Dwarf Gourami Research Articles

Experimental transmission and virulence of a megalocytivirus (Family Iridoviridae) of dwarf gourami ( Colisa lalia) from Asia in Murray cod ( Maccullochella peelii peelii) in Australia

In February 2003 there were 90% losses of Murray cod ( Maccullochella peelii peelii) fingerlings in a Victorian aquaculture facility. The disease was caused by a megalocytivirus (Family Iridoviridae) closely related to the recognized species Infectious Spleen and Kidney Necrosis Virus (ISKNV) and strain dwarf gourami iridovirus (DGIV), neither of which had previously been reported from farmed or wild fish in Australia. Experimental transmission trials were undertaken to test the hypothesis that the outbreak could have arisen through introduction of a virus with ornamental gouramis imported from South East Asia. Intraperitoneal injection of Murray cod fingerlings using filtered tissue homogenates from dwarf gourami ( Colisa lalia) positive for megalocytivirus DNA by polymerase chain reaction (PCR) resulted in > 90% mortality. Mortality was also induced by cohabitating Murray cod fingerlings and dwarf gourami; as the fish were physically separated, the virus spread between the two species via water. Histopathology revealed lesions identical to those reported in the Victorian outbreak, 125–130 nm icosahedral virions were observed in lesions and most exposed fish were PCR positive. DNA sequencing confirmed 99.9 to 100% homology of major capsid protein and ATPase nucleotide sequences between DGIV, ISKNV, the viral inoculum obtained from dwarf gourami and virus present in experimentally infected Murray cod. These findings confirm that Murray cod are highly susceptible to a megalocytivirus present in ornamental fish imported from South East Asia. The implications for aquaculture, conservation of native fish and quarantine policy are discussed.

Causal Agents of Mycobacterial Diseases in Freshwater Ornamental Fish and their Importance for Human Health in the Czech Republic

In the period from 1995 to 2004, the following mycobacterial species were isolated during the examination of the state of health of aquarium fish from the Moravian-Silesian region of the Czech Republic: Mycobacterium fortuitum (Paracheirodon axelrodi, Poecilia sphenops, Hemigrammus rhodostomus, Hyphessobrycon flammeus), M. chelonae (P. axelrodi, Betta splendens, Xiphophorus helleri, Puntius tetrazona), M. kansasii (P. axelrodi, B. splendens), M. simiae (Hyphessobrycon ecuadoriensis), and M. gordonae (P. axelrodi, Colisa lalia, B. splendens, Symphysodon discus). The first isolation from aquarium fish was recorded in the case of M. kansasii and the second isolation in the case of M. simiae; both should also be taken into account as aetiological agents of mycobacterial infections in humans. The measures to reduce mycobacterial infections in both fish and humans should include monitoring water mains as possible sources of M. kansasii infections, compliance with the sanitation principles in handling fish suspected of being infected and the content of tanks (molluscs, water, plants), and the introduction of strict veterinary inspection of imported fish, fish from aquarist shops and from large-scale distributors.

CDNA cloning and mRNA expression of growth hormone in Belontiidae (Anabantoidei suborder)

Complete cDNA encoding for the growth hormone (GH) of the Belontiidae fish family (Anabantoidei suborder) was cloned by RACE PCR using several sets of degenerate oligonu- cleotides. GH cDNA of the pearl gourami (Trichogaster leeri; pgGH), cloned from the pituitary, included the 5’ and 3’ noncoding sequences of 44 bp and 181 bp, respectively, and consisted of 840 bp that encoded for a prehormone of 204 amino acid (aa) residues. GH cDNA of the blue gourami (T. trichopterus; bgGH) and its deduced aa sequence had the same lengths as those of the pgGH, with nucleotide and aa identities of 97% and 99%, respectively. GH cDNA cloned from the dwarf gourami (Colisa lalia; dgGH) differed from both pgGH and bgGH. The identity of the dgGH cDNA nucleotides was 88%, compared to pgGH and bgGH. However, the identity of the deduced dgGH aa sequence was 97% when compared to bgGH and 96% when compared to pgGH. Nucleotides of GH cDNA of the fighting fish (Betta splendens; ffGH) had an identity of 82% to those of pgGH and bgGH, and 81% to the dwarf gourami. Higher identity was found among the aa sequences than among the nucleotide sequences. The identity of the cloned aa ffGH compared to bgGH, pgGH, and dgGH was 93%, 92%, and 91%, respectively. Higher levels of GH mRNA were found in females in immature, vitellogenic, and mature stages than in males in various stages of gonadal development. No significant differences in the GH transcription level were found between immature and mature females and males. However, the mRNA level decreased significantly during sexual behavior in males. GH sequence and expression may be used as systematic markers for Belontiidae fish and possibly other fish.

