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Depolama Süresi ve Koşullarının Entomopatojen Nematodların Canlılığı ve Patojenitesi Üzerindeki Rolü

Yıl 2023, Cilt: 9 Sayı: 2, 176 - 185, 21.08.2023
https://doi.org/10.24180/ijaws.1297111

Öz

Entomopatojen nematodlar (EPN) (Rhabditida: Heterorhabditidae ve Steinernematidae) birçok zararlı böceklerin etkili biyokontrol ajanlarıdır ve genellikle laboratuvarda veya arazide kullanılmadan önce belirli bir süre depolanırlar. Bununla birlikte, elverişsiz saklama koşullarının EPN'lerin hayatta kalması ve infektivitesi üzerinde büyük bir etkisi vardır. Bu çalışma, dört yerel EPN türünün (Heterorhabditis bacteriophora FLH-4H, H. indica 216-H, Steinernema feltiae KCS-4S ve S. bicornotum MGZ-4S) infektif juvenillerinin (IJ) laboratuvar koşullarında optimum saklama koşullarını belirlemek için gerçekleştirilmiştir. IJ'lerin canlılıkları, farklı konsantrasyonlarda (500, 1000, 1500 ve 2000 IJ), sıcaklıklarda (9 ve 25 °C) ve depolama ortamında (ddH2O, musluk suyu ve steril Ringer solüsyonu) test edilmiştir. Genel olarak, test edilen EPN türlere ait IJ'lerin canlılıkları, 1000 ve 1500 IJ konsantrasyonlarında depolamadan sonraki 1. ayda en yüksek iken artan depolama süreleri ile kademeli olarak azalmıştır. Genel olarak Steirnematidlerin IJ'lerinin hayatta kalma oranları Heterorhabditid türlerinden daha yüksek olduğu belirlenmiştir. IJ'lerin en yüksek hayatta kalma oranı, genellikle Ringer solüsyonunda 9°C'de 1 aylık depolamadan sonra elde edilirken, musluk suyu, test edilen her iki sıcaklıkta da IJ'ler canlılıklarında önemli bir düşüşe neden olmuştur. Patojenite testlerinde, 9°C'de depolanan IJ'ler, Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvalarında daha yüksek ölüm oranlarına neden olmuştur. Sonuçlar, test edilen EPN türlerine ait IJ'lerin, infektivitelerinde ciddi bir düşüş olmadan 9°C'de, 1000 ve 1500 IJs konsantrasyonlarında, Ringer solüsyonunda daha uzun süre canlı kalabildiğini göstermiştir.

