Regulation of melanocortin-5 receptor pharmacology by two isoforms of MRAP2 in ricefield eel (Monopterus albus).

The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation in mammals. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. To explore potential interaction between ricefield eel (Monopterus albus) MC5R and MRAP2s (maMC5R, maMRAP2X1, and maMRAP2X2), herein we studied the pharmacological characteristics of maMC5R and its modulation by maMRAP2s expressed in the human embryonic kidney cells. Three agonists, α-melanocyte-stimulating hormone (α-MSH), ACTH (1-24), and [Nle4, D-Phe7]-α-MSH, could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared with human MC5R (hMC5R), maMC5R displayed decreased maximal binding but higher binding affinity to α-MSH or ACTH (1-24). When stimulated with α-MSH or ACTH (1-24), maMC5R showed significantly lower EC50 and maximal response than hMC5R. Two maMRAP2s had no effect on cell surface expression of maMC5R, whereas they significantly increased maximal binding. Only maMRAP2X2 significantly decreased the binding affinity of ACTH (1-24). Both maMRAP2X1 and maMRAP2X2 significantly reduced maMC5R efficacy but did not affect ligand sensitivity. The availability of maMC5R pharmacological characteristics and modulation by maMRAP2s will assist the investigation of its roles in regulating diverse physiological processes in ricefield eel.

A new freshwater leech species from Asian Swamp Eel stocks in China.

Discoveries of new Hemiclepsis species (Hirudinea: Glossiphoniidae) in East and Southeast Asia were expected. A peculiar freshwater leech was found on the body surface of Asian Swamp Eel Monopterus albus (Synbranchiformes: Synbranchidae) in Jingzhou, Hubei Province, China. Here, we describe this leech species as Hemiclepsis yangtzenensis sp. nov. It could be distinguished from other congeners based on a combination of morphological features such as very small size, translucent body with dense green reticulate markings throughout dorsum, very large posterior sucker with a characteristic reticulate pattern, and two pairs of well-developed drop-like eyespots. It also represents a phylogenetic lineage that is distant from other members of the genus based on the COI and 18S rRNA sequences. This leech species seems to be a specialized fish parasite that could negatively affect farming and wild stocks of Asian Swamp Eel in China.

Expression patterns of kiss2 and gpr54-2 in Monopterus albus suggest these genes may play a role in sex reversal in fish.

Due to its exceptionally small genome size and protogynous hermaphroditism, Monopterus albus has been proposed as a model for vertebrate sexual development. The Kiss/GPR54 system is a central regulator of sexual development in most vertebrates, but its role in sex reversal remains hypothetical. In contrast to mammals, fishes often possess more than one copy of the kiss and gpr54 genes. Our objectives were to identify all kiss/gpr54 genes in the genome of M. albus and to assess their involvement in sex reversal via their expression patterns (qPCR) in females, males, and intersex specimens. We identified only two genes: kiss2 and gpr54-2. kiss2 expression was extremely high in the gonads of males, intermediate in females, and low in intersex; and reduced in all tissues of intersex. gpr54 expression was also extremely high in the gonads of males, high in intersex, but low in females. gpr54 expression in brain was high in all three sexes. In conclusion, (a) kiss1 has been functionally replaced in M. albus; (b) the functions of gpr54-2 in brain are not sex-specific; (c) kiss2 appears to undergo a 'reset' in the expression during the sex change; and (d) sex-specific expression patterns in the gonads indicate that these two genes may play a role in sex reversal in fish.

Melanocortin-4 receptor in swamp eel (Monopterus albus): Cloning, tissue distribution, and pharmacology.