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.

Characterization of an iridovirus detected from cultured turbot Scophthalmus maximus in Korea

Juvenile turbot Scophthalmus maximus that became sick during an outbreak of disease at mariculture facilities at Go-Chang, Korea, in 2003, were examined to identify the cause of the disease. The fish had pale body color, an enlarged abdomen, protruding eyes, an enlarged spleen and kidney, and pale gills and/or liver. Histopathogical examination revealed basophilic enlarged cells in the kidney, spleen, gills, heart, stomach, intestine, liver, pancreas and skin. Hexagonal viral particles with a diameter of 136 to 159 nm were observed in the enlarged cells. A specific 1299 bp fragment of the major capsid protein (MCP) gene of the turbot iridovirus (TBIV) was amplified by PCR. Sequence homology was greater than 93.76% between the MCP gene in TBIV and the same gene in 5 viruses in the tentatively proposed genus Tropivirus (family Iridoviridae): red sea bream iridovirus, sea bass iridovirus, grouper sleepy disease iridovirus, African lampeye iridovirus and dwarf gourami iridovirus. These results suggest that the virus detected from turbot is similar to the proposed genus Tropivirus.

Selective modulation of voltage-gated calcium channels in the terminal nerve gonadotropin-releasing hormone neurons of a teleost, the dwarf gourami (Colisa lalia).

GnRH neurons in the terminal nerve (TN) have been suggested to function as a neuromodulatory system that regulates long-lasting changes in the animal behavior. Here we examined electrophysiological properties of TN-GnRH neurons in a teleost (dwarf gourami, Colisa lalia), focusing on the voltage-gated Ca2+ channels, which are thought to be coupled to several cellular events such as GnRH release. TN-GnRH neurons showed low-voltage activated (LVA) currents and three types of pharmacologically distinct high-voltage activated (HVA) currents. The L- and N-type currents constituted 30.7 +/- 3.1 and 41.0 +/- 3.9%, respectively, of HVA currents, which was recorded at the holding potential of -60 mV to inactivate the LVA currents. Although P/Q-type current was small and negligible, R-type current accounted for the remaining 23.6 +/- 1.6% of HVA currents. Next we examined the possibility of Ca2+ channel modulation induced by GnRH released in a paracrine/autocrine manner. HVA currents of up to 40% was inhibited by the application of salmon GnRH, which is the same molecular species of GnRH as is synthesized by TN-GnRH neurons themselves. However, salmon GnRH had no measurable effects on LVA currents. The inhibition of HVA currents had a dose dependence (EC50 was 11.5 nm) and type specificity among different HVA currents; N- and R-type currents were preferentially inhibited, but L-type currents had by far lower sensitivity. The physiological significance of different Ca2+ influx pathways, and their paracrine/autocrine regulation mechanisms in TN-GnRH neurons are discussed.

Complete genomic DNA sequence of rock bream iridovirus

Iridovirus is a causative agent of epizootics among cultured rock bream ( Oplegnathus fasciatus) in Korea. Here, we report the complete genomic sequence of rock bream iridovirus (RBIV). The genome of RBIV was 112 080 bp long and contained at least 118 putative open reading frames (ORFs), and its genome organization was similar to that of infectious spleen and kidney necrosis virus (ISKNV). Of the RBIV's 118 ORFs, 85 ORFs showed 60–99% amino acid identity to those of ISKNV. Phylogenetic analysis of major capsid protein (MCP), DNA repair protein RAD2, and DNA polymerase type-B family indicated that RBIV is closely related to red sea bream iridovirus (RSIV), Grouper sleepy disease iridovirus (GSDIV), Dwarf gourami iridovirus (DGIV), and ISKNV. The genome sequence provides useful information concerning the evolution and divergence of iridoviruses in cultured fish.

Different modes of gonadotropin-releasing hormone (GnRH) release from multiple GnRH systems as revealed by radioimmunoassay using brain slices of a teleost, the dwarf gourami (Colisa lalia).

