Hypseleotris that includes parental species and hemiclonal hybrid lineages. These hemiclones can be difficult to distinguish from their parent taxa, making delineation of species unusually difficult. We approach this historical taxonomic problem by using single nucleotide polymorphism (SNP) genotyping to distinguish individuals of each species and hemiclones, enabling us to quantify the variation among evolutionary lineages and assign names to the species. Hypseleotris klunzingeri remains valid and does not have any hemiclones. We describe Hypseleotris bucephala and Hypseleotris gymnocephala from the Murray-Darling Basin and Hypseleotris acropinna from the Murray-Darling as well as eastern coastal streams north of the Mary River, part of the range attributed to H. galii. We further split H. galii to distinguish a species from the Mary River, Hypseleotris moolooboolaensis. We designate a neotype and redescribe H. galii due to uncertainty about the source and species identity of specimens used in the original description. We reconcile previous taxonomies, provide new common names for parental species, and advocate using the scientific names of both parents when referring to the hemiclone hybrids to avoid confusion with previous common names that did not distinguish parental taxa and hemiclones.The rivers of southeastern Australia host a species complex within the carp gudgeon genus  This fundamental premise is embedded in all but the most general species concepts and also serves as a ground assumption when constructing strictly bifurcating phylogenetic trees. However, in cases where lineages reticulate after diverging, species definitions for both the parent and daughter taxa are more difficult to assign. If lineages are interfertile they violate the central tenet of the biological species concept (reproductive isolation), and yet it is sometimes possible for parent lineages to generate a hybrid lineage and for both parents and hybrids to persist as cohesive lineages themselves. These lineages formed from hybridization may reproduce unisexually, a rare occurrence that has been documented in vertebrates including fishes, reptiles and amphibians, along with a diversity of invertebrate groups –6. Threeeleotris .Hypseleotris (carp gudgeons) are small fishes (most species attain adult lengths of 30–50 mm SL), which superficially resemble cyprinids  [Hypseleotris are not close relatives of other Australian Eleotridae, and neither morphological nor molecular phylogenetic studies have yielded a confident placement of Hypseleotris within the family [H. compressa, H. galii and H. klunzingeri are the only taxa that have been formally described [H. galii. With the exceptions of H. compressa and H. klunzingeri, all the southeastern Australian Hypseleotris have hybridized with each other to form multiple co-occurring stable hemiclonal unisexual lineages [Hypseleotris are genetically identifiable but morphologically range from intermediate too difficult to distinguish from one of their parental taxa. Without an understanding of the range of genotypes among southeastern Australian Hypseleotris, it has been impossible to unravel the morphological variation seen among individuals, correlate morphology with genetics and unambiguously assign taxonomic names [Hypseleotris hemiclonal species complex is given in Hypseleotris species and their hemiclones were first investigated using nuclear markers  in the Lower Murray River  in the Murray-Darling Basin (MDB) [H. galii/Murray-Darling carp gudgeon, Midgley's carp gudgeon and Lake's carp gudgeon, respectively. An additional allozyme genotype (HC) was assigned to H. klunzingeri, a southeastern species that does not form hemiclonal hybrids. In addition to the genetic distinctions, the species and hemiclones were separable in a discriminant function analysis based on scale counts, fin element counts and vertebral number, although the individual meristic counts may overlap [Hypseleotris from the Goulburn River system in Victoria (part of the MDB) based on cytochrome b sequence and microsatellite markers confirmed the presence of the genotypes delineated in [b as well as nuclear single nucleotide polymorphism (SNP) data to identify all of the parental and hemiclonal lineages in southeastern Hypseleotris including a remnant population of the pure parental HX genotype (Lake's carp gudgeon) in a small tributary of the upper Lachlan River (MDB) [ mouths) ,11. Theye family ,13. Amonescribed . Three aescribed : Midgleyescribed , a closelineages ,11,17. Hic names –21. Moreeated in . Most reer (MDB) . In thiser (MDB) .Table 1Hypseleotris and enabled assignment of morphotypes to genetic lineages. This allows discrimination of individuals representing pure parental species from their hemiclones, which is necessary in order to formally describe the species and clarify morphological distinctions among species and hemiclones. For this study, we examine representatives of all southeastern Australian Hypseleotris species and their hemiclones and confirm identity of all the examined individuals with SNP genotyping. Our aim is to update the taxonomy of the group and to compare pure and hemiclonal populations of known genotypes to evaluate whether and how the pure species and their hemiclones may be distinguished from one another morphologically. We then describe four Hypseleotris species, provide a redescription and neotype for H. galii, and discuss the use of names (common and scientific) in this group as it relates to their unusual reproductive biology [These studies have determined the geographical ranges of various genetic forms within southeastern Australian  biology ,17,22.. 2. 2.1Hypseleotris collected throughout Queensland, New South Wales, Victoria and South Australia between 2014 and 2018 that encompass many localities across the MDB, Cooper Creek (LEB) and east coast drainages. We collected fishes using a seine net and euthanized them humanely with an overdose of clove oil. Fishes were collected and processed under University of Canberra animal ethics approvals CEAE 13-06, CEAE 15-06 and 20180442. After collection, we provisionally sorted fishes to species and hybrid hemiclonal lineages in the field. We removed a small piece of muscle from the right side of the caudal peduncle for each fish and preserved it in liquid nitrogen for genotyping, then preserved individual fish in 5 ml tubes in buffered 10% formalin. After a few weeks we transferred the samples to 70% ethanol. First, fish had two soaks in water for approximately 12 h, followed by three changes of 30% ethanol, 50% ethanol and 70% ethanol for 2–12 h each and rinsed with a final replacement of 70% ethanol. A complete list of specimens examined for genotype and morphology, including their specimen codes, identification and collection data is given in electronic supplementary material, table S1.We base our descriptions and designate types from a large series of . 2.2Hypseleotris klunzingeri. We excluded hemiclone hybrids from the phylogenetic analyses because they violate the assumption of non-reticulate ancestry. All genotyped individuals present in the SNP analyses are listed in electronic supplementary material, table S2. Sequencing for the SNP datasets was performed using DArTseq  or DArTseqLD using medium- and low-density coverage, respectively. DArTseq is a variation of the double-digest RAD technique which combines next-generation sequencing, complexity reduction using restriction enzymes, and implicit fragment size selection [For each individual, we confirmed (or reassigned) the field identifications based on the nuclear SNP genotype, using two similar reference sets based on medium- and low-density SNP coverage. PCA analyses were conducted on the medium-density SNP reference set which included 278 individuals and the low-density set which included 167 individuals, spanning in both cases the taxonomic and geographical diversity of the hemiclonal complex. We performed phylogenetic analyses on the medium-density SNP reference dataset consisting of 240 individuals and the low-density SNP reference dataset consisting of 143 individuals, each including the non-hybrid representatives of the hemiclonal complex plus election .We filtered and error-checked the SNP datasets in two phases, one included automatically as part of DArTseq standard protocols , followe. 