Kaynakça

  • Andaló, V., Cavalcanti, R. S., Molina, J. P., & Moino Jr, A. (2010). Substrates for storing entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae). Scientia Agricola, 67(3), 342-347. https://doi.org/10.1590/S0103-90162010000300013.
  • Andalo, V., Moino, A., Maximiniano, C., Campos, V. P., & Mendonca, L. A. (2011). Influence of temperature and duration of storage on the lipid reserves of entomopathogenic nematodes. Revista Colombiana de Entomología, 37(2), 203-209.
  • Ansari, M. A., Shah, F. A., Tirry, L., & Moens, M. (2006). Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53. Biological Control, 39(3), 453-459. https://doi.org/10.1016/j.biocontrol.2006.07.004.
  • Bai, C., Shapiro-Ilan, D. I., Gaugler, R., & Yi, S. (2004). Effect of entomopathogenic nematode concentration on survival during cryopreservation in liquid nitrogen. Journal of nematology, 36(3), 281.
  • Bhat, A. H., Chaubey, A. K., & Askary, T. H. (2020). Global distribution of entomopathogenic nematodes, Steinernema and Heterorhabditis. Egyptian Journal of Biological Pest Control, 30(1), 1-15. https://doi.org/10.1186/s41938-020-0212-y.
  • Brown, I. M., & Gaugler, R. (1997). Temperature and humidity influence emergence and survival of entomopathogenic nematodes. Nematologica, 43(5), 363-375.
  • Canhilal, R., Waeyenberge, L., Yüksel, E., Koca, A. S., Deniz, Y., & İmren, M. (2017). Assessment of the natural presence of entomopathogenic nematodes in Kayseri soils, Turkey. Egyptian Journal of Biological Pest Control, 27(2).
  • Canhilal, R., Waeyenberge, L., Toktay, H., Bozbuga, R., Çerintas, R., & Imren, M. (2016). Distribution of Steinernematids and Heterorhabditids (Rhabditida: Steinernematidae and Heterorhabditidae) in the Southern Anatolia Region of Turkey. Egyptian Journal of Biological Pest Control, 26(4).
  • Caylak, E., & Tokar, M. (2012). Investigating chemical and microbiological contaminants in drinking water of Cankiri Province, Turkey. Environmental Earth Sciences, 67(7), 2015-2025. http://dx.doi.org/10.1007/s12665-012-1641-z .
  • Glazer, I. (1996). Survival mechanisms of entomopathogenic nematodes. Biocontrol Science and Technology, 6(3), 373-378. https://doi.org/10.1080/09583159631343 .
  • Gülcü, B., & Hazir, S. (2012). An alternative storage method for entomopathogenic nematodes. Turkish Journal of Zoology, 36(4), 562-565. https://doi.org/10.3906/zoo-1103-10 .
  • Grewal, P. S. (2000). Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae). International Journal for Parasitology, 30(9), 995-1000. https://doi.org/10.1016/S0020-7519(00)00080-1.
  • Grewal, P. S., Bornstein-Forst, S., Burnell, A. M., Glazer, I., & Jagdale, G. B. (2006). Physiological, genetic, and molecular mechanisms of chemoreception, thermobiosis, and anhydrobiosis in entomopathogenic nematodes. Biological Control, 38(1), 54-65. https://doi.org/10.1016/j.biocontrol.2005.09.004.
  • Griffin, C. T. (1993). Temperature responses of entomopathogenic nematodes: Implications for the success of biological control programmes. In R.A. Bedding, R.J. Akhurst & H.K. Kaya (Eds.), Nematodes and the biological control of insect pests (pp. 115-126). CSIRO Publications.
  • Hass, B., Downes, M. J., & Griffin, C. T. (2002). Persistence of four Heterorhabditis spp. isolates in soil: role of lipid reserves. Journal of Nematology, 34(2), 151.
  • Hatab, M. A. A., & Gaugler, R. (1999). Lipids of in vivo and in vitro cultured Heterorhabditis bacteriophora. Biological Control, 15(2), 113-118. https://doi.org/10.1006/bcon.1999.0701.
  • Hazir, S., Stock, S. P., Kaya, H. K., Koppenhöfer, A. M., & Keskin, N. (2001). Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae). Journal of Invertebrate Pathology, 77(4):243–250. https://doi.org/10.1006/jipa.2001.5029.
  • Hazir, S., Kaya, H. K., Stock, S. P., & Keskin, N. (2003). Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) for biological control of soil pests. Turkish journal of Biology, 27(4), 181-202.