It has become a general notion that there are multiple GnRH systems in the vertebrate brains. To measure GnRH release activities from different GnRH systems, we conducted a static incubation of brain-pituitary slices under various conditions, and GnRH released into the incubation medium was measured by RIA. The slices were divided into two parts, one containing GnRH neurons in the preoptic area and axon terminals in the pituitary (POA-GnRH slices), and the other containing the cell bodies and fibers of terminal nerve-GnRH neurons and midbrain tegmentum-GnRH neurons (TN-TEG-GnRH slices). We demonstrated that GnRH release was evoked by high [K(+)](o) depolarizing stimuli (in both POA-GnRH and TN-TEG-GnRH slices) via Ca(2+) influx through voltage-gated Ca(2+) channels. The most prominent result was the presence of conspicuous sexual difference in the amount of GnRH release in the POA-GnRH slices. The GnRH release from TN-TEG-GnRH slices also showed a small sexual difference, which was by far more inconspicuous than that of POA-GnRH slices. Immunohistochemical analysis using an antiserum specific to the seabream GnRH (sbGnRH; suggested to be specific to POA-GnRH neurons) revealed the presence of a much larger number of POA-GnRH neurons in males than in females. This clear morphological sexual difference is suggested to underlie that of GnRH release in the POA-GnRH slices.

Recent developments in the application of live feeds in the freshwater ornamental fish culture

The industrial development of freshwater ornamental fish culture has been hampered by the lack of suitable live feeds for feeding the fish at the various production stages. This paper reports the recent developments in the applications of the freshwater rotifers ( Brachionus calyciflorus), Artemia nauplii, decapsulated Artemia cysts and on-grown Artemia in the freshwater ornamental fish culture. Results demonstrate that the rotifers are an ideal starter feed for dwarf gourami ( Colisa lalia), a typical freshwater ornamental fish species with larvae that are too small to ingest Artemia nauplii or Moina at its first feeding. Compared with the conventional yolk food, the use of rotifers as a starter feed significantly improves the growth and survival of the gourami larvae (Days 2–12), and the beneficial effects are extended to the subsequent Artemia-feeding phase (Days 13–32). The freshwater rotifers and Artemia nauplii are also useful in raising Discus larvae in the absence of their parents, which would eliminate the risk of larvae being eaten by the parent fish. Work on decapsulated Artemia cysts indicates that the cysts could be used as a substitute for Artemia nauplii or Moina in freshwater ornamental fish culture. The fry of all the five common ornamental fish species tested (guppy Poecilia reticulata, molly Poecilia sphenops, platy Xiphophorus maculatus, swordtail Xiphophorus helleri and neon tetra Hyphessobrycon herbertaxelrodi) could readily feed on the decapsulated cysts, and their performances in terms of stress resistance, growth and survival are comparable to or better than those fed on Artemia nauplii or Moina. A culture system for production of on-grown Artemia has also been developed specifically for the use in freshwater ornamental fish farms. The system, using diluted artificial seawater of 20‰ for culture, has a mean production rate of 3 kg/m 3 of water in a 12-day cycle and a production capacity of 8 metric tons of on-grown Artemia a year. With the system, farmers could produce any specific size of on-grown Artemia of up to 5 mm to suit the age and size of their fish, by varying the time of harvesting. This characteristic, coupled with the use of bioencapsulation technique to enhance the quality of the on-grown Artemia, would make the organism an ideal nursery diet for freshwater ornamental fish. All these results show that the live feeds used in marine foodfish hatchery could be applied to freshwater ornamental fish culture to improve their performance.

Glutamate receptors in the terminal nerve gonadotropin-releasing hormone neurons of the dwarf gourami (teleost)

The terminal nerve (TN)-gonadotropin-releasing hormone (GnRH) system has been suggested to function as a neuromodulatory system that regulates the motivational state of the animal. To investigate the synaptic control of activities of the TN-GnRH neurons, we analyzed electrophysiologically the type of glutamate receptors (GluRs) in the TN-GnRH neurons. By using various specific GluR agonists and antagonists, we found that they have ionotropic GluRs (iGluR; non-NMDAR and NMDAR) and group 3 metabotropic GluRs. However, in the combined presence of supramaximal concentration of iGluR blockers in the perfusing solution and the GDPβS in the patch pipette, there were still residual Glu-induced depolarizing responses. These results suggest the presence of a novel type of iGluRs, in addition to the conventional GluRs, in the TN-GnRH neurons.

Existence of multiple isoforms of GnRH ligands and receptors in the dwarf gourami, Colisa lalia

GnRH (gonadotropin-releasing hormone) plays a pivotal role in the regulation of reproductive functions. We have identified genes encoding three GnRH precursors and three GnRH receptors (GnRHRs) in the dwarf gourami, Colisa lalia. All of their mRNAs were expressed in all the organs examined in the present study. The cloned GnRHR genes were divided into two distinct lineages: types 1 and 2.

