055/s-2006-961450. Pearce DW, Puroshothaman S (1992) Protecting Biological Diversity: The Economic Value of Pharmaceutical Plants. Sanni SS (2007) Pharmacological and Toxicological effects of CSERGE Global Environmental Change Working Paper 92- Ocimum basilicum LINN. extracts in rats. PhD Thesis, 27, Centre for Social and Economic Research on the Global submitted to the Department Veterinary Physiology and Environment, University College. Pharmacology, Usman Danfodiyo, University Sokoto, Nigeria.

Posey DA (1999) Cultural and Spiritual Values of Biodiversity Schippmann U, Leaman DJ, Cunningham AB (2002) Impact of A Complementary Contribution to the Global Biodiversity cultivation and gathering of medicinal plants on biodiversity: Assessment, UNEP. Global trends and issues. In: Biodiversity and the Ecosystem Approach in Agriculture, Forestry and Fisheries. Food and Principe PP (1991) Valuing the Biodiversity of Medicinal Agriculture Organization (FAO), Rome. Plants. In: Akerele O, Heywood V, Synge H (Eds.) The Conservation of Medicinal Plants. Proceedings of an Wadood A, Ghufran M, Jamal SB, Naeem M, Khan A, Ghaffar International Consultation held on 21-27th March, 1988 at R, Asnad (2013) Phytochemical Analysis of Medicinal Plants Chiang Mai, Thailand, Cambridge University Press, Pp. 79- Occurring in Local Area of Mardan. Biochemistry and 124. Analytical Biochemistry 2:144. doi: 10.4172/2161- 1009.1000144. Reddy HS., Chakravarthi M, Chandrashekara KN, Naidu CV (2012) Phytochemical Screening and Antibacterial Studies on

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EFFECT OF NITROGEN LEVEL, GREEN AND ANIMAL MANURE ON THE GROWTH ATTRIBUTE OF CORN CROP (Zea mays L.)

Khashayar Zakikhani, Ali Kashani* and Farzad Paknejad

Department of Agronomy, Karaj Branch, Islamic Azad University, Karaj, Iran

Received – April 01, 2016; Revision – April 22, 2016; Accepted – May 13, 2016 Available Online – May 13, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).225.231

KEYWORDS ABSTRACT Nitrogen This study was to conducted to evaluate the effects of individual or combined application chemical Fertilizer nitrogen fertilizer , green and animal manure on the various growth attributes such as number of seeds, weight of 1000 grains, grain yield, biomass and total grain protein in Single Cross hybrid (S.C 704) in Protein maize. This experiment was conducted in 2014 at the Agricultural Research Station of Islamic Azad University of Karaj, Iran. The study was conducted in split plot factorial in a randomized complete Green Manure block design with four replications. Results of the analysis of variance showed that the difference among grain number per cob among various treatments of cover crop levels at 1% and the effect of different Cover crops amounts of animal manure at the 5% level were significant, but there was no significant interaction among characters. But for other traits, the results of variance analysis table showed that the interaction of green manure and animal manure on 1000 grain weight at 5% and the nitrogen fertilizer on grain yield and biomass at 5%, interaction of different levels of nitrogen fertilizer and green manure at 5% and the interaction of different amounts of nitrogen, green manure and animal manure on grain proteins at the level of 1% were significant.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Ali Kashani) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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226 Zakikhani et al

