Haematological and Histopathological Examinations of African Catfish (Clarias gariepinus) Exposed to Sub-Lethal Concentrations of Paraquat

The study was designed to investigate the sub-lethal effects of paraquat on haematological parameters and histopathology of the gills, skin and liver of Clarias gariepinus juveniles of mean weight (38.26±1.20g) and length (17.50±1.55cm). The fish were exposed to 10, 20, 30, 40 and 50% of the 96hrsLC50 value of 107mg/l estimated from the 96 hours acute toxicity test. Blood samples were collected into heparinized tubes for the analyses of some haematological parameters, while the gills, skin and liver were also removed for histological examinations following standard procedures. The result revealed a significant reduction (P < 0.05) in the values of red blood cells (RBCs), haemoglobin (Hgb), packed cell volume (PCV) and erythrocytes indices from the control. The white blood cells (WBCs) and platelets (Plt) were however increasing significantly (P < 0.05) from those of the control as the test concentrations increased. The histology of the gills revealed some alterations such as epithelial proliferation, vacuolation of the mucus, hyperplasia of epithelial tissue of the gill filament, lifting and necrosis of the secondary lamellae. The exposed skin showed mucous cell proliferation, erosion of the epithelial lining, hypertrophy, necrosis of epithelial cells and widening of the epidermal and dermal layers. The liver exhibited cellular proliferation, sinusoid enlargement, congestion of the central vein, paranchymatous degeneration, vacuolar degeneration, pyknotic nucleic degeneration, legions and necrosis with severity as concentration of paraquat increases. The gills and skin were observed to be the most affected tissues in this study. The study also revealed that paraquat was toxic to C. gariepinus and causes some haematological and histopathological alterations in the fish blood and tissues at concentrations higher than 30mg/l. Therefore, the use of paraquat by farmers should be regulated particularly in area close to the aquatic environment.

Differential Expression of Hypothalamic and Gill-crh System With Osmotic Stress in the Euryhaline Black Porgy, Acanthopagrus schlegelii

The local gill production of corticotropin releasing hormone (crh) and crh-receptor (crhr) is hypothesized to play important roles during seawater (SW) and freshwater (FW) acclimation in euryhaline black porgy (Acanthopagrus schlegelii). The mRNA expression of crh, crhr, and Na+/K+-ATPase (a-nka) was examined in SW and FW diencephalon (Dien) and in the gills at different exposure time by Q-PCR analysis. The in situ hybridization results indicate that crh mRNA hybridization signals were more abundant in FW fish in the gigantocellular (PMgc) and parvocellular (PMpc) part of the magnocellular preoptic nucleus versus SW fish. The crh and crhr-expressing cells were located in basal cells of gill filament. Furthermore, in vitro dexamethasone (DEX) treatment could increase the crh-system in the gill. Increased transcripts of the crh-system in the gill via in vitro and in vivo CRH treatments suggest that CRH may regulate the system in a local manner. The a-Nka cells were localized in the filament and secondary lamellae mitochondria rich cells (MRCs) of FW fish at 8 h and 1 day. a-Nka cells were seen in both filament and lamellae in the FW but much less in SW fish indicating that gills play key roles in black porgy osmoregulation. Gill crh and crhr play important roles in the response to salinity stress.

PERKEMBANGAN JUMLAH EKTOPARASIT Pseudorhabdosynochus spp. PADA INSANG KERAPU HIBRIDA CANTIK (Epinephelus fuscoguttatus x E. polyphekadion) MELALUI METODE KOHABITASI