The terminal nerve ganglion cells project to the olfactory mucosa in the dwarf gourami.

Single- and double-label immunocytochemical studies were conducted using antisera to salmon gonadotropin-releasing hormone (sGnRH) and molluscan cardioexcitatory peptide (FMRFamide) to determine whether terminal nerve ganglion cells project to the olfactory mucosa in the dwarf gourami, Colisa lalia. Both peptides were present in terminal nerve ganglion perikarya and fibers in brain and nasal cavity. Labeled fibers were present in the olfactory nerve and could be traced to the olfactory mucosa. All terminal nerve ganglion cells contained both sGnRH and FMRFamide-like peptides. This study suggests that the terminal nerve ganglion cells can influence both brain and chemoreceptive structures.

Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries.

Tropical iridovirus infection causes severe epizootic resulting in mass mortalities and large economic losses in freshwater ornamental fishes cultured in Southeast Asian countries, in wild fish seedlings captured in South China Sea, and in marine fishes farmed in Japan, Singapore, and Thailand. All of tropical iridovirus-infected fishes histopathologically showed the systemic formation of inclusion body-bearing cells and necrosis of virus-infected splenocytes and hematopoietic cells. We designed primer sets for the ATPase gene and the major capsid protein (MCP) gene and sequenced the PCR products derived from 5 iridovirus isolates from sea bass in South China Sea, red sea bream in Japan, brown-spotted grouper with a grouper sleepy disease in Thailand, dwarf gourami from Malaysia and African lampeye from Sumatra Island, Indonesia. The ATPase gene and the MCP gene of these 5 viral isolates were highly homologous (> 95.8%, > 94.9% identity, respectively) and the deduced amino acid sequences of the ATPase and the MCP were also highly identical (> 98.1%, > 97.2% identity, respectively). Based on the high homology, these 5 isolates of tropical iridovirus from various fishes in geographically different regions were determined to have a single origin and to be native to Southeast Asian regions. However, these sequences were far different from those of members of the genera Ranavirus, Lymphocystivirus and Iridovirus in the Family Iridoviridae. We propose a new genus "Tropivirus" for tropical iridovirus in the Family Iridoviridae.

Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami.

Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.

High vitamin A content in some small indigenous fish species in Bangladesh: perspectives for food-based strategies to reduce vitamin A deficiency

Recognising the importance of fish in the Bangladeshi diet, the objective of the present study was to screen commonly consumed fish species for vitamin A content to evaluate the potential of fish as a vitamin A source in food-based strategies to combat vitamin A deficiency. Samples of 26 commonly consumed fish species and one crustacean were collected in Kishoreganj and Mymensingh, Bangladesh. To obtain edible parts, the fish were cleaned by Bangladeshi women according to traditional practices. Distribution of vitamin A in parts of the fish and the effect of the cleaning practices on the vitamin A content in edible parts were assessed. The content of vitamin A compounds was analysed by high-performance liquid chromatography. The vitamin A content in small fish ranged from 2680 retinol equivalents (RE)/100 g raw edible parts in mola (Amblypharyngodon mola) to 20 RE/100 g raw edible parts in chata (Colisa lalia; an alternative scientific name is Colisa lalius). The vitamin A content in cultured species, silver carp (Hypophthalmichthys molitrix), rui (Labeo rohita), mrigal (Cirrhinus mrigala) and tilapia (Oreochromis niloticus) was low, <30 RE/100 g raw edible parts. In mola, 90% of the vitamin A was found in the eyes and viscera. The vitamin A content in the screened fish species was highly variable, by more than a factor of 100. The existence of commonly consumed fish in Bangladesh belonging to the categories of very high and high vitamin A content offers a great unexploited potential for food-based strategies to improve the vitamin A intake by promoting the production and consumption of these species.

Mechanisms of the modulation of pacemaker activity by GnRH peptides in the terminal nerve-GnRH neurons.