1 Introduction Like green manures, animals manures is also an important Maize is one of the strategic crops because of its importance at sources of nitrogen and provide nutrition to the growing crops human and animal nutrition and also broad compatibility with (Kaveh Firooz et al., 2013; Baghdadi et al., 2016). Animal the temperate and tropical climate zones (Yazdani et al., 2009). manure also reduced environmental risk and excess use of Area under maize cultivation has been widely increased during chemical fertilizers. Although, individually use of these past some decades; higher production of maize needs higher manure work as an soil supplement but its combination with dose of nitrogen fertilizers. Excessive use of chemical green manure and chemical fertilizers shows synergetic effect substances, has led to increased production costs and and not only increase crop production but also reduced the environmental risks (Biari et al., 2008). Further, individual dose of chemical fertilizers. Further, these combinations can application of nitrogen has limited its efficiency because of its increase the production efficiency in addition to cost savings. loss through denitrification, leaching and sublimation of Therefore, present study was under taken to explore the ammonium. This loss not only reduced the efficiency of potential of the use of nitrogen fertilizers, green manure and applied nitrogen but also responsible for the causes of animal manure, synergistically and to decide the right environmental pollution. Increasing concerns about the combination which leads to the best efficiency in improving harmful effect of the excess use of chemicals fertilizers such as the quantitative and qualitative yield of maize crops. nitrogen fertilizers pushed agricultural scientist and farmers to explore the alternate sources of chemical fertilizers. Organic 2 Materials and Methods sources of nitrogen can be serve a better option than the chemical fertilizers and can also play an important role in This experiment was conducted in 2014 at the Agricultural maintaining soil organic compounds and improve plant health Research Station of Islamic Azad University of Karaj, Iran. (Ajvanzadeh, 2005; Kamkar & Mahdavi damghani, 2006; The study was conducted in split plot factorial in a randomized Mahdavi Damqani et al., 2008; Ochiai et al., 2008;). complete block design with four replications. The main treatments consisted of nitrogen (at three levels of 120, 240 Now in these days, role of cover crops in improving soil and 360 kg of urea) and green manure (Perko). Subsidiary characteristics, quality and preventing soil erosion are also well treatment involved the animal manure (25 tons per hectare). reported; these cover crops also play a significant role in the Nitrogen and green manure were placed in main plots and determination of weeds, pests and diseases. These plants not animal manure was used in subplots. Dimensions of subplots only enhance the soil organic matter but also reduced the were considered as 6/21 square meters (3.6× 6 m) with 5 lines dependency and consumption of chemical fertilizers and and the distance between the main plots were 3 meters as well preventing environmental pollution (Hashemi et al., as distance between repetitions was taken 4 meters. Pre- 2010).Therefore, pre-sowings of these green plants not only cultured Perko plants were planted in the middle of March and preserving the soil nitrogen but also adding nitrogen and were returned to ground on late May. Pre-cultured planting carbon in agricultural systems and improving C/N ratio and irrigation was performed by flooding method and the preventing soil erosion. Three major groups of plants, operations of maize planting was carried out 14 days after the including grasses, legumes and Brassica are used as green returning the pre-cultured Perko plant to the soil. For planting manure. Among these Brassica is considered as most suitable the maize plant, after reviewing the available varieties of the replacement for legumes and grasses to cultivate and in studied region, Single Cross maize cultivars SC 704 was addition to the effect of green manure can significantly selected for this study. This variety is a serotinous hybrid and increase soil organic carbon and soil porosity (Collins et al., has optimal yield in a wide range of weather conditions. Maize 2007). planting was carried out in the middle of July and the harvesting was performed on October in 2014. The lines Use of these cover crops not only increase production but also distance for planting was 65 cm with a density of 8 plants per reduced the risk associated with the modern crop production. square meter in plots with a size of 16 square meters. Further, it also showing superiority over the biofertilizers and avoid the limitation associated with the use of biofertilizers and Selected cover crop was Perko belongs to family Brassicaceae and chemical fertilizers. Among varieties of organic fertilizers, and are widely cultivated in temperate areas of Europe green manure has high importance because of economic and (Kashani et al., 1986). In this study, the effect of cover crop, environmental issues; the use of pre-cultured plants of Perko different levels of nitrogen and animal manure was evaluated can be considered as a green manure for maize (Baghdadi et on yield and components of maize. The planting, harvesting, al., 2016). Perko is an annual plant with wide and curved and sampling required during the life cycle of the maize plants leaves which are connected by thick petiole to the growing were continuously carried out. In this experiment, the numbers point near the crown and the ground. This plant is a hybrid and of seeds, weight of 1000 grain , grain yield and protein content artificially tetraploid which was produced by the crossing (%) were determined. Protein content was measured by between winter rapeseed (with 19 pairs of chromosomes) and infrared spectroscopy (NIR). Chinese cabbage (with 10 pairs of chromosomes) (Kashani et al., 1986).

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Effect of nitrogen level, green and animal manure on the growth attribute of corn crop (Zea mays L.) 227 Table 1 The Physico-chemical properties of the study area soil samples.

characteristics sampling salinity pH Lime organic Nitrogen Phosphorus Potassium tissue depth Ds/m % carbon % % ppm ppm 0-30 5.81 8.35 15.08 1.28 0.13 22.23 342.7 Clay Loam

Statistical analysis were performed using the software SAS and manure. Similarly, Yazdani et al. (2009) reported that the Duncan's multiple range tests were used to compare means at application of animal manure increased the number of grain the probability level of 5%. per pod and it showed superiority over the chemical nitrogen fertilizers. 2.1 Physical and chemical properties of the soil 3.2 Weight of 1000 maize seeds To determine the physical and chemical properties of soil at the site of the experiment, 20 soil samples were randomly Effect of various fertilizers on weight of 1000 seed was taken from 0-30 cm depth for each experimental and culture reported in this study and revealed that treatments containing conditions. Then samples were carefully mixed together and nitrogen fertilizers shows difference at the probability level of turned into a single sample and were transferred to the 1% (P ≤ 0.01 Table 2) while in case of various manures laboratory; its characteristics have been shown in table 1. interaction this significant difference was reported on the Chemical analysis of the collected soil samples was carried out probability level 5 percent (P ≤ 0.05). Among various tested by the method described by Hesse (1971). Soil salinity combination highest seed weight (230.63g/1000 seeds) was measurements were performed by EC meter. reported in the treatment containing highest dose (360Kg/ha) of chemical nitrogen fertilizer urea which suggested that the 3 Results and Discussion seed weight was increased with the increasing the value of nitrogen fertilizers and while the least weight (153.95g/1000 3.1 The number of seeds per cob seeds) was reported from treatment containing lowest amount of nitrogen fertilizer (120 kg/ha N). Furthermore, individual Result of study reported that the number of seeds per cob was application of animal manure also showed poor performance significant at P ≤ 0.01 probability level in the treatment and are not significantly differ from the lowest level of containing green manure (Table 2). The maximum number was nitrogen fertilizer (Figure 3). Although individual application 389.33 under green manure treatment and the lowest number of green manure shows superiority over the lowest level of reduced to 269.38 at the time of non-use of green manure nitrogen and animal manure but it performed poorly as (Figure 1). The significant effect of different amounts of compared to interaction. animal manures on this number was also observed at the P ≤ 0.05 probability level (Table 2). According to mean Among various combinations, highest weight of 1000 seeds comparisons for the traits using Duncan's method, maximum was reported from the interaction of green manure, animal number was obtained 351.41 in animal manure treatment and manure and lowest dose of nitrogen fertilizers (224.66g/1000 the minimum was 307.30 in the absence of manure (Figure 2). seeds) and it was almost at par to the highest dose of nitrogen Results of this study are in agreement with the findings of fertilizers while this weight was immediately followed by the Zadabdullah (2012) those who reported significant interaction of green and animal manures (201.10g/1000 improvement in the grain per cob on the application of green seeds).

