Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide,Hepcidin (<Emphasis Type="Italic">Le</Emphasis>-Hepc), from <Emphasis Type="Italic">Leiognathus equulus</Emphasis> and Analysis of Its Functional Properties In Silico

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.     

Characterization and Structural Analysis of Hepcidin Like Antimicrobial Peptide From Schizothorax richardsonii (Gray)

Innate immune system is a primary line of defense in fish that protects it from the invading pathogens. Antimicrobial peptides (AMPs) are widely distributed in nature and are essential components of innate immunity. These molecules enable the host’s innate immune system to fight against a variety of infectious agents. One such AMP, hepcidin, is a cysteine rich amphipathic peptide. We have amplified, cloned and characterized hepcidin like AMP from Schizothorax richardsonii that inhabits one of the most difficult aquatic ecosystems in the Indian Himalayas. The cDNA encoding hepcidin like peptide was amplified as a 371 bp fragment with an open reading frame (ORF) of 279 nucleotides flanked by 5′ and 3′ UTRs of 70 and 22 bases respectively. This ORF encodes a peptide of 93 amino acids with a signal peptide of 24 amino acids and a mature peptide of 25 amino acids. The mature hepcidin like peptide of S. richardsonii has eight cystine residues that participate in the formation of four disulfide bonds, a unique feature of hepcidin like AMPs. A 3D model of hepcidin like mature peptide was generated using Modeller 9.10 which was validated using PROCHECK and ERRAT. Phylogenetic analysis of hepcidin like AMP from S. richardsonii revealed that it was closely related to hepcidin from olive barb (Puntius sarana).     

Phylogenetic relationships of Eurasian Nuthatches (Sitta europaea Linnaeus, 1758) from the Alborz and Zagros Mountains,Iran

The Eurasian Nuthatch (Sitta europaea Linnaeus, 1758) is a resident bird in the Alborz and Zagros deciduous forests. We investigated the phylogenetic relationships and the taxonomic position of the Eurasian Nuthatch among other separated lineages of Eurasia with the help of blood samples collected from 19 individuals belonging to four populations in the Eastern and Western Alborz, as well as in the Northern and Southern Zagros forests. Genetic variation was then analysed using complete ND2 gene sequence (1041?bp) and phylogenetic analysis was done using Bayesian and maximum likelihood inference. Additionally, a median-joining algorithm was used to reveal the relationships among haplotypes. The results of the phylogenetic and haplo-type network analyses indicated that Eurasian Nuthatch haplotypes from the Alborz and Zagros Mountains form lineages distinct from the Asian, Caucasian and European haplotypes. Furthermore, an analysis of molecular variance (AMOVA) detected significant (P<0.001) genetic structure among the lineages. The Asian, European, Caucasian and Alborz lineages diverged from one another by an uncorrected genetic distance ranging from 0.029 to 0.039, while the Zagros lineage showed a slightly lower genetic divergence from the Caucasian lineage (0.006), but it did not share any haplotype with the Caucasian lineage. Thus, we suggest considering five Conservation Significant Units (CSU) for the Eurasian Nuthatches as the result of used dataset.     

中国翼手目新记录——马来假吸血蝠

2006年11月26日在云南省勐腊县勐仑镇翠鼻峰一无名洞捕捉到1只雄性蝙蝠,体型中等,前臂长60.7 mm,体重21.4 g。耳大,椭圆形,两耳于前额上方约15%的高度相连,耳屏细长双叉;鼻叶较简单,后鼻叶椭圆形,顶部钝圆、两侧缘向外隆凸,中央具一条显著的纵形隆脊,隆脊下部与隆起的间鼻叶相接,间鼻叶呈三角形,顶部W-形,前鼻叶呈马蹄状,前端紧接吻部;无尾,第二指仅具第一指骨;上门齿缺失,上犬齿前后基部均有一较大的副尖,Pm³缺失,下颚第二前臼齿（Pm₃）缺失。经鉴定,该蝙蝠为中国新记录——马来假吸血蝠（Megaderma spasma）。该文给出此蝙蝠的形态与头骨测量数据,并与国内分布的另一种假吸血蝠——印度假吸血蝠（M. lyra）进行了对比。标本保存在广东省昆虫研究所。     

Rearing of Lymnaea columella (Say, 1817), intermediate host of Fasciola hepatica (Linnaeus, 1758)