According to our working hypothesis, the terminal nerve (TN)-gonadotropin releasing hormone (GnRH) system functions as a neuromodulatory system that regulates many long-lasting changes in animal behaviors. We have already shown by using in vitro whole brain preparations of a small fish (dwarf gourami) that the pacemaker activities of TN-GnRH neurons are modulated biphasically by salmon GnRH, which is the same molecular species of GnRH produced by TN-GnRH neurons themselves; the modulation consists of initial transient decrease and late increase of firing frequency. In the present study, we investigated the possible involvement of Ca2+ release from intracellular store and voltage dependent Ca2+ currents in the modulation of pacemaker activities. Pharmacological blockade of Ca2+ release from intracellular stores or apamin-sensitive Ca(2+)-activated K+ current inhibited the initial transient decrease of firing frequency by sGnRH. On the other hand, bath application of Ca2+ channel blockers Ni2+ or La3+ slowed down the pacemaker frequency and attenuated the rate of the late increase of pacemaker frequency by GnRH. Furthermore, voltage-clamp experiments suggested that low-voltage-activated (LVA) Ca2+ current and hihg-voltage-activated (HVA) Ca2+ current were present in the TN-GnRH neurons, and bath application of GnRH shifted the activation threshold of HVA Ca2+ current to more negative potentials. These results suggest that (1) sGnRH induces Ca2+ release from intracellular stores and activates apaminsensitive Ca(2+)-activated K+ current so that it decreases the frequency of pacemaker activity in the initial phase, (2) some kinds of Ca2+ currents contribute to the generation and modulation of pacemaker activities, and (3) HVA Ca2+ current is facilitated by sGnRH so that it increases the frequency of pacemaker activity in the late phase.

Physiology and release activity of GnRH neurons

This chapter discusses the physiology and release activity of gonadotropin-releasing hormone (GnRH) neurons. The GnRH was first isolated from the mammalian hypothalamus and was originally identified as a hypophysiotropic decapeptide hormone that facilitates gonadotropin release from the pituitary gonadotropes. It is possible to study, in a semi-intact whole brain, the physiology, morphology, and cell biology of single GnRH neurons by taking advantage of the in vitro experimental conditions. Because the GnRH neuronal cell bodies in most vertebrate species are small and diffusely distributed in various brain regions, it is extremely difficult to study various features of single GnRH neurons. Therefore, the dwarf gourami clearly has experimental advantages over other vertebrates to study the cellular physiology and morphology of GnRH neurons. It has been recognized recently that there are also diversity of GnRH receptors, and the functional and evolutionary significance of such diversity of GnRH ligand and receptors is attracting more and more attention.

Hepatocyte Nuclear Factors-1α, -1β, and -3β Expressed in the Gonad of Tilapia (Oreochromis mossambicus)

Hepatocyte nuclear factors (HNFs) are upstream regulators of many liver-specific genes and are involved in many cellular functions in the body, but their existence, expression, and function in gonads are still poorly understood. Here we report on the first cloning of partial cDNAs of HNF-1α and -1β and full HNF-3β cDNA from a tilapia (Oreochromis mossambicus) liver cDNA library. The deduced amino acid sequence of tilapia HNF-3β has a 90 to 96% identity with those of other fishes (dwarf gourami, medaka, and zebrafish), 74% with mammals (human, rat, and mouse), and 82% with Xenopus. RT-PCR detected IGF-I and -II and HNF-1α, -1β, and -3β in both liver and gonads and the identity of the PCR fragments was confirmed by PCR hybridization. Immunoprecipitation and Western blotting also detected all three HNF proteins in both liver and gonads. Expression of HNFs in the gonads of the tilapia suggests that multi-HNFs may form a cascade to regulate gonadal physiology in the bony fish.

Erratum to “Amperometric recording of gonadotropin-releasing hormone release activity in the pituitary of the dwarf gourami (teleost) brain-pituitary slices” [Neurosci. Lett. 299 (2001) 121-l24

Erratum to “Amperometric recording of gonadotropin-releasing hormone release activity in the pituitary of the dwarf gourami (teleost) brain-pituitary slices” [Neurosci. Lett. 299 (2001) 121-l24

Amperometric recording of gonadotropin-releasing hormone release activity in the pituitary of the dwarf gourami (teleosat) brain-pituitary slices

We developed a method for real-time electrochemical recording of gonadotropin-releasing hormone (GnRH) release using carbon fiber electrodes (CFE). The oxidation current of GnRH was measured by amperometry. We recorded the release activity in the pituitary of the teleost brain-pituitary slice. The CFE was located in the part of the pituitary that receives dense innervation from GnRH neurons in the preoptic area. A bulk amperometric current was recorded in response to high [K +] o stimulation in a dose dependent manner. Decreasing the holding potential to below the oxidation potential of GnRH resulted in the loss of amperometric currents. Thus, it is suggested that the amperometric currents are mainly attributed to the oxidation currents of GnRH and reflect the GnRH release activity.