Table 2 Variance analysis of main and interaction effects of traits

Source of variations Degree of Freedom Grain No. in cob 1000 grain Grain yield Biological Grain S.O.V. d.f. weight yield protein Replication 3 1226.44 ns 304.64 ns 141652.05 ns 12.07 ns 2.17 ns Nitrogen (N) 2 1112.99 ns 1710.62** 9191109.19* 24.88* 1.20 ns Green manure (Gn) 1 172662.03** 986.36 ns 1055065.57 ns 2.95 ns 7.85* N×Gn 2 1501.84 ns 593.62 ns 1497759.56* 54.85 ** 0.64 ns Error N×Gn 9 6801.09 443.09 32226.62 5.93 0.83 Animal manure (An) 1 23352.27 * 882.45 ns 772668.62 ns 46.21* 3.86* N×An 2 577.50ns 109.20 ns 37922.73 ns 3.28 ns 0.82 ns Gn×An 1 226.85 ns 2589.70 ** 922410.88 ns 40.88* 0.09 ns N×Gn×An 2 2255.03ns 280.35 ns 276572.20 ns 13.72 ns 2.53** Error 18 4332.10 290.70 649811.29 6.06 0.54 C.V. (%) 14.98 11.38 13.58 15.03 8 n.s: not significant. *, **: Statistically significant at P < 0.05, 0.01, respectively.

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228 Zakikhani et al

Figure 1 The effect of green manure on the number Figure 2 The effect of animal manure on the number of seeds per cob. of seeds per cob.

The results correspond with the results of Kaveh Firooz et al. 3.3 Grain yield (2013) those have reported that the increase in soil organic matters with a combination of animal manure and green Results of grain yield study shows similarities with the results manure increased thousand seed weight and finally the yield. of 1000 seeds and it was reported highest (6227.58 kg/ha) in Generally, there is a positive relationship between the soil the treatment containing 360 kg/ha N ha while the lowest biomass and size of plant in maize grain and grain Number and (4863.40 kg/ha) was reported in the treatment containing least it increase with the increasing levels of green and organic nitrogen fertilizer 120 kg/ha nitrogen (Figure 4). Interaction material (Zadabdullah, 2012). Result of study revealed that effect between green manure and nitrogen fertilizer on seed application of green and animal manures reduced the yield was significant at 5% probability level (P≤0.05) (Table dependency on the chemical fertilizers and it can be helpful in 2). reducing the cost of production.

Figure 3 Effect of various treatments on the weight of 1000 seeds; values without common letter differ significantly (P ≤ 0.05) as per LSD test

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Effect of nitrogen level, green and animal manure on the growth attribute of corn crop (Zea mays L.) 229

Figure 4 Effect of individual and combined application of nitrogen fertilizer and green manures on the grain yield; ; values without common letter differ significantly (P ≤ 0.05) as per LSD test.

In interaction study, maximum yield (6351.55 kg/ha) was Grain yield study also suggested the reduction in the reported from combination of green manure and 360kg/ha dependency chemical fertilizers. nitrogen and it was followed (6251.55 kg/ha) by the combination of green manure and 240kg/ha nitrogen results, 3.4 Effect of various combinations on Biomass of crops these two treatments are not significantly differ from each other. Green manure and nitrogen fertilizer interactions were significant at the 5% level on biomass. Highest (23.34 t/ha) Least effective combination was green manure and 120kg/ha and lowest (18.2 t/ha) biomass was reported from the nitrogen combination and it resulted only 4658.77k/ha grain treatments containing green manure along with 240Kg/ha and yield (Figure 4). Individual and combined application of 120Kg/ha nitrogen respectively (Figure 5). The combined animal manure are not showing significant difference from effect of green and animal manures on biological yield was individual application of nitrogen fertilizers or green manures, significant at the 5% level. Green manure along with animal that’s why these treatment was not reported in the figures. The manure treatments had the greatest biomass about 25.66 t/ha results were consistent with the findings of Baghdadi et al and the least rate was 18.04 t/ha in the absence of both (2016), Kaveh Firooz et al. (2013) and Adesoji et al. (2013). manures (Figure 6). Results of present study are in agreement The researchers reported that the grain yield increased in maize with the findings of the Baghdadi et al. (2016). due to increasing nitrogen levels and use of organic fertilizers.

Figure 5 Interactions of nitrogen fertilizer and green Figure 6 Effect of animal and green manure on Maize manure on Maize Biomass; values without common Biomass; values without common letter differ letter differ significantly (P ≤ 0.05) as per LSD test significantly (P ≤ 0.05) as per LSD test

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Figure 5 Interactions of animal manure, green manure and various amounts of nitrogen on maize grain protein.

3.5 Grain protein References

Results of variance analysis revealed that the interaction of the Adesoji AG, Abubakar IU, Tanimu B, Labe DA (2013) three parameters including nitrogen fertilizers, green manure Influence of Incorporated short duration legume fallow and and animal manure on grain protein was significant at 1% nitrogen on maize (Zea mays L.) growth and development in probability level (Table 2). Based on the table the highest northern guinea savannah of Nigeria. American-Eurasian average grain protein content was 10.17% in the treatments Journal Agricultural Environment Science 13: 58-67. DOI: using green and animal manures with 120 kg N ha and the 10.5829/idosi.aejaes.2013.13.01.1885. lowest content was observed 7.95% in treatments with the use of animal manure and 120 kg N ha without green manure Ajvanzadeh M (2005) Effects of organic materials with (Figure 7). The results were aligned with the findings of different levels of quality and the physicochemical properties Campbell & Akhtar (1990); Tripolskaya & Romanovskaya of soil and potato yield. Ph.D. thesis submitted to University of (2006) and Talger et al. (2009). Zanjan, Iran, Pp. 30-37 (In Persian).