The intermediate host of Fasciola hepatica, Lymnaea columella, collected in Belo Horizonte, Minas Gerais, Brazil, was reared in our laboratory. The aim of the current study was to standardize a rearing and maintenance technique. Two kinds of diet were tested: fresh lettuce (A) and rodent ration + 10% CaCO3 plus fresh lettuce (B). The age for the beginning of oviposition ranged from 27 to 57 days. Ten days after oviposition at 24.7 degrees C, 100% eclosion occurred. The complete life cycle varied from 37 to 67 days. The average numbers of eggs per egg mass were 26.3 and 31.1 with diets (A) and (B), respectively. The lettuce and ration fed snails presented a increased growth although the difference was not statistically significant (p > 0.05). The mortality rate varied from 40 to 64% after 90 days. The maximum longevity was 183 days, 21.5 mm length and 11 mm wide. The methodology to mass breed and maintain these snails was found to be suitable in the laboratory     

Cytotaxonomic study of Cephalota hispanica (Gory, 1833) and Spiralia maura (Linnaeus, 1758), two cicindelids from Portugal (Coleoptera)

Cephalota hispanica and Spiralia maura have similar numbers of autosomes but distinct chromosome morphology and sex chromosome systems, (9II+X₁X₂Y/9II+X₁X₂ and 9II+X₁X₂X₃Y/9II+X₁X₂X₃, respectively). The former sex chromosome system follows the pattern of Nearctic and Indian groups and the latter the Palearctic type. The chromosomal polymorphism found suggests the occurrence of other structural rearrangements in the process of differentiation of cicindelids than those usually referred to robertsonian translocations. Moreover, the polyploidy observed in males of both species suggests pre-meiotic endoreduplication phenomena.     

DNA isolation of mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) as an alternative method to conventional fin‐clipping

The first objective of the actual project was to investigate if mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) is suitable for DNA extraction. In the second step, it was analyzed if the avoidance of environmentally hazardous chemicals could lead to sufficiently pure DNA isolation. Finally, it was examined if samples taken from different fish body parts yielded different grades of purity of the extracted DNA. Mucus was collected from 552 individuals of S. truttaand T. thymallusfrom 2012 to 2015. DNA was extracted with two different kits (E.Z.N.A.^® Insect DNA Kit and my‐Budget DNA Mini Kit) and two different swabs (my‐Budget roughened laminated sterile cotton swabs and common autoclaved cotton swabs). The results showed that the my‐Budget DNA Mini Kit in combination with the my‐Budget roughened laminated sterile cotton swabs was the most suitable method. It is a non‐destructive and swift technique that results in the concentration of DNA being high and pure.     

On the Record of a Spot-Fin Porcupine Fish,Diodon hystrix (Linnaeus, 1758) from Mandarmani,Bay of Bengal Coast of West Bengal,India

A dead specimen of a spot-fin porcupine fish Diodon hystrix was observed on the sea shore in Mandarmani, West Bengal, India. The fish weighed 2.7 kg, 76.5 cm in length and 2.54 cm in eye diameter. Although distributed circumtropically and recorded from Indian coastal waters, D. hystrix is not harvested at present, as a part of commercial capture fishery. Scars and symptoms ensuring trawler net trap caused death of the specimen. This incident warrants for a strict vigil on fishing gear used in this part of Bay of Bengal so as to ensure death escape of non-target marine resources.     

Biochemical and genetic characterization of European silver eels,Anguilla anguilla (Linnaeus, 1758), from the Comacchio lagoon (Italy)

The European eel Anguilla anguilla has declined significantly over recent decades, exceeding its safe biological limits and probably reaching a historical minimum (1% of the 1960 recruitment level). Twenty‐three migrating females at the IV stage of silvering were collected from the Comacchio lagoon (Northern Italy); they showed high levels of cholesterol and triglycerides, probably due to lipid accumulation before migration across the Atlantic Ocean. Electrophoretic patterns showed high amounts of putative α and β globulins, probably due to high concentrations of lipoproteins. Genetic results obtained from a total of 49 specimens (20 from the Comacchio lagoon and 29 from Val Dogà) agree with the panmixia hypothesis, lacking any genetic differentiation over time and space at neutral loci. In conclusion, despite the small sampling and the population decline in recent years, high levels of variability were recorded.     