2.3Based on the combination of field identifications and clustering patterns seen in the SNP PCAs and the ML phylogenies, we selected 255 genotyped individuals for morphological examination, focusing on external characters that can be easily measured and used for identification. For each individual, we counted dorsal, anal and pectoral fin elements; the final dorsal and anal elements were branched to the base and were counted as one element. We also counted the number of lateral scales from the pectoral fin axil to the centre of the caudal peduncle and the number of head scales at the dorsal midline from the base of the first dorsal fin anteriad to the interorbital region. For the pectoral fin and lateral scale counts we performed counts on the left side of the fish only because muscle samples from the caudal peduncle had been removed on the right side of each fish, except in rare cases where the left pectoral fin was damaged and so the right was counted. We present the counts for each species in . 3. 3.1Hypseleotris individuals in the medium-density SNP dataset and 1823 polymorphic SNP loci for 167 Hypseleotris individuals in the low-density SNP dataset. After filtering for repeatability (0.99) and call rate by locus (0.7), the medium-density dataset retained 24 883 and then 13 605 loci and all individuals had a call rate above 58%, with the missing data most likely due to mutations at the enzyme cutting sites rather than being present but not sequenced. The low-density dataset included 1497 loci after filtering for repeatability. One individual had a call rate of 31%, with all remaining samples being over 40%. The PCA plots for Hypseleotris species and hemiclones are shown in electronic supplementary material, figure S1.For the PCA analyses we recovered 28 207 polymorphic SNP loci for 278 Hypseleotris individuals in the medium-density SNP dataset and 2137 polymorphic SNP loci for 143 individuals in the low-density SNP dataset. After filtering for repeatability (0.99) and call rate by locus (0.7), the medium-density dataset retained 32 630 and then 14 745 loci and all individuals had a call rate above 55%. The low-density dataset included 1805 loci after filtering for repeatability, and 890 loci after filtering for call rate by locus (0.5). Two individuals had a call rate of 33 and 37%, with all remaining samples being over 47%. The concatenated SNP variants for the medium- and low-density datasets are given in electronic supplementary material, tables S3 and S4. Phylogenies of southeastern Hypseleotris individuals examined in both the medium- and low-density SNP datasets are given in For the phylogenetic analyses we recovered 35 976 polymorphic SNP loci for 240 Hypseleotris species, replacing the three informal names  and also distinguishing a fourth new species for individuals similar to but distinct from H. galii in the Mary River. These species are formally described below, but for clarity, we use the new scientific and common names throughout the rest of the paper: H. gymnocephala for the bald carp gudgeon (formerly Lake's carp gudgeon); H. bucephala for the boofhead carp gudgeon (formerly Midgley's carp gudgeon); H. acropinna for the cryptic carp gudgeon (formerly Murray-Darling carp gudgeon); and H. moolooboolaensis for the Mary carp gudgeon.Based on the genetic and morphological data, we describe four new . 3.2H. gymnocephala, 78 H. bucephala, 46 H. acropinna, 15 H. moolooboolaensis, 12 H. galii, 29 H. gymnocephala × H. bucephala hybrids, 26 H. acropinna × H. bucephala hybrids, 4 H. moolooboolaensis × H. bucephala hybrids, 3 H. galii × H. bucephala hybrids, 14 H. acropinna × H. gymnocephala hybrids and 8 H. klunzingeri. We did not examine hybrids between H. galii and H. gymnocephala, which are rare and may be the result of translocations given their native ranges have never been recorded to overlap. Hybrids between H. moolooboolaensis and H. gymnocephala have not been recorded, and are similarly unlikely since the species do not co-occur. Using only genotyped individuals for our morphological survey enables us to definitively assign character states to taxa, overcoming the primary difficulty in Hypseleotris identifications: uncertainty