Conclusion Baghdadi A, Kashani A, Vazan S (2016) The effect of pre- sowing treatments and nitrogen rates on yield of silage maize Evaluation of the overall results of this study suggested that (Zea mays L.) SC 704. International Journal of Advanced Life use of green manure and animal manure not only reduced the Sciences 9: 10-18. risks of chemical fertilizers but also increased crop production, grain yield and protein contents of maize grain. The most Biari A, Gholami A, Rahmani HA (2008) Growth promotion important sources of soil organic matters include livestock and enhanced nutrient uptake of maize by application of plant waste, plant residues and composts obtained from municipal growth promoting rhizobacteria in arid region of Iran. Journal garbage which today considering to the importance of organic Biological Science 8: 1015-1020. DOI: farming, their use has been greatly considered. It was also 10.3923/jbs.2008.1015.1020. reported that individual application of manure or chemical fertilizers cannot solve all the nutritional requirements of Campbell JA, Akhtar ME (1990) Impact of tillage on soil plants, therefore combination of these fertilizers and proper water regimes in the rainfed areas of Pakistan. Soil Physics management in the use of amount, the time and way of using 267-275. these fertilizers could have the best impact on productivity. Collins HP, Delgado JA, Alva AK, Follett RF (2007) Use of Conflict of Interest Nitrogen-15 Isotopic Techniques to Estimate Nitrogen Cycling from a Mustard Cover Crop to Potatoes. Agronomy Journals Authors would hereby like to declare that there is no conflict of 99 : 27-35. doi:10.2134/agronj2005.0357. interests that could possibly arise.

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Effect of nitrogen level, green and animal manure on the growth attribute of corn crop (Zea mays L.) 231 Hashemi M, Farsad A, Herbert SJ (2010) Managing cover crop wheat-cotton agroecosystem in Iran. Asian Journal of Plant for maximum nutrient recovery. Environmental Science Sciences 5: 559-562. DOI: 10.3923/ajps.2006.559.562. Research Institute, Shahid Beheshti University, Tehran., 248- 254. (In Persian) Ochiai N, Powelson ML, Crowe FJ, Dick RP (2008) Green manure effects on soil quality in relation to suppression of Hesse PR (1971). A Text Book of Soil Chemical Analysis. John Verticillium wilt of potatoes. Biology and Fertility of Soils 44: Nurray Williams Clowes and sons Ltd. London, 324 pp. 1013-1023. DOI: 10.1007/s00374-008-0289-z.

Kamkar B, Mahdavi Damghani A (2006) Principles of Talger L, Lauringson E, Roostalu H, Astover A (2009) The sustainable agriculture. First Edition. Publications University effects of green manures on yields and yield quality of spring of Mashhad, Iran., 62-74. (In Persian) wheat. Agronomy Research 7:125-132.

Kashani A, Bohrani J, Alemi Said Kh, Mesgarbashi M (1986) Tripolskaya L, Romanovskaya D (2006) A study of nitrogen Scientific Report of introduction of three varieties of forage migration affected by different plants for green manure in plants in the genus Brassica and report the results of their sandy loam soil. Ekologija 4: 89-97. preliminary research in Khuzestan. Journal of Agriculture 11: 74-78. (In Persian) Yazdani M, Bahmanyar MA, Pirdashti H, Esmaili MA (2009) Effect of Phosphate Solubilization Microorganisms (PSM) and Kaveh Firooz M, Kashani A, Paknejad F, Vazan S (2013) Plant Growth Promoting Rhizobacteria (PGPR) on Yield and Effects of pre-sowing and nitrogen fertilizer levels on Yield Components of Corn (Zea mays L.). International qualitative and quantitative characteristics of 640 corn Journal of Biological, Biomolecular, Agricultural, Food and varieties. M. Sc. thesis submitted to Islamic Azad University of Biotechnological Engineering 3:50-52. Karaj, Iran, Pp.100-108. (In Persian) Zadabdullah M, Vazan S, Kashani A (2014) Effects of Mahdavi Damqani A, Koocheki A, Rezvani Moqaddam P, Nitrogen Fertilizer on cover crops and weed control and crop Nassiri Mahallati M (2006) Studying the sustainability of a yield in Tehran. Ms.C. thesis submitted to Islamic Azad University of Karaj, Iran. 85-96. (In Persian)

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Journal of Experimental Biology and Agricultural Sciences, April - 2016; Volume – 4(2)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

PHENOTYPIC CHARACTERIZATION OF AN INDIGENOUS Bacillus thuringiensis STRAIN (B.t. LDC 501) EXPRESSING CANCER CELL KILLING PROTEIN

Abirami P1, Poornima Kkani2, Suguna P1, Saranya V1, Selvanayagam P1 and Shenbagarathai R1,*

1PG and Research Department of Zoology, Lady Doak College, Madurai-2. 2Department of Zoology, Thiagarajar College, Madurai-2.

Received – January 12, 2015; Revision – January 27, 2016; Accepted – May 10, 2016 Available Online – May 14, 2016

DOI: http://dx.doi.org/10.18006/2016.4(2).232.241

KEYWORDS ABSTRACT Bacillus thuringiensis Screening of coastal soil samples from Ramnad district, Tamil Nadu, South India, revealed the presence Antibiotic sensitivity of a novel non-haemolytic and non-insecticidal Bacillus thuringiensis strain that contained spherical inclusion body and exerted cytotoxic action on cultured human cancer cells. When protoplasmic extract Cytotoxicity of the B.t. LDC 501 isolate digest with proteinase K exerted significant toxicity on cell lines such as U- 937, HL-60 and primary leukemic cells. It also had mild influence on Jurkat (T cell lymphoma) but non- Biochemical tests toxic to normal human leukocytes. Majority of the biochemical tests of the strain revealed similar characteristics like that of insecticidal strain B. thuringiensis (BGSC-4Q2) with slight variation. Leukemic cells Moreover, antibiotic sensitivity assay discriminated the strain from B. anthracis due to its ability to resist ampicillin. Phylogenetic analysis revealed the distinctive nature of the strain as it forms a separate Cancer clade from other Bacillus strains.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (R.Shenbagarathai) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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233 Abirami et al

