Implications of absence of seawater-type mitochondria-rich cells and results of molecular analyses for derivation of the non-parasitic Ukrainian brook lamprey <Emphasis Type="Italic">Eudontomyzon mariae</Emphasis>

The Ukrainian brook lamprey Eudontomyzon mariae is the most widespread lamprey species in eastern Europe. Although E. mariae is generally considered a derivative of Eudontomyzon danfordi, an exclusively freshwater parasitic species, it has alternatively been suggested that it was recently derived from a now extinct anadromous Black Sea ancestor. Several non-parasitic lampreys and the landlocked sea lamprey, which have recently evolved from anadromous ancestors, still develop a seawater-type mitochondria-rich cell (SW-MRC) in their gills. In contrast, this cell type is not present in the gills of either Lampetra aepyptera, a non-parasitic lamprey of ancient origin, or the parasitic Ichthyomyzon unicuspis and I. castaneus that likewise have long evolutionary histories in fresh water. Eudontomyzon mariae from the Vistula River in the Baltic River basin does not possess SW-MRC, which is inconsistent with a recent origin from an anadromous ancestor. Mitochondrial DNA sequence data were thus used to infer the relationship between different populations of E. mariae and E. danfordi, and to reconstruct the transition from anadromy to freshwater residency. The results suggest that E. mariae evolved independently in the Baltic, Black, and Caspian Sea basins, and not recently from an anadromous ancestor. Although E. mariae in the Danube River may have arisen relatively recently from E. danfordi (differing by 0.7–1.1% in cytochrome b gene sequence), other E. mariae populations (including in the Vistula River) are genetically closer (0.6%) to the hypothetical ancestor of both E. mariae and E. danfordi. That ancestor was probably a freshwater resident, since SW-MRCs are not rapidly lost following confinement in fresh water.     

The Psilostomidae Looss, 1900 (<Emphasis Type="Italic">sensu stricto</Emphasis>) (Digenea: Echinostomatoidea): description of three new genera and a key to the genera of the family

Three new psilostomid genera, Byrdtrema n. g., Longisaccus n. g. and Macracetabulum n. g., each with a single species, are described from ducks, Aix sponsa (L.) and Bucephala albeola (L.) in North America. Byrdtrema n. g. and Macracetabulum n. g. possess a bipartite seminal vesicle and share this character with four psilostomid genera, Grysoma Byrd, Bogitsh & Maples, 1961, Neopsilotrema Kudlai, Pulis, Kostadinova & Tkach, 2016, Psilostomum Looss, 1899 and Psilotornus Byrd & Prestwood, 1969. Byrdtrema n. g. differs from Macracetabulum n. g. in the shape of the body (elongate vs elongate-oval); the position of the ventral sucker (in first third of body vs just pre-equatorial); the shorter forebody; as well as in the smaller size of the eggs in relation to body length. Both new genera differ from (i) Grysoma by the nature of the vitellarium (large, compact follicles with small vitelline cells vs weakly defined follicles with large vitelline cells, respectively) and the smaller size of the eggs in relation to body length; (ii) Psilostomum in the posterior extend of the cirrus-sac in relation to ventral sucker (slightly posterior vs more posterior), the location of the genital pore (at the level of oesophagus vs just postbifurcal), the shorter length of uterine and longer post-testicular fields in relation to body length, and the anterior limits of vitellarium (at the level of ventral sucker vs posterior to ventral sucker); (iii) Psilotornus by the presence of a muscular pharynx (vs absent or rudimentary) and the location of the cirrus-sac (antero-dorsal to ventral sucker or more posterior vs entirely anterior to ventral sucker) and ovary (in hindbody vs in forebody). Byrdtrema n. g. differs from Neopsilotrema in the shape of the body (elongate vs subspherical to elongate-oval) and ventral sucker (elongate-oval vs subspherical to transversely oval), the shorter forebody and smaller eggs in relation to body length. Macracetabulum n. g. differs from Neopsilotrema by the shape of the ventral sucker (elongate-oval vs subspherical to transversely oval), the anterior limits of vitellarium (level of middle of ventral sucker vs level of intestinal bifurcation or anterior testis); and the slightly smaller size of eggs in relation to body length. Among the psilostomid genera, Longisaccus n. g. shows close affinities to Psilochasmus Lühe, 1909 in the presence of the long cirrus-sac and tubular internal seminal vesicle but can be clearly distinguished from the latter by the absence of the retractile tail-like process. In combination with molecular data, the above differences justify the recognition of three new genera. A key to the genera of the Psilostomidae is provided.     