Hydrozoan and protozoan epibionts on two decapod species, Liocarcinus depurator (Linnaeus, 1758) and Pilumnus hirtellus (Linnaeus, 1761), from Scotland

Crabs of the species Liocarcinus depurator and Pilumnus hirtellus, collected on the west coast of Scotland, showed hydrozoan and protozoan epibionts. The epibionts of Liocarcinus depurator were the protozoan Ephelota plana and the hydrozoans Clytia gracilis and Leuckartiara sp. The epibionts of Pilumnus hirtellus were the protozoans E. plana and Zoothamnium sp. For Liocarcinus depurator the number of Ephelota per crab fluctuated between 0 and 47 and the greatest number of suctorians were located on the chelipeds, carapace and anterior pereiopods. The hydrozoans, for Liocarcinus depurator, showed densities of 0-20 (Clytia gracilis on the second pereiopod) and 0-507 individuals per crab (Leuckartiara sp., principally on chelipeds, carapace and the fourth right pereiopod). For Pilumnus hirtellus, Ephelota plana showed densities between 3 and 56 individuals per crab, the greatest number of suctorians being located on the same areas as on Liocarcinus depurator. There was a density of 10-69 individuals per crab of Zoothamnium sp. on Pilumnus hirtellus (located on the carapace). Ephelota plana has not been observed previously as an epibiont on crustacea, nor had Zoothamnium sp. as an epibiont on Pilumnus hirtellus. Both hydrozoans, Leukartiara and Clytia, have not been previously described as epibionts on Liocarcinus depurator. Data about the morphological characteristics and distribution of these epibionts are included.     

Complex symbiotic systems of two treehopper species: Centrotus cornutus (Linnaeus, 1758) and Gargara genistae (Fabricius, 1775) (Hemiptera: Cicadomorpha: Membracoidea: Membracidae)

The aim of the conducted study was to describe the symbiotic systems (the types of symbionts, distribution in the body of the host insect, the transovarial transmission between generations) of two treehoppers: Centrotus cornutus and Gargara genistae by means of microscopic and molecular techniques. We found that each of them is host to four species of bacteriome-inhabiting symbionts. In C. cornutus, ancestral bacterial symbionts Sulcia and Nasuia are accompanied by an additional symbiont—the bacterium Arsenophonus. In the bacteriomes of G. genistae, apart from Sulcia and Nasuia, bacterium Serratia is present. To our knowledge, this is the first report regarding the occurrence of Serratia as a symbiont in Hemiptera: Auchenorrhyncha. Bacteria Sulcia and Nasuia are harbored in their own bacteriocytes, whereas Arsenophonus and Serratia both inhabit their own bacteriocytes and also co-reside with bacteria Nasuia. We observed that both bacteria Arsenophonus and Serratia undergo autophagic degradation. We found that in both of the species examined, in the cytoplasm and nuclei of all of the cells of the bacteriome, bacteria Rickettsia are present. Our histological and ultrastructural observations revealed that all the bacteriome-associated symbionts of C. cornutus and G. genistae are transovarially transmitted from mother to offspring.     

Diet of young-of-the-year bluefin tuna, Thunnus thynnus (Linnaeus, 1758), in the southern Tyrrhenian (Mediterranean) Sea

The diet of juvenile bluefin tuna (Thunnus thynnus) caught 2 to 8 miles off NE Sicily was investigated in order to improve knowledge of the species’ early life history. From 1998 to 2000, 107 specimens ranging from 63 to 495 mm (total length) were fished between July and November. Fishes were caught by trolling line or purse seine in a Fish Aggregating Devices (FADs) area 2 to 8 miles offshore and as by‐catch of the purse‐seine sardine fishery 2 to 4 miles offshore. Most frequently found items in the T. thynnus stomachs were fishes (84.5%), crustaceans (54.6%) and cephalopods (50.5%). The largest contribution in weight was provided by cephalopods (47.3%) and fishes (46.5%), while the most abundant items were fishes (51%), cephalopods (27.2%) and crustaceans (21.1%). These results suggest that young‐of‐the‐year tuna have an essentially piscivorous diet, although invertebrate prey provide a substantial contribution to the food array. Prey show little relationship with FADs, although one prey species (blue runner, Caranx crysos) is associated with FADs in the Mediterranean.     

The ultrastructure of the epicardium epithelium of the slug, Arion ater (Linnaeus, 1758), (Pulmonata, Stylommatophora)

In the present paper we investigated the fine structure of terrestial slug Arion ater (L.) Interacisternal microtubules were formed in Arion ater, however they do not correspond to interacisternal polycylinders because they differ in size and they are not continuous with the membrane cistern. From our study it may be concluded that these intracisternal microtubules suggest an accumulation of protein.