  • Fitters, P. F., & Griffin, C. T. (2004). Spontaneous and induced activity of Heterorhabditis megidis infective juveniles during storage. Nematology, 6(6), 911-917. https://doi.org/10.1163/1568541044038597.
  • Kaya, H. K., & Stock, S. P. (1997). Techniques in insect nematology. In L. Lacey (Ed.), Manual of Techniques in Insect Pathology (pp. 281-324). Academic Press.
  • Kepenekci, I., Hazir, S., & Lewis, E. E. (2016). Evaluation of entomopathogenic nematodes and the supernatants of the in vitro culture medium of their mutualistic bacteria for the control of the root‐knot nematodes Meloidogyne incognita and M. arenaria. Pest management science, 72(2), 327-334. https://doi.org/10.1002/ps.3998.
  • Kour, S., Khurma, U., Brodie, G., & Singh, S. (2022). Modeling the potential global distribution of suitable habitat for the biological control agent Heterorhabditis indica. Ecology and Evolution, 12(6), e8997. https://doi.org/10.1002/ece3.8997.
  • Metwally, H. M., Hafez, G. A., Hussein, M. A., Hussein, M. A., Salem, H. A., & Saleh, M. M. E. (2012). Low cost artificial diet for rearing the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) as a host for entomopathogenic nematodes. Egyptian Journal of Biological Pest Control, 22(1), 15.
  • Mokrini, F., Laasli, S. E., Benseddik, Y., Joutei, A. B., Blenzar, A., Lakhal, H., Sbaghi, M., İmren, M., Özer, G., Paulitz, T., Lahlai, R., & Dababat, A. A. (2020). Potential of Moroccan entomopathogenic nematodes for the control of the Mediterranean fruit fly Ceratitis capitata Wiedemann (Diptera: Tephritidae). Scientific reports, 10(1), 1-11. https://doi.org/10.1038/s41598-020-76170-7.
  • Odendaal, D., Addison, M. F., & Malan, A. P. (2016). Entomopathogenic nematodes for the control of the codling moth (Cydia pomonella L.) in field and laboratory trials. Journal of Helminthology, 90(5), 615-623. https://doi.org/10.1017/s0022149x15000887.
  • Prabhuraj, A., Viraktamath, C. A., & Kumar, A. R. V. (2000). Modified trapping technique for the isolation of insect parasitic nematodes. Journal of Biological Control, 14(2) 83-85. https://doi.org/10.18311/jbc/2000/4169.
  • Selvan, S., Gaugler, R., & Lewis, E. E. (1993). Biochemical energy reserves of entomopathogenic nematodes. The Journal of parasitology, 79(2)167-172. https://doi.org/10.2307/3283503.
  • Singh, M., Rani, P., Prashad, H., & Nalini, C. (2023). Effect of storage media and temperature on viability and pathogenicity of North Indian populations of entomopathogenic nematodes. Journal of Entomological Research, 47(1), 209-214. https://doi.org/10.1007%2Fs12639-014-0639-8.
  • Sharmila, R., & Subramanian, S. (2016). Effect of low temperature on the activity of entomopathogenic nematodes. International Journal of Forestry and Crop Improvement, 7(1), 19-23. https://doi.org/10.15740/HAS/IJFCI/7.1/00-00.
  • Susurluk, I. A., Kumral, N. A., Peters, A., Bilgili, U., & Açıkgöz, E. (2009). Pathogenicity, reproduction and foraging behaviours of some entomopathogenic nematodes on a new turf pest, Dorcadion pseudopreissi (Coleoptera: Cerambycidae). Biocontrol Science and Technology, 19(6), 585-594. http://dx.doi.org/10.1080/09583150902957348.
  • Qiu, L. & Bedding, R. (2000). Energy metabolism and its relation to survival and infectivity of infective juveniles of Steinernema carpocapsae under aerobic conditions. Nematology 2, 551-559. http://dx.doi.org/10.1163/156854100509330.
  • Qiu, L., & Bedding, R. A. (2002). Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 131(4), 757-765. https://doi.org/10.1016/s1096-4959(02)00019-2.
  • Yadav, A. K. (2016). Effects of storage temperature on survival and infectivity of three indigenous entomopathogenic nematodes strains (Steinernematidae and Heterorhabditidae) from Meghalaya, India. Journal of Parasitic Diseases, (40), 1150-1154. https://doi.org/10.1007%2Fs12639-014-0639-8.
  • Yuksel, E., & Canhilal, R. (2019). Isolation, identification, and pathogenicity of entomopathogenic nematodes occurring in Cappadocia Region, Central Turkey. Egyptian Journal of Biological Pest Control, 29(1), 1-7. http://dx.doi.org/10.1186/s41938-019-0141-9.

