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Characterization of Bacillus thuringiensis isolat from several areas in Java and South Sulawesi for biological control of myasis, Chrysomya bezziana Muharsini, S; Wardhana, April H; Rijzaani, H; Amirhusein, B
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 4 (2003)
Publisher : Indonesian Animal Sciences Society

Bacillus thuringiensis is a species of bacteria which has potential use for biological control of parasite. The aim of the study is to characterize the local isolate of B. thuringiensis using PCR technique. Primer of cry gen was used in the study. Two hundred and twenty seven soil samples were collected from several areas in Java Island and South Sulawesi Province. Samples were obtained from surrounding soils of animal housing and paddocks. Samples were inoculated in Nutrient Agar and the suspect of bacilli were then cultured in Luria Bertani acetic buffer. Seventy-three pure isolates of Bacillus thuringiensis were obtained. Thirty-five isolates from Bogor, Yogyakarta and Majalengka regencies were amplified using PCR and consisted of a B. thuringiensis sporulation gene which has a 850 bp band in an agarose gel. Amplification using cryIVA,B primers demonstrated four isolates contain a faint band of 800 bp and 12 isolates consist of a distinctive band of 300-400 bp. The results showed that several B. thuringiensis isolates containing a cry IV gen and those isolates are needed to be tested as a biological control agent for Chrysomya bezziana. Key words: Bacillus thuringiensis, Chrysomya bezziana, biological control, cry IV gen

Reliable preservation methods of mitochondrial DNA analysis for larvae and adult flies of Chrysomya bezziana (Diptera: Calliphoridae). Wardhana, April H; Muharsini, S; ., Suhardono
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 4 (2003)
Publisher : Indonesian Animal Sciences Society

The Old World Screwworm fly, Chrysomya bezziana is known as a myiasis agent in Indonesia. The aim of the study is to establish a reliable preservation methods for molecular analysis of insect. Cytochrom b (CB) and Cytochrome Oxidase sub unit I (COI) of mitochondrial DNA were used as markers. Drying method was used to preserve adult flies, while the larvae were kept in 10% formalin and 80% ethanol with or without soaked using hot water prior preservation. Two samples of each preservation were tested. Four primers were used to amplify samples using Polymerase Chain Reaction (PCR). The PCR product were then purified and sequenced. Sequence data analysis of all samples suggested no differences between all preservation methods except using 10% formalin. Preservation samples using 10% formalin is not suitable for collecting mitochondrial DNA of insect. The best methods was using 80% ethanol with killed using hot water prior preservation. This method was able to extract a stabile mitochondrial DNA. Â Key words: Chrysomya bezziana, preservation, formalin, ethanol, mitochondrial DNA

diversity of myiasis fly, Chrysomya bezziana population in Indonesia based on mitochondrial DNA Wardhana, April H; Muharsini, S; Asmara, Widya
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 2 (2004)
Publisher : Indonesian Animal Sciences Society

Chrysomya bezziana is a fly causing myiasis in most livestock in Indonesia. To date, the genetic diversity of C. bezziana has been argued among researchers. The aim of this study was to analyse genetic diversity of C. bezziana population in Indonesia using mitochondrial cytochrome b gene (cyt b) as a marker. The preliminary study showed that the larvae instar III stadium (L3) was the most appropriate sample for molecular analysis and identification. Twenty-four L3 were collected from cattles, buffalos and horses in Bogor, Makassar and East Sumba. DNA samples were isolated from muscle tissue of the larvae. The fragment of cyt b gene (279 bp) was amplified using primer CB3FC-NINFA. The PCR product was subsequently purified, sequenced and analysed using PAUP version 4. The results showed that C. bezziana from Indonesia was different with those from Asia, basicaly for DNA sequences, however it had similar to those from Papua New Guinea. Two haplotypes were identified i.e. haplotype 6 (Makassar haplotype that similar to those from Papua New Guinea) and haplotype 7 (Bogor haplotype that similar to those from Makassar and East Sumba). Key words: Chrysomya bezziana, genetic diversity, mitochondrial DNA

icacy test of hexane extract of Annona squamosa L seeds for Chrysomya bezziana larvae growth in vitro Wardhana, April H; Widyastuti, E; Wiratmana, A.W.A; Muharsini, S; ., Darmono
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 4 (2004)
Publisher : Indonesian Animal Sciences Society

