Identification of sex linked molecular markers in Indonesian giant freshwater prawn Macrobrachium rosenbergii

Male giant freshwater prawn grows faster than its female. Therefore, male mono sex culture is one of the solutions to improve aquaculture production. The all-male population of giant freshwater prawns can be produced by mating the neo-females (sex-reversed males) with the normal males. This study was aimed to identify the molecular markers related to the giant freshwater prawn sex. Specific primers were designed based on female-specific AFLP marker sequences to distinguish male and female sex on the prawns. Three locations for obtaining the Indonesian prawns in this study were Aceh, Sukabumi, and Solo. Based on the PCR analysis with MrMKn primers, 30 samples of female prawns had 100 % occurred DNA bands, while no DNA bands were obtained in all-male prawns from Solo. Nevertheless, MrMKn primers still detected 10–16 % male prawns from Sukabumi and Aceh. This indicated that MrMKn primers could not yet distinguish the male prawns for all populations. Moreover, the results suggested that the three prawn samples were different based on female-specific gene sequence. The MrMKn primers have the opportunity to be used in the selection of the female ZZ (neo-female) prawns from Solo without progeny test, so that the determination of female ZZ candidates can be identified more quickly. However, the primer still needs to be redesigned to distinguish neo-female prawns from Sukabumi and Aceh.   Keyword: giant freshwater prawn, mono sex, neo-female, sex markers   ABSTRAK   Udang galah jantan lebih cepat tumbuh dibandingkan dengan betinanya sehingga budidaya udang galah monoseks jantan menjadi salah satu solusi untuk meningkatkan produksi budidaya. Populasi monoseks jantan udang galah dapat dihasilkan dengan mengawinkan neofemales (sex-reversed males) dengan jantan normal. Sistem kromosom pada udang galah berbeda dengan ikan. Individu betina bersifat heterogametik (WZ) dan jantan homogametik (ZZ). Dalam perkembangannya, terdapat kendala dalam menentukan individu neofemale yang memiliki kromosom ZZ. Berdasarkan pendekatan sistem kromosom tersebut, maka dapat dijadikan acuan untuk membuat marka molekuler terkait kelamin udang galah. Penelitian ini bertujuan mengidentifikasi marka molekuler terkait jenis kelamin pada udang galah. Primer spesifik didesain berdasarkan sekuen female specific AFLP marker untuk membedakan kelamin jantan dan betina pada udang galah. Tiga sumber udang galah digunakan dalam penelitian ini, yaitu Aceh, Sukabumi, dan Solo. Berdasarkan hasil analisis PCR dengan primer MrKNn, dari 30 sampel pada kelompok udang galah betina diperoleh hasil 100% pita DNA muncul, dan tidak terdapat pita DNA pada semua udang galah jantan asal Solo. Namun demikian, primer MrMKn tersebut masih mendeteksi sebesar 10–16% pada udang galah asal Sukabumi dan Aceh. Hal ini menunjukkan bahwa primer MrMKn belum dapat membedakan udang galah jantan dari semua populasi. Selain itu, dapat dikatakan bahwa ketiga udang galah uji adalah berbeda, khususnya sekuen gen spesifik betina. Primer MrMKn berpeluang digunakan dalam proses seleksi udang galah betina ZZ (neofemale) asal Solo tanpa harus melalui uji progeni sehingga penentuan kandidat betina ZZ lebih cepat teridentifikasi. Akan tetapi, primer masih perlu didesain ulang untuk membedakan neofemale asal Sukabumi dan Aceh.   Kata kunci: marka kelamin, monoseks, neo-female, udang gala

Genetic Characterization of Domesticated F1 Generation in Humpback Grouper (Cromileptes altivelis)

