Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

RNA interference is a mechanism for silencing specific genes. It has been applied in cell culture to inhibit expression of genes involved in disease including viral genes as recently shown for the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus or VHSV (Bohle et al., 2011). But evidence of specific siRNA inhibition in living fish is still needed. Using the small interfering RNAs (siRNAs), messenger RNA (mRNA) can be targeted resulting in degradation of targeted transcript or translational repression. Reporter genes such as luciferase and green fluorescence protein (GFP) can be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human embryonic kidney HEK293t cells was used because they are easy to transfect and generally show high expression of transfected genes. Various types of fish including albino trouts and transparent fish were used as animal models to get better visualization of reporter gene expression. High variability of reporter gene expression was found between individual fish but it seems that in glass catfish, siRNAs are able to reduce reporter gene expression in the muscle showing that it is possible to use siRNA as technology to target genes locally in living fish. In parallel experiments, which will not be reported here, we examine the delivery of siRNAs using pharmacological formulations in order to achieve systemic delivery and knock down effect

DNA Barcoding of Neolissochilus sumatranus and Tor douronensis to Support In Situ Conservation of Indonesian Mahseers

The fishes of genera Neolissochilus and Tor are locally known as Ikan Batak or ihan in North Sumatra. They are sacred and are usually served in Batak tribe traditional ceremonies. However, their population in the wild is declining because of habitat degradation, unwise exploitation, water pollution, and the introduction of invasive species. Therefore, suitable management is needed to maintain Ikan Batak's sustainability in their natural habitat, which can be conducted through in situ conservation. The habitat of Ikan Batak in the Bonan Dolok river of the Samosir Regency can potentially be developed as a conservation site. Information concerning this species in Bonan Dolok River is needed as a basis for effective in situ conservation management. Morphologically identified fish captured in Bonan Dolok River were validated using DNA barcoding of cytochrome oxidase I (COI) mitochondrial DNA segment. This study confirmed that Ikan Batak captured from Bonan Dolok River belongs to two genera, Neolissochilus and Tor. Furthermore, the phylogenetic tree construction using Maximum Likelihood showed that the specimens identified as N. sumatranus were identical to N. cf soroides, a species found in the Malaysian peninsula, Thailand, and Cambodia. &nbsp

In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important

Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine but the induction of non-specific immune responses following their delivery continues to be a serious problem. With the purpose of avoiding such effects chemically modified siRNAs are tested in screening assay but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate antiviral effect of siRNAs is functionally monitored as reduced mortality in challenge studies involving an interferon sensitive virus. Modifications with locked nucleic acid (LNA), altritol nucleic acid (ANA) and hexitol nucleic acid (HNA) reduced the antiviral protection in this model indicative of altered immunogenicity. For LNA modified siRNAs, the number and localization of modifications in the single strands was found to be important and a correlation between antiviral protection and the thermal stability of siRNAs was found. The previously published sisiRNA will in some sequences, but not all, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo

Distributional mapping and impacts of invasive alien fish in Indonesia : an alert to inland waters sustainability

Inland water ecosystems in Indonesia face an increasing threat of alien and invasive alien fish species. Their existence have been found in Indonesian waters since before 1900, and their introduction is rapid and ongoing. In this review, we mapped the distribution of alien and invasive alien fish in different inland waters ecosystem types and islands in Indonesia, based on available literature indexed by Google Scholar. Impacts of invasive alien fish and how to control them are also discussed. This data mining exercise resulted in the successful mapping of alien and invasive alien fish in 72 lakes and 57 river systems, covering 28 provinces. A total of 50 species have spread with different compositions in each type of ecosystem and island. Oreochromis niloticus is the most commonly encountered alien species. Moreover, invasive cichlid fish are most frequently found in lakes, while Pterygoplichthys spp. are more frequently found in rivers although they can also inhabit many lakes. Java Island is a hotspot of alien and invasive alien fish distribution, with 39 species reported. Invasive alien fish harm native and endemic fish in Indonesia through competition for food, space and direct predation. The gradual reduction of established populations of invasive alien species is necessary to protect Indonesia’s freshwater ecosystems

Unveiling the power of environmental DNA: A review of eDNA application to monitoring threatened fish in Indonesian Waters

This study examines environmental DNA (eDNA) techniques to detect and monitor threatened fish species in Indonesian waters. By analyzing 17 selected documents out of 284 papers and 310 metadata files, the study identified 30 Indonesian threatened fish species through eDNA technology which only assessed 8.5% of 352 threatened fish species in Indonesian waters based on Fishbase data, particularly in families such as Osphronemidae, Melanotaeniidae, Dasyatidae, Carcharhinidae, and Gobiidae. The paper discusses estimating fish abundance using eDNA analysis, including sample collection, eDNA concentration analysis, correlation with fish biomass, and field application. Challenges and limitations in estimating fish abundance through eDNA are addressed, such as misinterpretation, environmental factors, eDNA persistence, and sampling bias. The findings highlighted the potential of eDNA as a valuable tool for detecting and monitoring threatened fish species in Indonesian waters. However, further research and conservation efforts are needed in this area. By addressing the identified challenges and limitations, a better understanding of threatened fish populations can be gained, leading to effective conservation strategies and the long-term survival of these species in Indonesian waters

Range extension of Blackstripe Lizardfish, Synodus nigrotaeniatus Allen, Erdmann & Peristiwady, 2017 (Actinopterygii, Aulopiformes, Synodontidae), from Jakarta Bay, Indonesia

Three specimens (194.4–203.8 mm standard lengths) of the lizardfish Synodus nigrotaeniatus Allen, Erdmann & Peristiwady, 2017, collected from Jakarta Bay, western Indonesia, represent a significant range extension for the species, which was previously known from only six specimens collected in Lembeh Strait, North Sulawesi, eastern Indonesia. Morphological comparisons with available data from the type specimens and molecular data from the mitochondrial cytochrome oxidase subunit I (COI) gene are presented. The COI sequence data show a 4% genetic difference from the related Synodus sageneus Waite, 1905

Range extension of Blackstripe Lizardfish, Synodus nigrotaeniatus Allen, Erdmann & Peristiwady, 2017 (Actinopterygii, Aulopiformes, Synodontidae), from Jakarta Bay, Indonesia

Three specimens (194.4–203.8 mm standard lengths) of the lizardfish Synodus nigrotaeniatus Allen, Erdmann & Peristiwady, 2017, collected from Jakarta Bay, western Indonesia, represent a significant range extension for the species, which was previously known from only six specimens collected in Lembeh Strait, North Sulawesi, eastern Indonesia. Morphological comparisons with available data from the type specimens and molecular data from the mitochondrial cytochrome oxidase subunit I (COI) gene are presented. The COI sequence data show a 4% genetic difference from the related Synodus sageneus Waite, 1905