as to species boundaries. We summarize the characters and meristic counts of the pure Hypseleotris species in We selected and examined a total of 255 genotyped individuals for morphology: 20 H. acropinna and H. moolooboolaensis from H. galii restricts H. galii to eastern coastal drainages south of the Mary River. Additionally, no holotype was designated for H. galii when it was described, so for clarity we provide a redescription of H. galii here and select genotyped specimens for designation as neotype  and additional voucher , also deposited at the AMS.The species are sexually dimorphic, so for each one we designate a male holotype and female allotype and deposit these specimens at Museums Victoria (NMV). We additionally deposit sets of paratypes  in the Australian Museum, Sydney (AMS) and Natural History Museum of Los Angeles County (LACM). Separating . 3.3Hypseleotris Gill, 1863Hypseleotris Gill, 1863: 270 Genus of Eleotridae with compressed head, body, mouth small, oblique, not extending beyond middle of eye. Gill opening extending to below posterior end of preoperculum. Pectoral base narrow, with dark pigment blotch on dorsal margin. Pelvic fins separate, each pelvic fin with one spine and five segmented rays. Body scales large, ctenoid; predorsal area (nape) and cheek variably scaled with cycloid scales. Head pores present or absent . Teeth small, monomorphic, in several rows in both jaws. First dorsal VI–IX; dorsal fin insertion pattern 3-1221 or 3-12210; second dorsal I, 10–13; anal I, 10–13, pectoral rays 13–17. Anal fin originating directly below origin of second dorsal fin. Anal fin elements 6–11 preceding the first haemal spine. Vertebrae 25–33. Species considered here sexually dimorphic, with mature males exhibiting heads enlarged in the dorsal/nape region, brightly coloured dorsal anal fins during the breeding season.This description combines our observations with the most prominent characters given in , along wHypseleotris acropinna Thacker, Geiger & Unmack sp. nov.urn:lsid:zoobank.org:pub:7ECCD2C4-302E-447F-BFCA-AE3C9E57F228Cryptic carp gudgeonFigures Hypseleotris sp. 3 [is sp. 3 ,11,31,32Hypseleotris sp. 4 (in part) [in part) ,21Hypseleotris sp. [tris sp. Holotype. NMV A 32246-001, 38.9 mm male (H0525) collected at Little Sideling Creek, a Condamine River tributary north of Barakula, Queensland, Australia (PU15-10), Lat/Long: −26.2452 150.4231Allotype. NMV A 32246-002, 39.8 mm female (H0529), from type localityParatypes. LACM 60028-1, 29.0 mm male (H0676) & 33.7 mm female (H0679), collected at Castle Creek, a Goulburn River tributary at Euroa, Victoria, Australia (PU15-95), Lat/Long: −36.7839 145.5701AMS I.47751-008, 31.8 mm male (H1266) & AMS I.47751-009, 32.9 mm female (H1253), collected at Sandy Creek, upper Gregory River, west of Childers, Queensland, Australia (PU17-63), Lat/Long: −25.2120 152.1111Diagnosis.Hypseleotris acropinna distinguished among southeastern Hypseleotris except H. galii and H. moolooboolaensis by adult males possessing elongated rays in posterior portions of the second dorsal, anal fins with fin tips extending along length of caudal peduncle. In breeding males, median fins with distal bands of pale orange. Distinguished from H. galii by absence of dark pigment around genital papillae in females; H. galii females with prominent dark spot ventrally surrounding genital papilla (Hypseleotris acropinna distinguished from H. moolooboolaensis in the Mary River (described below) by H. acropinna generally having fewer dorsal spines (VI–VII rather than VII–VIII), fewer dorsal rays (10–11 rather than 11–12), fewer lateral scales (31–33 rather than 33–34). Species very similar, additionally distinguished by their genotypes, geographical ranges. papilla . HypseleDescription. Counts based on individuals of known SNP genotype HA; counts of holotype indicated with asterisk. Hypseleotris with first dorsal spines VI–VII* (rarely VIII), second dorsal fin elements I, 10–11* (rarely 12); anal fin elements I, 11–12 ; pectoral fin rays 13–15 (14*); lateral scales 31*–34; head scales 12*–18 on dorsal midline. Head pores absent. Head pointed, with V-shaped snout in profile, unlike rounder profiles of