1 Introduction 2.2.1 Solubilization and activation of spore crystal complex Bacillus thuringiensis was first isolated from the diseased silkworm larvae by Ishiwata (1901). During its stationary The isolated B.t. LDC 501 strain was inoculated in Arret and growth phase, it produces parasporal crystalline inclusions that Kirshbaum Medium (sporulating agar) (Himedia, India) and are categorized into Cry and Cyt proteins (Van Rie & Ferre incubated at 28°C for 3-4 days. After observing the maximum 2000). Cry proteins are solubilized and activated by the parasporal inclusions, the spore crystal mixture was harvested alkaline environment of the host midgut followed by protease using 1 M NaCl followed by centrifugation (5810 R, activation. This facilitates binding of the toxin to the epithelial Eppendorf, Germany) at 12000×g for 10 min at 4°C. The pellet cells with the help of host-specific receptors like washed twice with distilled water supplemented 1 mM PMSF aminopeptidase N or cadherin, leading to oligomerization of (phenyl methyl sulphonyl fluoride). The spore crystal pellet the toxin monomers followed by osmotic cell lysis and death was solubilized in 50 mM Na2CO3 buffer of pH 10.5 of the host (Knowles, 1994). However, Cyt proteins exert in containing 10 mM DTT and 1 mM EDTA for 1 h at 37 °C vitro cytolytic and haemolytic activity on mammalian cell (Mizuki et al., 1999). The solubilized protein in the supernatant lines, however, without the involvement of a specific receptor was separated by centrifugation at 12,000×g for 20 min at 4 (Drobniewski, 1993). °C. It was then digested with 10 µg/ml of proteinase K (Merck-Genei, Bangalore, India) for 1.5h at 37 °C and 1 mM There were also reports on occurrence of non-insecticidal PMSF (Himedia, India) was added to stop the proteolytic strains in the natural environment which elicited interest on reaction. other possible functions (Ohba & Aizawa, 1986; Hastowo et al., 1992; Maeda et al., 2000; Lee et al., 2003). Outcomes of 2.2.2 Estimation of protein concentration these studies helps in the identification of another class of Cry protein named parasporin (PS) (Mizuki et al., 1999). It was Protein concentration of the parasporal inclusion was estimated non-haemolytic and exerted cytocidal activity against human by Bradford’s method using BSA (Bovine Serum Albumin) as cancer cells without affecting normal cells at the same time. So a standard (Bradford, 1976). Both the standard and test protein far, a total of six such parasporins which kills a plethora of of increasing concentration was added to the test tubes and cultured cancer cells were discovered (Wong, 2010). In made up to 0.1 mL with sodium carbonate buffer. Tube addition to that, some of the B.t. strains also harbors without protein sample was kept as blank. About 5 mL of parasporin-like genes (Ammons et al., 2016). Major Bradford reagent (Coomassie Brilliant Blue G-250 -100 mg, phenotypic characters of the eight PS1 producers, PS2, PS3 Ethanol (95%) - 50 mL, Phosphoric acid (85%) - 100 mL) was and PS4 were compared and found to be similar with the added to each tube and incubated at room temperature for 5 insecticidal B. thuringiensis (B.t.) strains (Ichikawa et al., min. Then absorbance was read at 595 nm and the values were 2007; Ichikawa et al., 2008). Due to the selective cytotoxic plotted with that of standard. nature, this organism can be exploited as potential therapeutic agent against cancer. 2.3 Maintenance of the cell lines

With regard to classification of B.t. strains, H-serotyping has The cell lines U-937 (histiocytic lymphoma), HL-60 been widely used. But, there is lack of information about (myelogenous leukemia), Jurkat (T cell lymphoma), primary genetic relatedness and phylogenetic relationships (Joung & leukemic cells from a patient and peripheral blood leukocytes Cote, 2001). Thus, there is a need to differentiate the cytocidal were cultured in media prescribed by the supplier with 10%

B.t. strain from other Bacillus species at biochemical and FBS and 1% antibiotic at 37°C with 5% CO2 in humidified molecular levels. Hence, the present study focuses on the incubator (SCO10W, Sheldon Manufacturing Inc, USA). phenotypic characterization of the isolate termed as B.t. LDC 501 which has a unique potent cell-killing activity on human 2.4 One dose cytotoxic assay cancer cell lines. The proteinase K activated parasporal protein of the potential 2 Materials and Methods strain B.t. LDC 501 was tested against the suspension cancer cell lines U-937, HL-60, Jurkat and primary leukemic cells for 2.1 Bacterial strains and culture media cytotoxic activity (Mizuki et al., 1999). Aliquots of 90 μl containing 2 x 105 cells were added to wells in a microtitre B.t.LDC 501 was isolated and identified by screening of plate in triplicates. About 100 μl of the parasporal proteins several coastal soil samples (Abirami et al., 2016) and (100 µg/ml) was added to each of the experimental wells and compared with reference strain B. thuringiensis israelensis incubated at 37°C with 5 % CO2 for 16 h. The solubilising (BGSC-4Q2) and were used in this study. The bacterial strains buffer containing Proteinase K and PMSF was used as mock were lyophilized for long term storage. control. The cyto-morphological changes on these cells were monitored under inverted phase contrast microscope 2.2 Processing of the parasporal inclusions (Olympus, CKX41, Tokyo, Japan).