Revision of <Emphasis Type="Italic">Podocotyloides</Emphasis> Yamaguti, 1934 (Digenea: Opecoelidae), resurrection of <Emphasis Type="Italic">Pedunculacetabulum</Emphasis> Yamaguti, 1934 and the naming of a cryptic opecoelid species

Despite morphological and ecological inconsistencies among species, all plagioporine opecoelids with a pedunculate ventral sucker are currently considered to belong in the genus Podocotyloides Yamaguti, 1934. We revise the genus based on combined morphological and phylogenetic analyses of novel material collected from haemulid fishes in Queensland waters that we interpret to represent species congeneric with the type-species, Pod. petalophallus Yamaguti, 1934, also known from a haemulid, off Japan. Our phylogenetic analysis demonstrates polyphyly of Podocotyloides; prompts us to resurrect Pedunculacetabulum Yamaguti, 1934; and suggests that Pod. brevis Andres & Overstreet, 2013, from a deep-sea congrid in the Caribbean, and Pod. parupenei (Manter, 1963) Pritchard, 1966 and Pod. stenometra Pritchard, 1966, from mullids and chaetodontids, respectively, on the Great Barrier Reef, may each represent a distinct genus awaiting recognition. Our revised concept of Podocotyloides requires a pedunculate ventral sucker, but also a uterine sphincter prior to the genital atrium, a petalloid cirrus appendage, restriction of the vitelline follicles to the hindbody, and for the excretory vesicle to reach to the level of the ventral sucker. Of about 20 nominal species, we recognise just three in Podocotyloides (sensu stricto): Pod. petalophallus, Pod. gracilis (Yamaguti, 1952) Pritchard, 1966 and Pod. magnatestes Aleshkina & Gaevskaya, 1985. We provide new records for Pod. gracilis, and propose two new species of Podocotyloides, Pod. australis n. sp. and Pod. brevivesiculatus n. sp., and one new Pedunculacetabulum species, Ped. inopinipugnus n. sp., all from haemulids. Podocotyloides australis is morphologically indistinguishable from Pod. gracilis, and exploits the same definitive host, but is genetically and biogeographically distinct. It is thus a cryptic species, the first such opecoelid to be formally named.     

Immunoreactivity of synapses on primary afferent axons and sensory neurons in lamprey spinal cord (<Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>)

The ultrastructure and immunospecificity of synapses on primary afferents and dorsal sensory cells (DCs) were studied in lamprey (Lampetra fluviatilis) spinal cords. Using the postembedding immunogold method with a combination of antibodies—polyclonal antibodies to glutamate and monoclonal antibodies to gamma-aminobutyric acid (GABA)—the presence of GABA-positive on the primary afferent axons and GABA-and glutamate-immunopositive synapses on the DC somatic membranes have been shown. Thus, it is obvious that sensory information in the lamprey is controlled by both presynaptic inhibition via synapses on the primary afferent axons and by direct synaptic influence on the body of the sensory neuron.     

Role of the <Emphasis Type="Italic">porcupine</Emphasis> gene in the development of the wing imaginal disk of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.     

Three new species of <Emphasis Type="Italic">Plagioporus</Emphasis> Stafford, 1904 from darters (Perciformes: Percidae), with a redescription of <Emphasis Type="Italic">Plagioporus boleosomi</Emphasis> (Pearse, 1924) Peters, 1957