H. bucephala and H. gymnocephala. Coloration overall pale with dark-edged scales; dark scale margins more intense at lateral midline. Fins clear to dusky except in breeding males exhibiting bright bands on fins as described above. Urogenital papillae in both males, females blunt, with lateral edges of papilla longer than incised centre, yielding w-shaped outline. No dark pigment around the genital papilla of females.Distribution. Found patchily throughout the MDB as well as eastern coastal streams north of the Mary River to Agnes Waters, with a more northerly disjunct population in Waterpark Creek. Hemiclonal lineages involving this species and both H. bucephala and H. gymnocephala are known from the MDB, Inman River  and east coast drainages. Introduced populations are also present in several coastal catchments in Victoria and South Australia. A map showing the native distributions of southeastern Australian Hypseleotris is given in Remarks. The meristic counts agree well with those tabulated in [H. acropinna parent. Hypseleotris acropinna is very similar to H. galii and new species H. moolooboolaensis, and corresponds to the HA allozyme genotype [H. acropinna. The Mary River population is a new species and is described below as H. moolooboolaensis. Similar individuals south of the Mary are H. galii, and may be distinguished by the presence of black pigmentation around the genital papillae of females that is lacking in the northern and inland populations  of the species boundary at the Mary River. In the Elliott River, females of H. acropinna have the black pigmentation surrounding the genital papillae that is characteristic of H. galii. In all other respects, including their SNP genotypes, they are concordant with H. acropinna, and it is unclear why the characteristic is present in only that population.lated in , with thlated in  are due genotype ,17. Therulations . The onlEtymology. The species name is formed from the Latin roots ‘acro’, meaning pointed, and ‘pinnis’, meaning fin, describing the elongate, pointed second dorsal and anal fins which help to distinguish them from H. bucephala. The term ‘acropinna’ is used as a noun in apposition. The common name ‘cryptic’ refers to the extreme difficulty in distinguishing this species from its hemiclone with H. bucephala. In addition, it was also long confused with H. bucephala in the Murray-Darling Basin, not being recognized until identified by [H. acropinna being common and widespread.ified by ,17 despiHypseleotris galiiFiretail gudgeonFigures Hypseleotris galii [is galii ,15,21,31Hypseleotris spp. [ris spp. Neotype. AMS I.49481-001, 28.6 mm female (H2287) collected from Hickeys Creek, a Macleay River tributary near Millbank, New South Wales, Australia (PU18-24), Lat/Long: −30.8645 152.6313Male voucher. AMS I.49482-001, 30.3 mm male (H2266) collected from the Corindi River, west of Corindi Beach, New South Wales, Australia (PU18-22), Lat/Long: −30.0391 153.1197Diagnosis.Hypseleotris galii distinguished among southeastern Hypseleotris except H. acropinna and H. moolooboolaensis by adult males possessing elongated rays in posterior portions of second dorsal, anal fins, with fin tips extending along length of caudal peduncle. In breeding males, median fins with distal bands of pale orange, caudal fin dusky red. Distinguished from H. acropinna by presence of dark pigment around genital papillae in females; H. galii females with prominent dark spot ventrally that surrounds genital papilla  by H. galii generally having fewer lateral scales (31–33 rather than 33–34). Species very similar, additionally distinguished by their genotypes and geographical ranges. papilla . HypseleDescription. Counts based on individuals of known SNP genotype HG; counts of holotype indicated with asterisk. Hypseleotris with first dorsal spines VII*, second dorsal fin elements I, 11*–12; anal fin elements I, 11*–12; pectoral fin rays 15–16*; lateral scales 31*–33; head scales 12–14 (13*) on dorsal midline. Head pores absent. Head pointed, with V-shaped snout in profile, unlike rounder profiles of H. bucephala, H. gymnocephala. Coloration overall pale with narrow dark parenthesis-shaped marks between scales