Chrysomya bezziana is primer agent causing myiasis in livestock and human throughout Africa to Asia. At present, treatment of myiasis using chemical synthetic insecticide causing environment and livestock production contaminations. The aim of this study was to evaluate the effect of hexane extract of Annona squamosa L seed to kill C. bezziana as botanical insecticide. Three level and method preparation of larvae (L1, L2 and L3) were carried out. Meat blood mixture (MBM) and larval rearing media (LRM) were mixed with hexane extract of A. squamosa L seed and tested to 625 and 750 larvae (L1and L2 respectively), while for L3 625 larvae were soaked in the solution containing the hexane extract. This last preparation was carried out to observe the effect of toxic contact. Each larvae treatment was divided into 5 levels such as negative control using distillation water (PO), given 0.25% hexane extract (P I); 0.50% (P II); 0.75% (P III) and positive control was given with 0.10% AsuntolÂ®. This assay was to know digest toxic effect of hexane extract of A. squamosa L seed for those larvae. Number of 625 instar III larvae (L3) was soaked in the solution containing the hexane extract of A. squamosa L seed for 10 second, placed into vermicullite and incubated on 36oC. This assay was to know contact toxic effect of them. All of larvae were allowed to become pupae. Parameters oberserve were number of larvae death, pupae weight and number of pupae become into fly. The data was analyzed using Anova (5%) and Z test (5%) then smallest significant difference test (BNT 5%). The results showed that 0.50% of hexane extract of A. squamosa L seed was able to decrease pupae weight for L1 and L2 and to cause fail pupae become fly (P<0.05). Instar III larvae (L3) soaked in PI until P III and not effect to pupae weight, pupae become fly and survival of adult flies (P>0.05). Â Key words: Chrysomya bezziana, myasis, srikaya, A. squamosa L, in vitro

In vitro study of larvacidal effect of Chrysomya bezziana between atsiri oil (Piper betle leaf) from Sri Lanka and Bogor Wardhana, April H; Muharsini, S; Santosa, S; Arambewela, L.S.R; Kumarasinghe, S.P.W
Indonesian Journal of Animal and Veterinary Sciences Vol 15, No 4 (2010)
Publisher : Indonesian Animal Sciences Society

Treatment of myiasis caused by the larvae of Chrysomya bezziana resulted variation results. The aim of the experiment are to compare larvacidal effect of atsiri oil of Piper betle leaf originated from Sri Lanka and Bogor agains Chrysomya bezziana larvae and to identify atsiri oil using Thin Layer Chromatography (TLC).Â Silica gel plate GF254 with toluena and ethyl ecetate as diluents was used in TLC. L1 and L2 of Chrysomya bezziana larvae were used for in vitro assay using agar plate contained atsiri oil of three concentrations ie. 2, 3 and 4%. Asuntol 1% and aquadest sterile were used as positive and negative control, respectively. Ten larvae were used in five replicates. The larvae were observed every 30 minutes for four hours. Larval mortality were counted and probit analysed using POLO-PC software, therefore the lethal concentration (LC50 and LC95) and lethal time (LT50 and LT95) were defined. The results showed there was a difference on migration distance between atsiri oil from Sri Lanka and Bogor on nRf 79-88 analysed using TLC. In vitro assay showed that both lethal concentration and lethal time of atsiri oil from Sri Lanka was higher than from Bogor. This results indicated that larvacidal effect of atsiri oil from Bogor was stronger than atsiri oil from Sri Lanka in in vitro both on L1 and L2. Key Words: Piper Bitle Leaf, Atsiri Oil, Chrysomya bezziana, Sri Lanka, Bogor