Genus Pseudorhabdosynocus merupakan Monogenea yang sering menginfeksi ikan kerapu. Pengaruh perbedaan jarak antara ikan sakit dengan ikan sehat terhadap perkembangan jumlah Pseudorhabdosynochus spp. pada ikan kerapu hibrida “cantik” dipelajari dalam penelitian ini melalui metode kohabitasi. Kohabitasi dilakukan dengan dua metode yaitu (a) menempatkan lima ekor ikan sakit ke dalam keranjang dengan jarak 25 cm dari dasar bak, dan (b) menempatkan lima ekor ikan sakit ke dalam keranjang dengan jarak 10 cm dari dasar bak. Kedua keranjang tersebut diapungkan ke dalam bak plastik berbeda dengan volume 100 L air laut (33 ppt) yang masing-masing telah berisi 30 ekor ikan kerapu hibrida “cantik” sehat. Masing-masing lima ekor ikan dari kedua metode kohabitasi diambil pada hari ke-2, 4, 6, 8, 10, dan 15 pemeliharaan. Hasil penelitian menunjukkan bahwa infeksi buatan menggunakan metode kohabitasi (b) lebih cepat menyebarkan Pseudorhabdosynochus spp. dari ikan sakit ke ikan sehat dibandingkan dengan metode kohabitasi (a). Perkembangan populasi Pseudorhabdosynochus spp. dan telurnya pada metode kohabitasi (b) lebih tinggi yaitu 1.495 ± 206,3 ekor/ikan dan 18,6 ± 3,8 telur/ikan dibandingkan dengan metode kohabitasi (a) yaitu 163,2 ± 16,3 ekor/ikan dan 3,8 ± 0,7 telur/ikan pasca 15 hari kohabitasi. Secara histopatologi, lamela insang yang terinfeksi Pseudorhabdosynochus spp. menunjukkan adanya hyperplasia epitel sel filamen insang yang menimbulkan fusi filamen. Kerusakan filamen di hampir semua lamela insang menyebabkan terganggunya sistem pernapasan ikan kerapu. Hasil tersebut dapat disimpulkan bahwa penyebaran Pseudorhabdosynochus spp. semakin cepat dengan semakin dekat jarak kontak antara ikan sakit dengan ikan sehat.Pseudorhabdosynocus is a genus of Monogenea that frequently infect grouper fish. This study aimed to observe the changes of density patterns of Pseudorhabdosynochus spp. in hybrid grouper gill through cohabitation. Two cohabitation methods were applied to understand the effects of distance between sick and healthy fish in terms of parasite infection. The cohabitation methos were arranged as follows: (a) five fish infected with Pseudorhabdosynochus spp. were placed into a basket at a distance of 25 cm from the bottom of the tank, and (b) five fish infected with Pseudorhabdosynochus spp. were placed into a basket at a distance of 10 cm from the bottom of the tank. The two baskets were floated into different plastic tanks of 100 L of seawater (33 ppt), each of which contained 30 healthy hybrid groupers. Each of the five fish from the two cohabitation methods was sampled on day 2, 4, 6, 8, 10, and 15 after cohabitation. The results showed that the spread of Pseudorhabdosynochus spp. from sick fish to healthy fish with the cohabitation method b was faster than the cohabitation method a. The development number of Pseudorhabdosynochus spp. and its eggs in the cohabitation method b were higher, reaching 1,495 ± 206.3 parasite/fish and 18.6 ± 3.8 eggs/fish than the cohabitation method a, 163.2 ± 16.3 parasite/fish and 3.8 ± 0.7 eggs/fish after 15 days of cohabitation. Histopathologically, gill lamella infected with Pseudorhabdosynochus spp. showed the presence of epithelial hyperplasia of gill filament cells causing fusion. Damage of the gill filament in all of gill lamella has caused disruption of the grouper breathing system. From these findings, it can be concluded that the spread of Pseudorhabdosynochus spp. was faster if the distance of direct contact between sick and healthy fish was closer.

Identification of a Novel Pattern Recognition Receptor DM9 Domain Containing Protein 4 as a Marker for Pro-Hemocyte of Pacific Oyster Crassostrea gigas

DM9 refers to an uncharacterized protein domain that is originally discovered in Drosophila melanogaster. Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as CgDM9CP-4) was identified from C. gigas. CgDM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of CgDM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, β-glucan and Mannose) and microorganisms (Staphylococcus aureus, Micrococcus luteus, V. splendidus, V. anguillarum, Escherichia coli, Pichia pastoris and Yarrowia lipolytica). CgDM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. CgDM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that in CgDM9CP-4-negative hemocytes. The mRNA expression level of CgDM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors (CgGATA, CgRunt, CgSCL, and CgNotch). In addition, CgDM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that CgDM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.

Description of the larva of Cinygmula levanidovi Tshernova & Belov, 1982(Ephemeroptera, Heptageniidae) with redescription of the male adult from the Russian Far East

The larvae, male and female imagines, and eggs of Cinygmula levanidovi Tshernova & Belov 1982 are described based on reared specimens from the Russian Far East. The larvae, female imago and eggs are described and illustrated for the first time. The larva of C. levanidovi is similar to the one of C. hirasana Imanishi, 1935 and C. kurenzovi (Bajkova, 1965). However, it can be distinguished from these species and from all other Far Eastern Cinygmula by the shape of its tergalius I, which has a heart-shape and bears a single short gill filament. Tergalius I of C. hirasana and C. kurenzovi possess a similar shape, but there are no gill filaments on the first and the other tergalii.