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Phenotypic characterization of an indigenous Bacillus thuringiensis strain (B.t. LDC 501) expressing cancer cell killing protein 234 Cultures were inoculated by stab method to the tubes 2.5 MTT-assay containing SIM agar. After 24-48 h of incubation at 37 °C, Kovac’s reagent was added and observed for the color change. Proteinase K activated parasporal toxin (0.01 µg/ml, 0.1 µg/ml, 1 µg/ml, 10 µg/ml and 100 µg/ml) was added to the cancer 2.6.5 Urease test cells (~2 x 105) in a 96-well plate for 16 h. After incubation, 10 μl of MTT solution (from 5 mg/ml stock) was added to the This test was performed to check the production of urease cells and kept in dark for 4 h. After centrifugation at 1100 ×g enzyme by the organism. The urea broth was prepared and for 5 min, the cells were solubilised in 100 μl of DMSO. The cultures were inoculated aseptically. The tubes were then kept absorbance was measured at 570 nm in the ELISA plate reader for 24-48 h incubation at 37 °C and observed for the change in (VersaMax, Molecular Devices, CA, USA). The viability colour from phenol red to deep pink. percentage was calculated from the cells treated with buffer alone. The protein concentration inflicting 50% cell death was 2.6.6 Hydrogen sulphide considered as median lethal dose (LC50). Cultures were inoculated by stab method to the SIM agar deep 2.6 Biochemical characterization tubes and kept for 24-48 h incubation. The tubes were observed for the presence of black precipitate due to the

Biochemical characterization of the B.t. LDC 501 strain was production of H2S gas. carried out using HiBacillusTM Identification Kit (HiMedia, India) for the Voges-Proskauer, citrate utilization, malonate 2.6.7 Carbohydrate Fermentation utilization, arginine utilization, catalase, nitrate reduction, ortho-Nitrophenyl-β-galactoside (ONPG), carbohydrate 2.6.7.1 Triple Sugar Iron fermentation tests (Jung et al., 2007). B. thuringiensis israelensis (BGSC-4Q2) and E. coli (DH5 α) were used as The TSI agar slants containing 1% each of lactose and sucrose positive and negative control respectively. These tests were as well as 0.1% glucose with phenol red indicator. The culture performed by inoculating a loopful of culture on each well and inoculated by stab and streak method was incubated for 24 h incubated for 24 h at 37 °C. After incubation, Voges- and observed for the change in colour from red to yellow. Proskauer test was performed by adding 1-2 drops of Baritt Reagent A and B. For the nitrate reduction test, 1-2 drops of 2.6.8 Phenotypic characterization sulfanilic acid and one drop of α-naphthylamine was added and observed for its change in color. To determine the catalase The sporulated cells of B.t. LDC 501 strain were smeared on a action, 3% hydrogen peroxide solution was added and glass slide and heat fixed. It was stained with Coomassie production of effervescence was observed. All the biochemical Brilliant Blue R-250 (Appendix-8) for 3 min (Rampersad & tests were performed as described in Bergey’s manual of Ammons, 2005). Excess stain was removed by washing with Systematic Bacteriology (Garrity & Holt, 2001). tap water, dried and observed under bright field microscope using 100X oil immersion (Olympus-DP 12, Japan). 2.6.1 Gelatin hydrolysis 2.7 Antibiotic sensitivity test The cultures were inoculated in the medium containing nutrient gelatin stab and incubated at 25°C for 48 h. The tubes Antibiotic sensitivity test was carried out using disc diffusion were then kept at 4°C for 30 min to check gelatin liquefaction. method (Doern, 2011). B.t. LDC 501 was spread on to the plates containing Muller Hinton agar in triplicates. Antibiotic 2.6.2 Starch hydrolysis diffusion discs such as ampicillin (10 mcg/disc), bacitracin (10 mcg/disc), chloramphenical (30 mcg/disc), kanamycin (10 The cultures were streaked as a single line on the starch agar mcg/disc), penicillin (20 U/disc), polymixin (300 U/disc), and incubated for 24 h. A solution of iodine was added to find streptomycin (10 mcg/disc), tetracycline (30 mcg/disc) and out the starch utilization. rifampicin (5 mcg/disc) were kept in each corner as three per plate. The plates were incubated for 24 h at 37 °C. 2.6.3 Casein hydrolysis 2.8 Phylogenetic analysis To the milk agar plate, cultures were inoculated to check the casein hydrolyzing property. The formation of clear zone The genomic DNA from B.t. was extracted by the method of around the growth of the organism was observed. Ceron et al., (1994) with slight modifications. 16S rDNA sequence analysis was performed to confirm the strain belongs 2.6.4 Indole production to Bacillus thuringiensis. Bacteria-specific forward (5’- AGAGTTTGATCCTGGCTCAG-3’) and reverse primers (5’- ACGGCTACCTTGTTACGACTT-3’) (27F/1492R) were used for PCR amplification (Poornima et al., 2010).

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2.9 Statistical Analysis of the toxic effects on the normal human leukocytes serving as control, when observed under similar time intervals Data obtained for MTT values were represented as mean ± (Figure.1c). standard error of the mean and the significant differences between the groups were tested using one-way ANOVA 3.2 MTT assay (SigmaStat 2.0, Systat Software Inc, USA). When the level of significance was p≤0.05, then a posterori post hoc pairwise The activated proteins of B.t. LDC 501 exhibited dose- comparsion was performed using Tukey test. dependent action on primary leukemic cells, U-937 and HL-60

with LC50 value of 0.05 µg/ml, 0.1 µg/ml and 20 µg/ml 3 Results respectively (Figure. 1b). The resistant Jurkat cell line showed cell death at 100 µg/ml only whereas normal human leukocytes 3.1 Cytotoxicity assay remain intact at all concentrations of the protein.