Geographical characteristics of Chrsyomya bezziana based on external morphology study Wardhana, April H.; Muharsini, S; Ready, P.D; Cameron, M.M; Hall, M.J.R
Indonesian Journal of Animal and Veterinary Sciences Vol 17, No 1 (2012)
Publisher : Indonesian Animal Sciences Society

Correct identification of Chrysomya bezziana is a fundamental step to evaluate the successfulness of the eradication program based on Sterile Insect Techinque (SIT). However, geographical variation of the fly is being controversial among scientists. The aim of the study was to investigate the effect of preservation method on visualisation of characters of external morphology and to analyse geographical variation of C. bezziana populations throughout their distribution regions. A total of 88 flies collected from 7 populations in Indonesia, 2 populations in Africa and each 1 population from Oman, India, Malaysia and Papua New Guinea (PNG) were tested in the study. All larvae were removed from natural myiasis cases. The larvae were reared at laboratory until they became adult flies. The samples were preserved into two methods, wet (80% ethanol) and dried (pin) methods. Ten external characters of head and body were observed.Â Data were subjected to principal components and hierarchical cluster analyses in UNISTATÃ software. The Euclid distance measure was used for the cluster analysis, and the linking method used was the unweighted pair-group method using arithmetic average (UPGMA), to compute the distance between clusters. Results indicated that dried preservation (pinned samples) provided better external character than ethanol preservation. Based on external morphology of adult stages revealed that C. bezziana occurs as two geographical races, African and Asian races. There was no geographical variation of C. bezziana throughout Indonesian archipelago, except for the population from PNG forming its own cluster. Key Words: Myiasis, Chrysomya bezziana, Morphology, Geographical Variaton

In vitro study of larvacidal effect of Chrysomya bezziana between atsiri oil (Piper betle leaf) from Sri Lanka and Bogor April H Wardhana; S Muharsini; S Santosa; L.S.R Arambewela; S.P.W Kumarasinghe
Jurnal Ilmu Ternak dan Veteriner Vol 15, No 4 (2010): DECEMBER 2010
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Treatment of myiasis caused by the larvae of Chrysomya bezziana resulted variation results. The aim of the experiment are to compare larvacidal effect of atsiri oil of Piper betle leaf originated from Sri Lanka and Bogor agains Chrysomya bezziana larvae and to identify atsiri oil using Thin Layer Chromatography (TLC). Silica gel plate GF254 with toluena and ethyl ecetate as diluents was used in TLC. L1 and L2 of Chrysomya bezziana larvae were used for in vitro assay using agar plate contained atsiri oil of three concentrations ie. 2, 3 and 4%. Asuntol 1% and aquadest sterile were used as positive and negative control, respectively. Ten larvae were used in five replicates. The larvae were observed every 30 minutes for four hours. Larval mortality were counted and probit analysed using POLO-PC software, therefore the lethal concentration (LC50 and LC95) and lethal time (LT50 and LT95) were defined. The results showed there was a difference on migration distance between atsiri oil from Sri Lanka and Bogor on nRf 79-88 analysed using TLC. In vitro assay showed that both lethal concentration and lethal time of atsiri oil from Sri Lanka was higher than from Bogor. This results indicated that larvacidal effect of atsiri oil from Bogor was stronger than atsiri oil from Sri Lanka in in vitro both on L1 and L2. Key Words: Piper Bitle Leaf, Atsiri Oil, Chrysomya bezziana, Sri Lanka, Bogor