A form of Plagioporus Stafford, 1904 is described from the intestine of three North American species of darters (Perciformes: Percidae) from River West Twin, Wisconsin, USA, that we consider to be conspecific with Plagioporus boleosomi (Pearse, 1924) Peters, 1957 based on similarities in the sucker ratio, extent of the forebody, shape and position of the testes, vitellarium distribution and terminal genitalia. Three new species of Plagioporus are described from the intestine of darters as follows: Plagioporus fonti n. sp. from Percina nigrofasciata Agassiz in Florida, USA, Plagioporus limus n. sp. from Etheostoma squamosum Distler in Arkansas, USA and Plagioporus aliffi n. sp. from Etheostoma blennioides newmanni Miller in Arkansas, USA. Morphologically Plagioporus fonti n. sp., Plagioporus limus n. sp. and Plagioporus aliffi n. sp. are most similar to one another and to P. boleosomi, Plagioporus lepomis Dobrovolny, 1939 and ‘P. etheostomae’, a nomen nudum for a species described from Etheostoma blennioides Rafinesque in Kentucky, USA, all of which are collectively distinguished from congeners in having a combination of confluent vitellarium in the post-testicular space and absence of vitelline follicles with their entire length distributed in the forebody. Plagioporus fonti n. sp., P. limus n. sp. and P. aliffi n. sp. are respectively distinguished from one another and their closest congeners in having the anterior extent of the vitellarium in the anterior half of forebody to slightly anterior to the ventral sucker as opposed to one approximately at the level of the posterior margin of the ventral sucker, possession of an excretory vesicle reaching the anterior testis as opposed to one only reaching the posterior testis and having a longer than wide oral sucker and a wider than long ventral sucker. A Bayesian inference (BI) analysis of partial 28S rDNA sequences was conducted using the three new species and 24 sequences of opecoelids retrieved from GenBank, including ten species of Plagioporus. Plagioporus aliffi n. sp., Plagioporus fonti n. sp. and P. boleosomi comprised a moderately supported sister group to a clade containing all species of Plagioporus except Plagioporus limus n. sp. and Plagioporus shawi (Mcintosh, 1939) Margolis, 1970. Plagioporus limus and in turn P. shawi were resolved as sister to all other congeners with high and moderate support, respectively.     

Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

     

Phylogenetic relationships of sculpin <Emphasis Type="Italic">Cottus volki</Emphasis> Taranetz, 1933 (Scorpaeniformes,Cottidae) according to the results of analysis of control region in mitochondrial DNA

Sequencing of the fragment of control region in mitochondrial DNA in sculpin Cottus volki and the comparison of obtained data with homologous nucleotide sequences in the other species from genus Cottus demonstrated that C. volki occupies the basal position in the “poecilopus” group, which includes also typical sculpin C. poecilopus Heckel, 1837 from water bodies of Europe, sculpin C. szanaga Dybowski, 1869 from the Amur River basin, and sculpin Cottus cf. poecilopus from the Lena River basin. Early separation of C. volki line from common trunk of “poecilopus” group explains the presence in C. volki of several primitive (plesiomorphic for this group) morphological characters: long interior ray of ventral fin, practically complete lateral body line, and well-developed dentition of palatine bones.     

Descriptions of eight new species of <Emphasis Type="Italic">Ligophorus</Emphasis> Euzet &; Suriano, 1977 (Monogenea: Ancyrocephalidae) from Red Sea mullets

Eight new species of Ligophorus Euzet &; Suriano, 1977 (Monogenea: Ancyrocephalidae) are described from two species of mullets from the Red Sea. Ligophorus bykhowskyi n. sp. and L. zhangi n. sp. from Crenimugil crenilabris (Forsskål) differ from other species of the genus in the structure of the male copulatory organ, which has a simple accessory piece and a wide copulatory tube that arises from a large, single-chambered, expanded base. Ligophorus simpliciformis n. sp., L. bipartitus n. sp., L. campanulatus n. sp., L. mamaevi n. sp., L. lebedevi n. sp. and L. surianoae n. sp. from Liza carinata (Valenciennes) are differentiated on the basis of the morphometrics of the hard parts of the haptor and male copulatory organ. The eight species represent the first records of species directly attributed to Ligophorus from the Red Sea. Measurements of the haptoral hard-parts and the male copulatory organ of the new species are analysed with the aid of Principal Component Analysis. Three morphological types of male copulatory organ, five types of anchor, and two types of ventral and three types of dorsal bars were distinguished among these species. L. bykhowskyi and L. zhangi from C. crenilabris have the same type of male copulatory organ and anchors. Those species from Liza carinata have only one common morphological character, a thick copulatory tube, but have two types of accessory piece, four types of anchors and three types of bars. All species of Ligophorus found on mullets in the Red Sea have an accessory piece without a distal bifurcation and thus differ from most species of this genus from other regions of the world’s oceans.     