First generation (F1) of hatchery produced humpback grouper (Cromileptes altivelis) has been characterized genetically in order to serve the information of their status in related to their breeding strategy. PCR-RFLP method was used to detect the variation of mtDNA D-loop region of F1 population at BBPBL Lampung and BBAP Situbondo. The result of study showed that reducing of haplotype diversity had been arised from broodstock (0.8548) to F1 generation population (0.7473; 0.7273; and 0.6947, respectively).  Genetic divergence that had found between population BBPBL Lampung and BBAP Situbondo make it possible to do outbreeding in order to get its heterosis's effect. Keywords: mtDNA, haplotype diversity, genetic differentiation, Cromileptes altivelis   ABSTRAK Ikan kerapu tikus (Cromileptes altivelis) generasi pertama (F1) hasil domestikasi di hatchery telah dikarakterisasi secara genetik untuk menyediakan informasi status sehubungan dengan program pemuliaannya.  Metode PCR-RFLP digunakan untuk mendeteksi variasi sekuens D-loop mtDNA ikan kerapu tikus F1 yang diproduksi di BBPBL Lampung dan BBAP Situbondo.  Hasil penelitian menunjukkan bahwa telah terjadi penurunan keragaman haplotipe dari induk (0,8548) ke populasi generasi F1 (masing-masing 0,7473; 0,7273; dan 0,6947).  Adanya keragaman genetik antara populasi ikan kerapu tikus di BBPBL dan BBAP Situbondo memungkinkan dilakukannya outbreeding untuk mendapatkan efek heterosis. Kata kunci: mtDNA, keragaman haplotipe, diferensiasi genetik, Cromileptes altiveli

Genetic variability of the fifth generation of nile tilapia Oreochromis niloticus using microsatellite DNA markers

ABSTRACTFifth generations of Nile tilapia from several strains have been produced by using selective breeding program in Main Centre for Freshwater Aquaculture Development (MCFAD) Sukabumi, West Java. This research was aimed to evaluate the impact of family selection program of some highly economic traits on its genetic variability using microsatellite DNA markers. The total of 180 specimens have been collected from fifth generation of nine reciprocal mating between three families selected from fourth generation of Nile tilapia and were screened for genetic variability at three microsatellite loci (UNH 123*, UNH 172*, UNH 216*). The results showed that the amount of genetic variability on fifth generations of Nile tilapia from three strains was ranged between 33 to 100% and the highest genetic distance relationship between families was 0.3875. This research approved that females and males issued from the family which have more amount of genetic variability and higher distance to others could be considered as genetic materials to produce the next generation.Keywords: microsatellite DNA, genotype, genetic variability, genetic distance, Oreochromis nilotiocus ABSTRAKBeberapa strain ikan nila generasi kelima telah dihasilkan dalam program pemuliaan di Main Centre untuk Freshwater Aquaculture Development (MCFAD) Sukabumi, Barat Jawa. Riset ini bertujuan untuk mengevaluasi pengaruh seleksi famili terhadap performa karakter ekonomis penting berdasarkan keragaman genetiknya menggunakan penanda microsatellite DNA. Spesimen dari 180 individu generasi kelima hasil persilangan resiprokal antara tiga famili generasi keempat dianalisis dengan penanda tiga microsatellite loci (UNH 123*, UNH 172*, UNH 216*). Hasil penelitian menunjukkan bahwa keragaman genetik ikan nila generasi kelima berkisar antara 33 sampai 100% dan hubungan kekerabatan genetik antar famili yang paling jauh adalah 0,3875. Individu betina dan jantan yang berasal dari famili dengan tingkat keragaman genetik dan kekerabatan yang lebih tinggi dapat dipertimbangkan sebagai sumber genetik berkualitas untuk menghasilkan generasi berikutnya.Kata kunci: microsatellite DNA, genotipe, keragaman genetik, jarak genetik, Oreochromis nilotiocu