Melanocortin-4 receptor (MC4R) plays critical roles in the regulation of various physiological processes, such as energy homeostasis, reproduction and sexual function, cardiovascular function, and other functions in mammals. Although the functions of the MC4R in fish have not been extensively studied, the importance of MC4R in regulation of piscine energy expenditure and sexual functions is emerging. Swamp eel (Monopterus albus) is an economically and evolutionarily important fish widely distributed in tropics and subtropics. We cloned swamp eel mc4r (mamc4r), consisting of a 981 bp open reading frame encoding a protein of 326 amino acids. The sequence of maMC4R was homologous to those of several teleost MC4Rs. Phylogenetic and chromosomal synteny analyses showed that maMC4R was closely related to piscine MC4Rs. qRT-PCR revealed that mc4r transcripts were highly expressed in brain and gonads of swamp eel. The maMC4R was further demonstrated to be a functional receptor by pharmacological studies. Four agonists, α-melanocyte stimulating hormone (α-MSH), ß-MSH, [Nle4, D-Phe7]-α-MSH (NDP-MSH), and adrenocorticotropin, could bind to maMC4R and induce intracellular cAMP production dose-dependently. Small molecule agonist THIQ allosterically bound to maMC4R and exerted its effect. Similar to other fish MC4Rs, maMC4R also exhibited significantly increased basal activity compared with that of human MC4R. The high basal activity of maMC4R could be decreased by inverse agonist ML00253764, suggesting that maMC4R was indeed constitutively active. The availability of maMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in swamp eel.

Sequence and Expression Analysis of Interferon Regulatory Factor 10 (IRF10) in Three Diverse Teleost Fish Reveals Its Role in Antiviral Defense.

BACKGROUND: Interferon regulatory factor (IRF) 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice), as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity. MOLECULAR CHARACTERIZATION OF IRF10 IN THREE FISH SPECIES: In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF) in the 5'-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells. EXPRESSION ANALYSIS OF IRF10 IN VIVO AND IN VITRO: Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10 in antiviral defense.

Gene cloning and induced expression pattern of IRF4 and IRF10 in the Asian swamp eel (Monopterus albus).

The Asian swamp eel (Monopterus albus) is one of the most economically important freshwater fish in East Asia, but data on the immune genes of M. albus are scarce compared to other commercially important fish. A better understanding of the eel's immune responses may help in developing strategies for disease management, potentially improving yields and mitigating losses. In mammals, interferon regulatory factors (IRFs) play a vital role in both the innate and adaptive immune system; though among teleosts IRF4 and IRF10 have seldom been studied. In this study, we characterized IRF4 and IRF10 from M. albus (maIRF4 and maIRF10) and found that maIRF4 cDNA consists of 1 716 nucleotides encoding a 451 amino acid (aa) protein, while maIRF10 consists of 1 744 nucleotides including an open reading frame (ORF) of 1 236 nt encoding 411 aa. The maIRF10 gene was constitutively expressed at high levels in a variety of tissues, while maIRF4 showed a very limited expression pattern. Expression of maIRF4 and maIRF10 in head kidney, and spleen tissues was significantly up-regulated from 12 h to 48 h post-stimulation with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) and a common pathogenic bacteria Aeromonas hydrophila. These results suggest that IRF4 and IRF10 play roles in immune responses to both viral and bacterial infections in M. albus.

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis and Ureaplasma parvum.

OBJECTIVES: Chlamydia trachomatis and Ureaplasma urealyticum are common pathogens of sexually transmitted diseases. The majority of human ureaplasma isolates belong to the new species U. parvum. Clinically, C. trachomatis and U. parvum usually double infect in the nongonococcal urethritis patients. A novel method for simultaneous detection of C. trachomatis and U. parvum was set up in the present work. DESIGN AND METHODS: Multiple real-time quantitative PCR was developed to allow for rapid, sensitive, specific and quantitative detection of C. trachomatis and U. parvum, simultaneously. To evaluate the applicability of the multiplex real-time quantitative PCR assay to clinical specimens, 64 samples of cervical swabs collected were studied. RESULTS: Compared to the results obtained from single real-time quantitative PCR of C. trachomatis and U. parvum, the specificity, sensitivity and quantitative detection results of multiple real-time quantitative PCR are approximately identical with those of the former. CONCLUSIONS: This assay will be of great value in the simultaneous and rapid diagnosis of C. trachomatis and U. parvum in the future.

[Effects of starvation on digestive enzyme activities of Monopterus albus].

Starvation is a major environmental stress, which has a broad effect on the physiology and ecology of aquatic animals. In this study, Monopterus albus was starved for 30 days at (20 +/- 0.5) degrees C, and the activities of protease, trypsin, amylase and lipase in its digestive organs were measured on the 0, 3rd, 5th, 10th, 15th, 20th, and 30th day of starvation. The results showed that starvation had definite effects on the activities of all test enzymes. With the prolongation of starvation, the activities of test enzymes decreased, which was most significant when the fish was starved for 5-10 days. After 10 days of starvation, the decreasing trend of the enzyme activities became less obvious.