The Role of Storage Duration and Conditions on the Survival and Pathogenicity of Entomopathogenic Nematodes

Yıl 2023, Cilt: 9 Sayı: 2, 176 - 185, 21.08.2023
https://doi.org/10.24180/ijaws.1297111

Öz

Entomopathogenic nematodes (EPNs) (Rhabditida: Heterorhabditidae and Steinernematidae) are effective biocontrol agents for many insect pests and are generally stored for a period of time prior to their use in the laboratory or field. However, unfavorable storage conditions have a great impact on the survival and infectivity of EPNs. This study was conducted to determine the optimum storage conditions of infective juveniles (IJs) of four native EPN species (Heterorhabditis bacteriophora FLH-4H, H. indica 216-H, Steinernema feltiae KCS-S, and S. bicornotum MGZ-4S) under laboratory conditions. The survival capability of the IJs was tested at different concentrations (500, 1000, 1500, and 2000 IJs), temperatures (9 and 25 °C) and storage media [double-distilled water (ddH2O), tap water, and sterile Ringer solution]. In general, the survival of IJs of tested EPN species was the highest at the 1st month after treatment (MAT) at the concentrations of 1000 and 1500 IJs and gradually decreased with the increasing storage periods. The survival rates of the IJs of Steirnematids were generally higher than Heterorhabditid species. The highest survival of IJs was generally obtained after 1-month storage in Ringer solution at 9°C while tap water led to poor survival for the IJs at both temperatures tested. The IJs that were stored at 9°C induced higher mortalities on the larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). The results showed that the IJs of tested EPN species can remain viable for a longer period of time in Ringer solution at 1000 and 1500 IJs concentrations at 9°C without losing much of their infectivity.