COMPARISON OF THREE DIFFERENT TECHNIQUES OF GENE TRANSFER IN HUMPBACK GROUPER ( ) CROMILEPTES ALTIVELIS

Humpback grouper is one of the most cultured fishes in Asia, including Indonesia. Themain problemfaced by humpback culture is its slow growth rate.One of themethods that willbe more effective and efficient to solve the problem is using transgenic technique. This studywas conducted to determine the effectiveness of transfection,microinjection and electroporationtechniques on gene transfer in humpback grouper. transfection was performed byincubating sperm to the foreign DNA (pktBP-ktGH gene construct)-transfectant complexsolution, while was by injecting those complex solution into testis of mature males.Microinjection was conducted in 2-4 cell stage embryos using 25 μg/ml of foreign DNAsolution, and duration of injection was 1, 2 and 3 seconds. Electroporation by 50 V, 30 ms ofpulse length, 5 of pulse number and 0.1 of pulse interval was performed to sperm using threeDNA concentration of 5, 10 and 20 μg/ml. The incorporation of foreign DNA in sperm andembryos were analyzed using PCR method. Based on PCR analysis, an optimum DNAconcentration for electroporation was 10 μg/ml. Limited number of embryos could bemicroinjected during 20-30 min to reach 2-4 cell stage. Microinjection for 1 second showedhigher survival rate of embryos, although none or very low number of larvae was hatched.Transfast was an effective DNA delivery reagent for humpback grouper sperm. Foreign DNAcould be detected in sperm from two out of ten transfected fish at least 36 hours posttransfection (hpt). By transfection, foreign DNA was detected in sperm at 48 hpt 25 Cincubation temperature. Our study revealed that transfection, microinjection as well aselectroporation could be used as transgenesis methods in humpback grouper. By means ofsimplicity and efficacy, however, electroporationwas an appropriate gene transfermethod.oIn vitroin vivoin vivoin vitr

PRODUKSI PROTEIN REKOMBINAN HORMON PERTUMBUHAN IKAN KERAPU

Salah satu spesies ikan yang menjadi target produksi perikanan budidaya nasional adalah ikan kerapu tikus (Cromileptes altivelis). Ikan kerapu tikus merupakan ikan laut budidaya komoditas ekspor, namun laju pertumbuhannya sangat lambat. Oleh karena itu, perlu dilakukan usaha budidaya yang mampu menaikkan laju pertumbuhan ikan kerapu tikus. Pendekatan nutrisi melalui penggunaan hormon pertumbuhan (Growth Hormone, GH) pada usaha budidaya diyakini mampu meningkatkan kecepatan tumbuh ikan budidaya. Pada penelitian ini, melalui teknologi DNA rekombinan telah dilakukan isolasi dan identifikasi gen GH yang selanjutnya dilakukan produksi protein rekombinan GH (recombinant Growth Hormone, rGH) dengan memanfaatkan penggunaan bakteri E. coli. Dalam pelaksanaan penelitian dilakukan konstruksi rGH yang menghasilkan bakteri E. coli BL21 (DE3) yang mampu memproduksi protein rGH. Produksi rGH dilakukan pada skala bioreaktor. Proses isolasi produk rGH-nya dalam bentuk pellet inclusion bodies yang selanjutnya dicampur dengan pelet pakan komersil hingga konsentrasi akhir protein dalam pakan mencapai 1 ng/6 mg pakan, di mana setelah dikering-anginkan, pelet pakan protein rekombinan GH dapat diaplikasikan untuk budidaya ikan kerapu tikus dan ikan budidaya lainnya

ISOLASI DAN KARAKTERISASI PROMOTER β-ACTIN DARI IKAN KERAPU BEBEK (Cromileptes altivelis)

Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasilan transgenesis.  Penelitian isolasi dan karakterisasi promoter β-actin (ktBA) dari ikan kerapu bebek (Cromileptes altivelis) dalam rangka pembuatan ikan kerapu autotransgenik telah dilakukan. Promoter β-actin memiliki aktivitas tinggi pada jaringan otot. Sekuens promoter ktBA diisolasi menggunakan metode degenerate PCR. Sekuensing dilakukan menggunakan mesin ABI PRISM 3100. Analisis sekuens menggunakan software BLAST, GENETYX versi 7 dan TFBind. Fragment DNA hasil amplifikasi PCR yang dipotong dari vektor kloning selanjutnya diligasi dengan pEGFPN1 untuk membuat konstruksi pktBA-EGFP. Konstruksi pktBA-EGFP dimikroinjeksi ke embrio ikan zebra (Danio rerio) fase 1 sel untuk menguji aktivitas promoter ktBA. Ekspresi gen EGFP diamati menggunakan mikroskop fluoresens. Analisis sekuens menunjukkan bahwa panjang fragmen DNA hasil amplifikasi PCR sekitar 1,6 kb dan memiliki faktor transkripsi yang conserved pada promoter β-actin, yaitu CCAAT, CArG dan boks TATA. Selanjutnya, sekuens ktBA dalam konstruksi pktBA-EGFP mampu mengendalikan ekspresi gen EGFP pada jaringan otot embrio ikan zebra yang dimikroinjeksi dengan konstruksi tersebut. Dengan demikian dapat disimpulkan bahwa fragmen DNA hasil amplifikasi PCR tersebut merupakan sekuens promoter β-actin ikan kerapu bebek. Pembuatan ikan kerapu autotransgenik selanjutnya dapat dilakukan dengan mengganti gen EGFP pada pktBA-EGFP dengan gen-gen asal ikan kerapu yang mengkodekan karakter penting dalam budi daya ikan.Promoter as gene expression regulator is one of the factors affecting the successful of transgenesis. Isolation and characterization of β -actin promoter (ktBA) from humpback grouper (Cromileptes altivelis) towards generation of autotransgenic grouper have been conducted.  β -actin promoter has high activity in muscle. Sequence of ktBA promoter was isolated by using degenerate PCR method. Sequencing was performed using ABI PRISM 3100 machine. Analysis of sequences was conducted using BLAST, GENETYX version 7 and TFBind softwares. DNA fragment of PCR amplification product digested from the vector cloning was then ligated with pEGFPN1 to generate pktBA-GFP construct. The construct was microinjected into one-cell stage of zebrafish (Danio rerio) embryos to test the ktBA promoter activity. EGFP gene expression was observed by fluorescence microscope. The result of sequence analysis showed that the length of DNA fragment obtained is about 1.6 kb and containing the evolutionary conserved sequences of transcription factor for β -actin promoter including CCAAT, CArG and TATA boxes. Furthermore, ktBA sequence in pktBA-EGFP construct could drove GFP expression in muscle of zebrafish embryos injected with the construct. The results suggested that PCR amplification product is the regulator sequence of humpback grouper β -actin gene. Autotransgenic grouper can be then produced by changing GFP gene fragment of pktBA-EGFP construct with genes from grouper encoding important traits in aquaculture

ECONOMIC VALUATION AND THE DETERMINANTS OF DEMAND FACTORS OF BANDUNG STRAWBERRY AGROTOURISM, WEST JAVA, INDONESIA

Strawberry agrotourism is the most extensive strawberry farming in Bandung, West Java. This agrotourism has the potential for nature, tourism, conservation, and regional economic potential. This research aims to identify the characteristics and assessment of visitors, identify factors influencing tourism demand, and estimate the value of the agrotourism economy. The economic value is analyzed using the method of TCM (Travel Cost Method). This method is calculated based on total consumer surplus, travel cost coefficient, and respondent visits. The average travel cost incurred by 379 respondents during the trip is IDR. 413,699, and the total cost of the entire journey is IDR.156,792,000. The surplus of consumer visits is IDR. 12,670, and the individual consumer surplus visits are IDR.33. The economic value generated for one year is IDR. 237,600. The factors that influence the demand are income, distance, and time to know the location. At the same time, the factors that do not affect the market are travel cost, education, number of dependents in the family, gender, and age of visitors