at lateral midline. Fins clear to dusky except in breeding males exhibiting bright bands on fins as described above. Urogenital papillae in males, females blunt, with lateral edges of papilla longer than incised centre, yielding w-shaped outline. Prominent dark pigment spot on, around the genital papilla of females.Distribution. Found in eastern coastal streams from south of the Mary River to the Karuah River, with populations further south to the Georges River probably being introduced. Hemiclonal lineages involving this species and H. bucephala or H. gymnocephala are also known from east coast drainages, although the H. gymnocephala hemiclones could be translocations. A map showing the native distributions of southeastern Australian Hypseleotris is given in Remarks. This is a well-known species, which we redescribe here in order to facilitate its distinction from the other southeastern Australian species and in order to designate a new type specimen. The species was described as Carassiops galii [H. galii and deposit an additional male voucher specimen. Both of these specimens are of confirmed SNP genotype HG.ps galii , from a ps galii  describeEtymology. Named by J. Douglas Ogilby [s Ogilby  in honouHypseleotris bucephala Thacker, Geiger & Unmack sp. nov.urn:lsid:zoobank.org:pub:7ECCD2C4-302E-447F-BFCA-AE3C9E57F228Boofhead carp gudgeonHypseleotris sp. 1 [is sp. 1   collected at Lachlan River, below Cargellio Weir, New South Wales, Australia (PU16-08), Lat/Long: −33.2012 146.4523Allotype. NMV A 32244-002, 31.8 mm female (H0727), from type localityParatypes. LACM 60026-1, 28.0 mm male (H0714) & 27.7 mm female (H0728), from type localityAMS I.47086-002, 29.7 mm male (H0572) & AMS I.47086-003, 23.4 mm female (H0583), collected in a billabong on the upper Murray River near Tintaldra, New South Wales (PU15-87), Lat/Long: −36.0375 147.9728Diagnosis.Hypseleotris bucephala distinguished among southeastern Hypseleotris in possessing blunt head, snout rounded in profile, with dorsal scales; lacking pointed median fin tips of H. acropinna, H. galii, H. mooloboolaensis; unique median fin coloration in breeding males with distal half of fin bearing bands of red, black, orange, often with thin edge of white.Description. Counts are based on individuals of known SNP genotype HB; counts of holotype indicated with asterisk. A Hypseleotris with first dorsal spines VII*–VIII (rarely VI), second dorsal fin elements I, 10–12*; anal fin elements I, 10–12*; pectoral fin rays 13–15*; lateral scales 30–35*; head scales 0–19 (18*) on dorsal midline. All specimens examined with scales on dorsal head, nape, ranging from well-developed, imbricate to scattered, small, cycloid. In some poorly scaled specimens, no scales present at dorsal midline, but always present elsewhere on head. Head pores absent. Coloration overall tan to brown, with alternating light, dark bands on dorsal half of body. Ventral half paler, with silvery throat, belly. Fins clear to dusky except in breeding males exhibiting bright bands on fins as described above. Urogenital papillae in males, females blunt, with lateral edges of papilla longer than incised centre, yielding w-shaped outline.Distribution. Found throughout the MDB, Bulloo River , Cooper Creek (LEB), and eastern coastal streams from the Tully River south to around the Brisbane River. Recently confirmed to occur in the upper Einasleigh River (draining into the Gulf of Carpentaria) [H. acropinna, H. galii, H. moolooboolaensis, or H. gymnocephala are known from the MDB, BBB, LEB and east coast drainages. A map showing the native distributions of southeastern Australian Hypseleotris is given in entaria) . HemicloRemarks. The meristic counts agree well with those previously tabulated by [Hypseleotris bucephala and the other southeastern Australian Hypseleotris species. Hypseleotris bucephala can be distinguished as one of the two species  with a blunted snout, particularly in mature males which develop an expanded hump on the forehead. Hypseleotris bucephala is distinguished from H. gymnocephala by having scales on the predorsal (nape) region of the head, as opposed to lacking scales on the nape. Hypseleotris bucephala