The activated proteins of B.t. LDC 501 exerted significant 3.3 Biochemical characterization cytotoxic action on primary leukemic cells followed by U-937 and HL-60. The cells readily collapsed after toxin exposure The results of biochemical characterization for the strain that resulted in clump formation and finally cell death B.t.LDC 501 was illustrated in Figure 2 & Table 1. However, (Figure.1a). Among the cell lines tested, the T cell lymphoma tests such as arginine utilization, triple sugar iron and sucrose cell line Jurkat was resistant to the B.t. LDC 501 protein utilization were positive for B.t. LDC 501 strain which is in action. The activated protein of B.t. LDC 501 did not show any contrast to the negative values obtained with BGSC-4Q2.

Figure1a: Cytopathic effect of activated B.t.LDC 501 proteins on HL-60, U-937, primary leukemic cells and Jurkat under phase contrast microscope at 16 h postinoculation.

Figure1C: Non-toxic effect of B.t.LDC 501 proteins on normal human leukocytes at regular time intervals

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Phenotypic characterization of an indigenous Bacillus thuringiensis strain (B.t. LDC 501) expressing cancer cell killing protein 236

Figure1B: Dose response curve of B.t.LDC 501 activated proteins on HL-60, U-937, primary leukemic cells and Jurkat under phase contrast microscope at 16 h postinoculation. The level of cytotoxicity was assessed with MTT assay after 16 h post inoculation; Symbols: black circle shaded, HL 60; black circle without shade, U-937; black triangle shaded, Human Leukemic cells; black triangle without shade, Jurkat; respectively. Each point represents mean± standard error of triplicates. Different alphabets represent statistical significance p≤0.05, between the groups tested by Tukey test.

3.4 Phenotypic characterization 3.6 Phylogenetic analysis

The Coomassie stained crystals appeared better than unstained The amplified product (1400 bp) of 16S rDNA of B.t.LDC crystals. As observed in Figure 3, the crystals were spherical in 501 strain was sequenced (Figure 5a). BLAST analysis of 16S shape and smaller than spores. rDNA sequence showed maximum score for B. thuringiensis followed by B. cereus and B. anthracis. The sequence was 3.5 Antibiotic sensitivity test submitted to GenBank and accession number: JQ988062 was obtained. Phylogenetic analysis revealed that the strain Antibiotic sensitivity test revealed that B.t. LDC 501 strain was originates from B. cereus group and then forms separate clade resistant to ampicillin, penicillin and bacitracin but highly with B. thuringiensis (Figure 5b). sensitive to kanamycin and streptomycin (Figure 4 &Table 2).

Figure 2: Biochemical test performed using Bacillus Identification kit. Arrow head in the 8th well inoculated with B.t.LDC 501 culture denotes the ability to ferment the carbohydrate whereas it is not observed in BGSC-4Q2 strain.

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Table 1: Results of various biochemical tests performed for B.t.LDC 501 strain

S.No Name of the test B.t.LDC 501 B.t.israelensis 4Q2 (Positive control) E.coli (Negative control)

1. Gram’s stain Gram positive Gram positive Gram negative 2. Spore forming Positive Positive Negative 3 Cell shape Long rod Long rod Small rod 4 Density Opaque Opaque Translucent 5 Elevation Convex Convex Raised 6 Margin Irregular Irregular Entire 7 Motility Positive Positive Positive 8 Malonate utilization Negative Negative Negative 9 Catalase Positive Positive Negative 10 Voges Proskauer Negative Negative Negative 11 Citrate utilization Negative Negative Negative 12 ONPG Negative Negative Positive 13 Nitrate reduction Positive Positive Positive 14 Arginine utilization Positive Negative Negative 15 Triple Sugar Iron Positive Negative Negative 16 Hydrogen sulfide Negative Negative Negative 17 Indole Negative Negative Positive 18 Urease Positive Positive Positive 19 Starch hydrolysis Positive Positive Negative 20 Casein hydrolysis Negative Negative Negative 21 Carbohydrate utilization Sucrose Positive Negative Positive Mannitol Negative Negative Negative Glucose Positive Positive Positive Arabinose Negative Negative Negative Trehalose Positive Positive Negative

4 Discussion for the disease anthrax in animals and humans (Ombui et al., 1997). Thus, there is a need to differentiate the cytocidal B.t Since B. thuringiensis strains are more similar to B.cereus strain from other Bacillus species in biochemical and taxonomically, the production of crystalline inclusions is the molecular levels. In addition to that, number of phenotypic key phenotypic feature to distinguish both the types. B. cereus tests also required to separate single species (Drobniewski, is a well-known food-borne pathogen which produces 1993). diarrhoeal type of enterotoxin. B.anthracis is a causative agent

Figure 3 Microscopic images of B.t.LDC 501 parasporal inclusions under bright field; S-Spores, C -Crystals.

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Phenotypic characterization of an indigenous Bacillus thuringiensis strain (B.t. LDC 501) expressing cancer cell killing protein 238

Figure 4 Plates showing antibiotic sensitivity of B.t.LDC 501 against nine different antibiotics discs. Amp-Ampicillin, B-Bacitracin, C- Chloramphenical, K-Kanamycin, P-Penicillin, PB-Polymixin, S-Streptomycin, TE-Tetracycline, RF-Rifampicin.