Two new species of <Emphasis Type="Italic">Bacciger</Emphasis> Nicoll, 1914 (Trematoda: Faustulidae) in species of <Emphasis Type="Italic">Herklotsichthys</Emphasis> Whitley (Clupeidae) from Queensland waters

Two new species of Bacciger Nicoll, 1914 (Faustulidae) are described infecting clupeids collected from the waters off Queensland, Australia; Bacciger minor n. sp. is described from Herklotsichthys castelnaui (Ogilby) in Moreton Bay, southern Queensland and Bacciger major n. sp. is described from Herklotsichthys quadrimaculatus (Rüppell) collected off Lizard Island, on the northern Great Barrier Reef. The two species both differ from previously described species of Bacciger in the combination of their generally elongate bodies, an entire rather than deeply lobed ovary, vitelline follicles that reach to at least the intestinal bifurcation, instead of restricted to further posteriorly but principally distributed in the hindbody, and intestinal caeca extending posteriorly well past the ventral sucker. The two new species have non-overlapping size ranges and differ in their sucker ratios, the distribution of the vitelline follicles and in the shape of the cirrus-sac. ITS2 and 28S rDNA sequence data distinguish the two new species unambiguously. Phylogenetic analysis of available 28S data show they are most closely related to Pseudobacciger cheneyae Sun, Bray, Yong, Cutmore & Cribb, 2014, also recorded off Lizard Island. These are the first faustulids reported from species of Herklotsichthys Whitley, but overall members of the Clupeidae undoubtedly harbours the richest faustulid fauna of any fish family. Baccigeroides ovatus (Price, 1934) n. comb. is proposed for Bacciger ovatus (Price, 1934) Bray & Gibson, 1980 (syn. B. opisthonema Nahhas & Cable, 1964) based on the position of the genital pore being far anteriorly removed from the ventral sucker.     

Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand,with descriptions of <Emphasis Type="Italic">Echinostoma novaezealandense</Emphasis> n. sp. and <Emphasis Type="Italic">Echinoparyphium poulini</Emphasis> n. sp.

Morphological and molecular characterisation of echinostome specimens (Digenea: Echinostomatidae) recovered in one Anas platyrhynchos L. and one Cygnus atratus (Latham) (Anseriformes: Anatidae) from New Zealand revealed the presence of two known species, Echinostoma miyagawai Ishii, 1932 and Echinoparyphium ellisi (Johnston & Simpson, 1944) and two species new to science. Comparative morphological and phylogenetic analyses supported the distinct species status of Echinostoma novaezealandense n. sp. ex Branta canadensis (L.), A. platyrhynchos and C. atratus, and Echinoparyphium poulini n. sp. ex C. atratus. Echinostoma novaezealandense n. sp., a species of the “revolutum” species complex characterised by the possession of a head collar armed with 37 spines, keyed down to E. revolutum but was distinguished from the latter in having a much narrower body with almost parallel margins, longer oesophagus, wider cirrus-sac, larger seminal vesicle, much smaller ventral sucker, ovary, Mehlis’ gland and testes, more anteriorly located ovary and testes, and distinctly smaller eggs (81–87 × 42–53 vs 106–136 × 55–70 µm). This new species appears similar to Echinostoma acuticauda Nicoll, 1914 described in Australia but differs in having a longer forebody, more posteriorly located ovary and testes, and much smaller eggs (81–87 × 42–53 vs 112–126 × 63–75 µm). Echinoparyphium poulini n. sp. is differentiated from the four species of Echinoparyphium possessing 37 collar spines considered valid as follows: from E. chinensis Ku, Li & Chu, 1964 in having a much smaller body, four (vs five) angle spines and simple seminal vesicle (vs bipartite); from E. schulzi Matevosyan, 1951 in having a less robust body at a comparable body length, much smaller ventral sucker, ovary and testes, and longer but narrower eggs (87–109 × 50–59 vs 70–85 × 60–84 µm); and from the two smaller forms, E. serratum Howell, 1968 and E. aconiatum Dietz, 1909, in a number of additional metrical features correlated with body size and especially in the possession of much larger collar spines. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for representative isolates of the four species and analysed together with sequences for Echinostoma spp. and Echinoparyphium spp. available on GenBank. Phylogenetic analyses based on the mitochondrial nad1 gene revealed congruence between the molecular data and species identification/delineation based on morphology; this was corroborated by the 28S rDNA sequence data.     