PERFORMA IKAN NILA (Oreochromis niloticus) HASIL SEX REVERSAL, GENETICALLY MALE DAN YY PADA FASE PENDEDERAN PERTAMA

Penelitian ini dilakukan untuk mengkaji performa ikan nila hasil sex reversal (SRV), genetically male tilapia (GMT), dan YY pada fase pendederan pertama di akuarium. Benih ikan dipelihara selama 22 hari, dari umur 6 hari hingga 28 hari. Parameter yang diamati meliputi tingkat sintasan, persentase ikan jantan, laju pertumbuhan, dan biomassa. Hasil penelitian menunjukkan bahwa tingkat sintasan tidak berbeda (P>0,05) antar ketiga kelompok ikan dan kontrol (KN), berkisar antara 85,30%--86,20%. Persentase ikan jantan antara SRV (94,5% ± 1,32%) vs. GMT (93,8% ± 1,25%) dan GMT vs. YY (90,2% ± 1,83%) tidak berbeda (P>0,05), sedangkan antara SRV lebih tinggi daripada YY (P<0,05). Persentase ikan jantan pada ketiga kelompok ikan tersebut lebih tinggi (P<0,05) dibandingkan dengan KN (56,9% ± 3,62%). Pertumbuhan ikan YY dan GMT lebih cepat (P<0,05) dibandingkan dengan ikan SRV dan kontrol (KN). Bobot rata-rata ikan YY pada akhir penelitian mencapai 485 mg, ikan GMT 456 mg, ikan SRV 379 mg dan kontrol 342 mg. Produksi biomassa ikan YY, GMT, dan SRV masing-masing sebesar 41,3%; 32,9%; dan 10,3% lebih tinggi dibandingkan dengan KN. Dengan performa yang tinggi dan pertimbangan teknis di lapangan, benih GMT merupakan alternatif yang baik untuk dibudidayakan dalam rangka meningkatkan produksi ikan nila.The experiment was conducted to determine the performance of sex reversed (SRV), genetically male tilapia (GMT), and YY tilapia on first nursery phase in aquarium. Fry were reared for 22 days, from 6 to 28 days-old. Survival rate, percentage of male fish, growth rate and biomass were observed. The result of the study showed that survival rate among fish group and control were similar (P>0.05), ranged from 85.30%-86.20%. Percentage of male fish between SRV (94.5% ± 1.32%) versus GMT (93.8% ± 1.25%) and GMT versus YY (90.2% ± 1.83%) were also similar (P>0.05), while SRV is higher than YY (P<0.05). Percentage of male fish in the three fish groups was higher than that of control (56.9% ± 3.62%). Growth of YY fish and GMT were higher compared to SRV and control fish (KN). The mean weight of YY fish at the end of the experiment reached 476 mg, GMT fish 447 mg, SRV fish 379 mg and control 342 mg. Biomass of YY, GMT and SRV fish were respectively higher by 41.3%, 32.9%, and 10.3% compared to control. With high performance and technical consideration in farm, GMT fish can be a potential alternative to be cultured in fish farm in order to increase aquaculture production of nile tilapia

Sustainable aquaculture to improve productivity and water quality of marginal brackishwater pond

An Integrated Multi Trophic Aquaculture (IMTA) technology by using blacktiger shrimp, tilapia, algae/seaweed (Gracillaria sp.) and oyster green mussel (Pernapiridis sp.) that are cultivated integrately in one pond has demonstrated high productivity on the biomass and environmental stability of water quality compared to the monoculture and polyculture without algae and benthic organism. The availability of seaweed and benthic organism in an integrated aquaculture system have contributed on the improving water quality by reducing DIN and increasing DO concentration. In the future, developing aquaculture models using the biorecycle system to reduce and minimize the inorganic and organic waste from the remaining feed, faeces and the other sources will be useful to maintain sustainable aquaculture in the coastal area. Particularly when supported by coastal communities involvement as denoted on the Sato Umi-GEMPITA-SPL/SFiCoMS concept, it will be successful