Kaynakça

  • Andaló, V., Cavalcanti, R. S., Molina, J. P., & Moino Jr, A. (2010). Substrates for storing entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae). Scientia Agricola, 67(3), 342-347. https://doi.org/10.1590/S0103-90162010000300013.
  • Andalo, V., Moino, A., Maximiniano, C., Campos, V. P., & Mendonca, L. A. (2011). Influence of temperature and duration of storage on the lipid reserves of entomopathogenic nematodes. Revista Colombiana de Entomología, 37(2), 203-209.
  • Ansari, M. A., Shah, F. A., Tirry, L., & Moens, M. (2006). Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53. Biological Control, 39(3), 453-459. https://doi.org/10.1016/j.biocontrol.2006.07.004.
  • Bai, C., Shapiro-Ilan, D. I., Gaugler, R., & Yi, S. (2004). Effect of entomopathogenic nematode concentration on survival during cryopreservation in liquid nitrogen. Journal of nematology, 36(3), 281.
  • Bhat, A. H., Chaubey, A. K., & Askary, T. H. (2020). Global distribution of entomopathogenic nematodes, Steinernema and Heterorhabditis. Egyptian Journal of Biological Pest Control, 30(1), 1-15. https://doi.org/10.1186/s41938-020-0212-y.
  • Brown, I. M., & Gaugler, R. (1997). Temperature and humidity influence emergence and survival of entomopathogenic nematodes. Nematologica, 43(5), 363-375.
  • Canhilal, R., Waeyenberge, L., Yüksel, E., Koca, A. S., Deniz, Y., & İmren, M. (2017). Assessment of the natural presence of entomopathogenic nematodes in Kayseri soils, Turkey. Egyptian Journal of Biological Pest Control, 27(2).
  • Canhilal, R., Waeyenberge, L., Toktay, H., Bozbuga, R., Çerintas, R., & Imren, M. (2016). Distribution of Steinernematids and Heterorhabditids (Rhabditida: Steinernematidae and Heterorhabditidae) in the Southern Anatolia Region of Turkey. Egyptian Journal of Biological Pest Control, 26(4).
  • Caylak, E., & Tokar, M. (2012). Investigating chemical and microbiological contaminants in drinking water of Cankiri Province, Turkey. Environmental Earth Sciences, 67(7), 2015-2025. http://dx.doi.org/10.1007/s12665-012-1641-z .
  • Glazer, I. (1996). Survival mechanisms of entomopathogenic nematodes. Biocontrol Science and Technology, 6(3), 373-378. https://doi.org/10.1080/09583159631343 .
  • Gülcü, B., & Hazir, S. (2012). An alternative storage method for entomopathogenic nematodes. Turkish Journal of Zoology, 36(4), 562-565. https://doi.org/10.3906/zoo-1103-10 .
  • Grewal, P. S. (2000). Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae). International Journal for Parasitology, 30(9), 995-1000. https://doi.org/10.1016/S0020-7519(00)00080-1.
  • Grewal, P. S., Bornstein-Forst, S., Burnell, A. M., Glazer, I., & Jagdale, G. B. (2006). Physiological, genetic, and molecular mechanisms of chemoreception, thermobiosis, and anhydrobiosis in entomopathogenic nematodes. Biological Control, 38(1), 54-65. https://doi.org/10.1016/j.biocontrol.2005.09.004.
  • Griffin, C. T. (1993). Temperature responses of entomopathogenic nematodes: Implications for the success of biological control programmes. In R.A. Bedding, R.J. Akhurst & H.K. Kaya (Eds.), Nematodes and the biological control of insect pests (pp. 115-126). CSIRO Publications.
  • Hass, B., Downes, M. J., & Griffin, C. T. (2002). Persistence of four Heterorhabditis spp. isolates in soil: role of lipid reserves. Journal of Nematology, 34(2), 151.
  • Hatab, M. A. A., & Gaugler, R. (1999). Lipids of in vivo and in vitro cultured Heterorhabditis bacteriophora. Biological Control, 15(2), 113-118. https://doi.org/10.1006/bcon.1999.0701.
  • Hazir, S., Stock, S. P., Kaya, H. K., Koppenhöfer, A. M., & Keskin, N. (2001). Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae). Journal of Invertebrate Pathology, 77(4):243–250. https://doi.org/10.1006/jipa.2001.5029.
  • Hazir, S., Kaya, H. K., Stock, S. P., & Keskin, N. (2003). Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) for biological control of soil pests. Turkish journal of Biology, 27(4), 181-202.
  • Fitters, P. F., & Griffin, C. T. (2004). Spontaneous and induced activity of Heterorhabditis megidis infective juveniles during storage. Nematology, 6(6), 911-917. https://doi.org/10.1163/1568541044038597.
  • Kaya, H. K., & Stock, S. P. (1997). Techniques in insect nematology. In L. Lacey (Ed.), Manual of Techniques in Insect Pathology (pp. 281-324). Academic Press.