<Emphasis Type="Italic">Paralepidapedon variabile</Emphasis> sp. n. (Trematoda,Lepocreadioidea, Lepidapedidae) and other representatives of the genus <Emphasis Type="Italic">Paralepidapedon</Emphasis> from Antarctic fish

In the Ross Sea and Amundsen Sea, four representatives of the genus Paralepidapedon—Paralepidapedon cf. dubium Prudhoe et Bray 1973 sensu Sokolov et Gordeev 2013, P. lepidum (Gaevskaya et Rodyuk 1988), Paralepidapedon sp., and P. variabile sp. n.—were found in demersal fishes Muraenolepis marmorata and Macrourus whitsoni. Paralepidapedon variabile sp. n. is described from Muraenolepis marmorata in the Amundsen Sea. Paralepidapedon variabile sp. n. differs from other species of the genus Paralepidapedon by the position of the anterior border of the vitellarium at the level of the anterior edge of the ventral sucker or genital pore and by the highly variable shape of the testes: from roundish with a smooth edge to sinuate–lobate. Paralepidapedon lepidum was found for the first time in the Antarctic.     

Revision of <Emphasis Type="Italic">Rhabdolepis macropterus</Emphasis> (Bronn, 1829) (Osteichthyes,lower Actinopterygii; Lower Permian,SW Germany)

The neotype of Rhabdolepis macropterus is revisited. Together with additional material, a new reconstruction of the skull in lateral view is presented. Main characters of this species of the family Elonichthyidae are a ventral accessory operculum which completely separates the operculum from the suboperculum, and further the border between the frontal (parietal) and the parietal (postparietal) bones lying anterior to the border between the dermosphenotic and the dermopterotic bones. The diagnosis of genus Rhabdolepis is renewed. The species Rh. macropterus is compared with the sympatric elonichthyid species Rhabdolepis saarbrueckensis and Elonichthys fritschi.     

A New Species of <Emphasis Type="Italic">Prolibytherium</Emphasis> (Ruminantia,Mammalia) from Pakistan,and the Functional Implications of an Atypical Atlanto-Occipital Morphology

We describe a new species of Prolibytherium, P. fusus, sp. nov., from the lower Miocene of Pakistan, thus extending the genus to Asia. Prolibytherium is otherwise known only from Libya. This species differs from Prolibytherium magnieri in several basioccipital and atlanto-occipital morphologies. Namely, the posterior basioccipital tuberosities are continuous at the midline and lack the elevated transverse ridge seen in P. magnieri, and the notch formed between the lateral occipital condyles and paraoccipital process is lower. Both species of Prolibytherium have a characteristic ventrally fused occipital condyle at the midline, with a notably fuller circumferential articular surface. Prolibytherium magnieri also has thickened dorsal and ventral arches of the atlas. These specimens also possess a longitudinal groove for the Eustachian tube extending from the alisphenoid canal to the bullae, and a second deep grove isolating the basisphenoid bone from the temporal bone. These, plus several other atlanto-occipital morphologies strengthen the cervical support of the head. This is especially important for Prolibytherium, as the taxon possesses massive aliform cranial appendages. We relate the approximation of the occipital condyles to a convergent state in two giraffids (Giraffokeryx punjabiensis and Schansitherium tafeli), each of which possesses multiple pairs of ossicones, presumably necessitating a strengthened atlanto-occipital joint.     

A new species of <Emphasis Type="Italic">Pempheris</Emphasis> (Perciformes: Pempheridae) endemic to the Ogasawara Islands,Japan

Pempheris familia sp. nov. is described on the basis of two specimens collected from the Ogasawara Islands, Japan. The new species is most similar to Pempheris japonica Döderlein in Steindachner and Döderlein 1883, endemic to Japanese and Korean waters, in having adherent scales with strong ctenii on the lateral and ventral surfaces of the body, each scale expanded basally and distally due to central narrowing, the abdomen with a U-shaped cross-sectional outline, a large ventral fenestra between the coracoid and cleithrum, 10 dorsal-fin soft rays and 35 or 36 anal-fin soft rays. However, Pempheris familia can be distinguished from P. japonica by the following combination of characters: 84–88 pored lateral-line scales; 14 or 15 scale rows above the lateral line; 50–55 predorsal scales; 26 circumpeduncular scales; and a distinct blackish blotch on the pectoral-fin base. Pempheris familia appears to be endemic to the Ogasawara Islands.