Geographical characteristics of Chrsyomya bezziana based on external morphology study April H. Wardhana; S Muharsini; P.D Ready; M.M Cameron; M.J.R Hall
Jurnal Ilmu Ternak dan Veteriner Vol 17, No 1 (2012): MARCH 2012
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Correct identification of Chrysomya bezziana is a fundamental step to evaluate the successfulness of the eradication program based on Sterile Insect Techinque (SIT). However, geographical variation of the fly is being controversial among scientists. The aim of the study was to investigate the effect of preservation method on visualisation of characters of external morphology and to analyse geographical variation of C. bezziana populations throughout their distribution regions. A total of 88 flies collected from 7 populations in Indonesia, 2 populations in Africa and each 1 population from Oman, India, Malaysia and Papua New Guinea (PNG) were tested in the study. All larvae were removed from natural myiasis cases. The larvae were reared at laboratory until they became adult flies. The samples were preserved into two methods, wet (80% ethanol) and dried (pin) methods. Ten external characters of head and body were observed. Data were subjected to principal components and hierarchical cluster analyses in UNISTATÒ software. The Euclid distance measure was used for the cluster analysis, and the linking method used was the unweighted pair-group method using arithmetic average (UPGMA), to compute the distance between clusters. Results indicated that dried preservation (pinned samples) provided better external character than ethanol preservation. Based on external morphology of adult stages revealed that C. bezziana occurs as two geographical races, African and Asian races. There was no geographical variation of C. bezziana throughout Indonesian archipelago, except for the population from PNG forming its own cluster. Key Words: Myiasis, Chrysomya bezziana, Morphology, Geographical Variaton

Characterization of Bacillus thuringiensis isolat from several areas in Java and South Sulawesi for biological control of myasis, Chrysomya bezziana S Muharsini; April H Wardhana; H Rijzaani; B Amirhusein
Jurnal Ilmu Ternak dan Veteriner Vol 8, No 4 (2003): DECEMBER 2003
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Bacillus thuringiensis is a species of bacteria which has potential use for biological control of parasite. The aim of the study is to characterize the local isolate of B. thuringiensis using PCR technique. Primer of cry gen was used in the study. Two hundred and twenty seven soil samples were collected from several areas in Java Island and South Sulawesi Province. Samples were obtained from surrounding soils of animal housing and paddocks. Samples were inoculated in Nutrient Agar and the suspect of bacilli were then cultured in Luria Bertani acetic buffer. Seventy-three pure isolates of Bacillus thuringiensis were obtained. Thirty-five isolates from Bogor, Yogyakarta and Majalengka regencies were amplified using PCR and consisted of a B. thuringiensis sporulation gene which has a 850 bp band in an agarose gel. Amplification using cryIVA,B primers demonstrated four isolates contain a faint band of 800 bp and 12 isolates consist of a distinctive band of 300-400 bp. The results showed that several B. thuringiensis isolates containing a cry IV gen and those isolates are needed to be tested as a biological control agent for Chrysomya bezziana. Key words: Bacillus thuringiensis, Chrysomya bezziana, biological control, cry IV gen

Reliable preservation methods of mitochondrial DNA analysis for larvae and adult flies of Chrysomya bezziana (Diptera: Calliphoridae). April H Wardhana; S Muharsini; Suhardono .
Jurnal Ilmu Ternak dan Veteriner Vol 8, No 4 (2003): DECEMBER 2003
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The Old World Screwworm fly, Chrysomya bezziana is known as a myiasis agent in Indonesia. The aim of the study is to establish a reliable preservation methods for molecular analysis of insect. Cytochrom b (CB) and Cytochrome Oxidase sub unit I (COI) of mitochondrial DNA were used as markers. Drying method was used to preserve adult flies, while the larvae were kept in 10% formalin and 80% ethanol with or without soaked using hot water prior preservation. Two samples of each preservation were tested. Four primers were used to amplify samples using Polymerase Chain Reaction (PCR). The PCR product were then purified and sequenced. Sequence data analysis of all samples suggested no differences between all preservation methods except using 10% formalin. Preservation samples using 10% formalin is not suitable for collecting mitochondrial DNA of insect. The best methods was using 80% ethanol with killed using hot water prior preservation. This method was able to extract a stabile mitochondrial DNA. Key words: Chrysomya bezziana, preservation, formalin, ethanol, mitochondrial DNA

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