Histopathology and molecular identification of Henneguya pseudoplatystoma

The present study proposes to characterize the parasites isolated during the initial phase of production in fish farms located in Mato Grosso do Sul in the central-western region of Brazil, using histopathology analysis and molecular techniques. A total of 340 hybrid surubim fish (Pseudoplatystoma reticulatum × P. corruscans) from four farms were examined during the co-feeding phase. Histopathology analysis showed that 10.9% (n = 37) of the fish were infected with parasites. Branchitis, lifting epithelium, hypertrophy of epithelial cells, lamellar fusion, aneurisms and infection in the bone tissue of the gill filament was observed. The parasite species was determined by amplification of the 18S rRNA gene followed by sequencing. The phylogenetic analysis of nucleotide sequences indicates a close relationship (99.6%) with Henneguya pseudoplatystoma reported to be infecting the hybrid Pseudoplatystoma. This study demonstrates the occurrence of H. pseudoplatystoma in hybrid surubim (P. reticulatum × P. corruscans) during the co-feeding phase in fish farms in Mato Grosso do Sul. Also, molecular techniques provide a faster and sensitive method to identify fish parasites, and may assist in the development of new management techniques aimed at improving the sanitary conditions contributing to the reduction of mortality rates in these animals.

Black sea bass are a host in the developmental cycle of Lernaeenicus radiatus (Copepoda: Pennellidae): insights into parasite morphology, gill pathology and genetics

Lernaeenicus radiatus, a mesoparasitic pennellid copepod, has long been known in the northwest Atlantic with metamorphosed females infecting the muscle of marine fish. The study herein is the first to identify a definitive first host, black sea bass Centropristis striata, for L. radiatus supporting larval development to adults and sexual reproduction in the gills. This finding suggests a two-host life cycle for L. radiatus, with black sea bass as the first host. Heavy infections in the gill were associated with considerable pathology related to a unique and invasive attachment process that penetrated the gill and selectively attached to the gill filament cartilage. The morphology of the developing copepod was highly conserved with that of a related pennellid copepod, Lernaeocera branchialis, though was distinguished by the attachment process, unique pigmentation and other morphologic features described herein. Sequencing the small and large subunits of the ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes demonstrated L. radiatus to share closer identities with Lernaeocera and Haemobaphes spp. pennellid copepods rather than other Lernaeenicus spp. available in GenBank to date. Taxonomy of L. radiatus is discussed in relation to life cycles, tissue tropism, morphology and genetics of other closely related pennellid copepods.

Differential Expression and Localization of Branchial AQP1 and AQP3 in Japanese Medaka (Oryzias latipes)

Aquaporins (AQPs) facilitate transmembrane water and solute transport, and in addition to contributing to transepithelial water transport, they safeguard cell volume homeostasis. This study examined the expression and localization of AQP1 and AQP3 in the gills of Japanese medaka (Oryzias latipes) in response to osmotic challenges and osmoregulatory hormones, cortisol, and prolactin (PRL). AQP3 mRNA was inversely regulated in response to salinity with high levels in ion-poor water (IPW), intermediate levels in freshwater (FW), and low levels in seawater (SW). AQP3 protein levels decreased upon SW acclimation. By comparison, AQP1 expression was unaffected by salinity. In ex vivo gill incubation experiments, AQP3 mRNA was stimulated by PRL in a time- and dose-dependent manner but was unaffected by cortisol. In contrast, AQP1 was unaffected by both PRL and cortisol. Confocal microscopy revealed that AQP3 was abundant in the periphery of gill filament epithelial cells and co-localized at low intensity with Na+,K+-ATPase in ionocytes. AQP1 was present at a very low intensity in most filament epithelial cells and red blood cells. No epithelial cells in the gill lamellae showed immunoreactivity to AQP3 or AQP1. We suggest that both AQPs contribute to cellular volume regulation in the gill epithelium and that AQP3 is particularly important under hypo-osmotic conditions, while expression of AQP1 is constitutive.

The Anatomy of Respiratory Organ of Climbing Perch (Anabas testudineus)

The study aims to find out about the anatomical and histological structure of gill organ and labyrinth in Anabas Testudineus. The organ used is gill and labyrinth of climbing perch. The histological observation is done by making the histological preparation applied to a Hematoxylin-Eosin staining. The result of this study shows that the anatomical gill of climbing perch consists of the gill filaments and gill rakers attaching to the gill arch and the cartilage fold which are shaped like a rose. Then, the histological observation of climbing perch gill consists of the gill filament and gill arch. The gill arch is composed of gill rakers, mucosal epithelium, adipose tissue, basal membrane, mucous cell, submucosa, artery, and bone tissue. The gill filament consists of primary and secondary lamella. Primary lamella is composed of cartilage coated by perichondrium, central venous sinus, chloride cell, while secondary lamella is composed of squamous epithelium cell, mucous cell, pillar cell, and erythrocyte. Labyrinth is made up of adipose tissue, elastic cartilage, perichondrium, epithelium, blood vessel, connective tissue, and mucous cell.