The spherical crystalline inclusion of the potential B.t.LDC The specific cytocidal action of B.t. LDC 501 proteins on 501 strain was smaller than the spores which is formed during colon cancer cell lines was reported (Abirami et al., 2016). In sporulation stage (Figure 3). Generally, spherical shaped this study, it showed significant toxicity towards suspension crystals are associated with Dipteran toxicity. Interestingly, cell lines such as U- 937, HL-60 and primary leukemic cell B.t.LDC 501 strain was non-toxic against two Dipteran lines. The only exception was the T-lymphoma cell line Jurkat mosquito species (Culex quinquefasciatus and Aedes aegypti) which was completely resistant with LC50 of 110 µg/mL (Abirami et al., 2016). The ability to act specifically on cancer (Figure 1a & 1b). The normal peripheral blood leukocytes cells by discriminating normal cells is the distinguished (Figure 1c) were not susceptible to the cytotoxic action of characteristic of parasporins (Mizuki et al., 1999). This action B.t.LDC 501 proteins when tested at different time intervals. differs from broad cytolytic Cyt proteins that kill both normal This is in contrast to an earlier reported parasporin protein PS- and cancer cells (Thomas & Ellar, 1983). This is correlated 2 with broad cell-killing action on various human cancer cell with the earlier studies that denoted the association of cyt lines and also on normal T lymphocytes (Ito et al., 2004). The genes with haemolysis and mosquitocidal action (Guerchicoff other parasporins reported acted only on a few cell types, not et al., 2001). ubiquitously, which is also a striking feature of B.t.LDC 501 protein.

Table.2 Zone of inhibition exhibited by B.t.LDC 501 against various antibiotics

S.No Name of the antibiotic diffusion disc Zone of inhibition (in cm) Susceptibility

1. Ampicillin 0.0 Resistant 2. Bacitracin 0.0 Resistant 3. Chloramphenical 0.9 Sensitive 4. Kanamycin 1.0 Sensitive 5. Penicillin 0.0 Resistant 6. Polymixin 0.4 Mild Resistant 7. Streptomycin 1.0 Sensitive 8. Tetracycline 0.8 Sensitive 9. Rifampicin 0.7 Mild Sensitive

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Biochemical analysis was performed to assess the ability to 2005). Like B.t.LDC 501, parasporin (PS2, PS3 and PS4) utilize, reduce some compounds, and to ferment specific producing strains and other insecticidal strains were also carbon sources. It is also useful to characterize and distinguish highly resistant to ampicillin (Figure 4). Further resistance to B. thuringiensis strains from other Bacillus species. B.t.LDC bacitracin exhibited by B.t. LDC 501 strain is in contradiction 501 isolate exhibits similar biochemical characteristics like that to the PS1, PS2, PS3 and PS4 producers (Ichikawa et al., 2007; of the reference strain BGSC-4Q2 (Figure 2). Ichikawa et al., 2008).

However, it differs in the following tests such as arginine These results illustrated that there is no major dissimilarity in utilization, triple sugar iron and sucrose utilization (Table 1). the phenotypic characteristics of the non-insecticidal strains This might be due to the variation in speciation process as from that of insecticidal ones (Ichikawa et al., 2008). Thus, described by Martin & Travers (1989). Carbohydrate biochemical analysis must be accompanied with molecular utilization and spore aspect ratio of PS2, PS3 and PS4 characterization in order to classify the microorganism. The producers were similar to that of insecticidal B.t. strains unique cytotoxic South Indian isolate, B.t. LDC 391 was also (Ichikawa et al., 2008). It is imperative to note that the found to be similar in phenotypic and genotypic characteristics cytotoxic Canadian isolate (M15) was unable to ferment starch like that of insecticidal strains (Poornima et al., 2012). like B.cereus (Jung et al., 2007). The diversity and phylogenetic relationships between the organisms was decoded by the gene sequences (Zuckerkandl & Pauling, 1965). The phylogenetic analysis of the potential strain B.t.LDC 501 was done by 16S rDNA analysis. The sequence showed high similarity to that of B. thuringiensis (Figure.5a & b). Bacillus species such as B. cereus, B. thuringiensis and B. anthracis shared high levels of 16S rDNA sequence similarity (Thorne, 1993).

Conclusion

In general, the existing parasporins react on only a limited set of cancer cell types, each with its own specificity. Protein of this study is also specific against certain cells, thus showing a tendency similar to that of other parasporins. The newly identified cytotoxic strain B.t.LDC 501 was phenotypically characterized and found to be different from other parasporin producers. Thus, further cytotoxic characterization of the parasporin-like protein enumerates its mode of action and uniqueness among the reported parasporins.

Acknowledgement

We thank Department of Science and Technology (SR/SO/HS- 48/2008), Council of Scientific and Industrial Research Figure 5a: PCR analysis of 16S rDNA of B.t.LDC 501 strain. (37(1480)/11/EMR-II), Science and Engineering Research Lane1-100 bp -3000bp DNA marker, Lane2- BGSC-4Q2, Board (SR/SO/HS-0116/2012) and Department of Lane3- B.t.LDC 501 Biotechnology-Bioinformatics Infrastructure Facilities vide reference 102/IFD/SAN/1125/2006-07 for the financial support. Bacillus species were also identified based on the level of susceptibility against different groups of antibiotics (Reva et Conflict of interest al., 1995). The result of antibiotic sensitivity test (Table 2) was similar to that of earlier reports (Luna et al., 2007). Penicillin Authors would hereby like to declare that there is no conflict of resistance of B.t.LDC 501 is comparable with that of B.cereus interests that could possibly arise. and B.thuringiensis strains but not with B.anthracis (Logan,

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Phenotypic characterization of an indigenous Bacillus thuringiensis strain (B.t. LDC 501) expressing cancer cell killing protein 240

Figure 5b: Phylogenetic analysis of 16S rDNA of B.t.LDC 501. Among the 11 different Bacillus strains analyzed using ClustalW, B.t.LDC 501 strain formed separate clade with the Bacillus thuringiensis strain.

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