  • Kepenekci, I., Hazir, S., & Lewis, E. E. (2016). Evaluation of entomopathogenic nematodes and the supernatants of the in vitro culture medium of their mutualistic bacteria for the control of the root‐knot nematodes Meloidogyne incognita and M. arenaria. Pest management science, 72(2), 327-334. https://doi.org/10.1002/ps.3998.
  • Kour, S., Khurma, U., Brodie, G., & Singh, S. (2022). Modeling the potential global distribution of suitable habitat for the biological control agent Heterorhabditis indica. Ecology and Evolution, 12(6), e8997. https://doi.org/10.1002/ece3.8997.
  • Metwally, H. M., Hafez, G. A., Hussein, M. A., Hussein, M. A., Salem, H. A., & Saleh, M. M. E. (2012). Low cost artificial diet for rearing the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) as a host for entomopathogenic nematodes. Egyptian Journal of Biological Pest Control, 22(1), 15.
  • Mokrini, F., Laasli, S. E., Benseddik, Y., Joutei, A. B., Blenzar, A., Lakhal, H., Sbaghi, M., İmren, M., Özer, G., Paulitz, T., Lahlai, R., & Dababat, A. A. (2020). Potential of Moroccan entomopathogenic nematodes for the control of the Mediterranean fruit fly Ceratitis capitata Wiedemann (Diptera: Tephritidae). Scientific reports, 10(1), 1-11. https://doi.org/10.1038/s41598-020-76170-7.
  • Odendaal, D., Addison, M. F., & Malan, A. P. (2016). Entomopathogenic nematodes for the control of the codling moth (Cydia pomonella L.) in field and laboratory trials. Journal of Helminthology, 90(5), 615-623. https://doi.org/10.1017/s0022149x15000887.
  • Prabhuraj, A., Viraktamath, C. A., & Kumar, A. R. V. (2000). Modified trapping technique for the isolation of insect parasitic nematodes. Journal of Biological Control, 14(2) 83-85. https://doi.org/10.18311/jbc/2000/4169.
  • Selvan, S., Gaugler, R., & Lewis, E. E. (1993). Biochemical energy reserves of entomopathogenic nematodes. The Journal of parasitology, 79(2)167-172. https://doi.org/10.2307/3283503.
  • Singh, M., Rani, P., Prashad, H., & Nalini, C. (2023). Effect of storage media and temperature on viability and pathogenicity of North Indian populations of entomopathogenic nematodes. Journal of Entomological Research, 47(1), 209-214. https://doi.org/10.1007%2Fs12639-014-0639-8.
  • Sharmila, R., & Subramanian, S. (2016). Effect of low temperature on the activity of entomopathogenic nematodes. International Journal of Forestry and Crop Improvement, 7(1), 19-23. https://doi.org/10.15740/HAS/IJFCI/7.1/00-00.
  • Susurluk, I. A., Kumral, N. A., Peters, A., Bilgili, U., & Açıkgöz, E. (2009). Pathogenicity, reproduction and foraging behaviours of some entomopathogenic nematodes on a new turf pest, Dorcadion pseudopreissi (Coleoptera: Cerambycidae). Biocontrol Science and Technology, 19(6), 585-594. http://dx.doi.org/10.1080/09583150902957348.
  • Qiu, L. & Bedding, R. (2000). Energy metabolism and its relation to survival and infectivity of infective juveniles of Steinernema carpocapsae under aerobic conditions. Nematology 2, 551-559. http://dx.doi.org/10.1163/156854100509330.
  • Qiu, L., & Bedding, R. A. (2002). Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 131(4), 757-765. https://doi.org/10.1016/s1096-4959(02)00019-2.
  • Yadav, A. K. (2016). Effects of storage temperature on survival and infectivity of three indigenous entomopathogenic nematodes strains (Steinernematidae and Heterorhabditidae) from Meghalaya, India. Journal of Parasitic Diseases, (40), 1150-1154. https://doi.org/10.1007%2Fs12639-014-0639-8.
  • Yuksel, E., & Canhilal, R. (2019). Isolation, identification, and pathogenicity of entomopathogenic nematodes occurring in Cappadocia Region, Central Turkey. Egyptian Journal of Biological Pest Control, 29(1), 1-7. http://dx.doi.org/10.1186/s41938-019-0141-9.
Toplam 34 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Entomoloji
Bölüm Bitki Koruma
Yazarlar

Onur Akı 0000-0002-9724-8883

Ebubekir Yüksel 0000-0002-6982-5874

Mustafa İmren 0000-0002-7217-9092

Refik Bozbuğa 0000-0001-9201-5725

Ramazan Canhilal 0000-0002-0753-0077

Erken Görünüm Tarihi 14 Ağustos 2023
Yayımlanma Tarihi 21 Ağustos 2023
Gönderilme Tarihi 15 Mayıs 2023
Kabul Tarihi 14 Haziran 2023
Yayımlandığı Sayı Yıl 2023 Cilt: 9 Sayı: 2

Kaynak Göster

APA Akı, O., Yüksel, E., İmren, M., Bozbuğa, R., vd. (2023). The Role of Storage Duration and Conditions on the Survival and Pathogenicity of Entomopathogenic Nematodes. International Journal of Agricultural and Wildlife Sciences, 9(2), 176-185. https://doi.org/10.24180/ijaws.1297111

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