corresponds to the HB allozyme genotype [lated by ,15. Coungenotype ,17.Etymology. The species name is formed from the Greek roots ‘bu’ meaning large, and ‘kephale’, Latinized to ‘cephalus’ meaning head, describing the blunt profile and enlarged forehead seen particularly in the males. The name takes a feminine ending, as an adjective, in agreement with the gender of the genus name Hypseleotris.Hypseleotris gymnocephala Thacker, Geiger & Unmack sp. nov.urn:lsid:zoobank.org:pub:7ECCD2C4-302E-447F-BFCA-AE3C9E57F228Bald carp gudgeonHypseleotris sp. 1 [is sp. 1   collected at Meadow Creek, a Lachlan River tributary at Gunning, New South Wales, Australia (PU14-170), Lat/Long: −34.7789 149.2686Allotype. NMV A 32245-002, 31.4 mm female (H0381), from type localityParatypes. LACM 60027-1, 26.8 mm male (H0373) & 29.1 mm female (H0391), from type localityAMS I.47540-001, 28.7 mm male (H0377) & AMS I.47540-002, 30.1 mm female (H0387), from type localityDiagnosis.Hypseleotris gymnocephala distinguished among southeastern Hypseleotris in possessing blunt head with dorsal scales absent as far posteriad as middle of second dorsal fin; lacking pointed median fin tips of H. acropinna, H. galii, H. mooloboolaensis; unique median fin coloration in mature males: distal portion of fin bearing band of pale orange, shading into thin band of white.Description. Counts based on individuals of known SNP genotype HX; counts of holotype indicated with asterisk. Hypseleotris with first dorsal spines VII–VIII*, second dorsal fin elements I, 11*–12 (rarely 10); anal fin elements I, 11*–12; pectoral fin rays 15–16* (rarely 17); lateral scales 28–35 (32*); head scales, head pores absent. Coloration overall pale with 6–7 alternating darker patches often visible along length of body. Fins clear to dusky except in breeding males exhibiting bright bands on fins as described above. Urogenital papillae in males, females blunt, with lateral edges of papilla longer than incised centre, yielding w-shaped outline.Distribution.Hypseleotris gymnocephala is currently only known from two small creeks in the upper Lachlan River in the MDB. Hemiclonal lineages involving this species and H. bucephala are widespread across the MDB as well as Bulloo River (BBB) and Cooper Creek (LEB). Hemiclones with H. acropinna are known from the MDB. Some hemiclones have also been recorded from coastal drainages south of the Mary River, including the Brisbane, Clarence and Georges rivers, all within the range of H. galii, although these populations could have introduced from elsewhere. A map showing the native distributions of southeastern Australian Hypseleotris is given in Remarks. The meristic counts agree well with those previously tabulated by [H. gymnocephala parent, with the exceptions of dorsal spines ranging up to IX, anal fin elements ranging from I, 10–13, and slightly more longitudinal scales (31–36) in those accounts. Counts for fin spines, fin rays and longitudinal scales overlap among H. gymnocephala and the other southeastern Australian Hypseleotris species. Hypseleotris gymnocephala can be distinguished as one of the two species  with a blunter snout, particularly in breeding males, which also develop an expanded hump on the forehead. By contrast to H. bucephala, which may lack scales on the predorsal midline but always has them elsewhere on the nape, H. gymnocephala never has scales on the predorsal region. Hypseleotris gymnocephala corresponds to the HX allozyme genotype [lated by ,15, all genotype ,17,22.Etymology. The species name is formed from the Greek roots ‘gymnos’ meaning naked, and ‘kephale’, Latinized to ‘cephalus’ meaning head, describing the scaleless skin of the forehead, nape and dorsal midline anterior to the first dorsal fin characteristic of this species. The name takes a feminine ending, as an adjective, in agreement with the gender of the genus name Hypseleotris.Hypseleotris moolooboolaensis Thacker, Geiger & Unmack sp. nov.urn:lsid:zoobank.org:pub:7ECCD2C4-302E-447F-BFCA-AE3C9E57F228Mary carp gudgeonHolotype. NMV A 32247-001, 33.0 mm male (H1294) collected at Gutchy Creek, tributary of the Mary River, near Tiaro Queensland, Australia (PU17-66), Lat/Long: −25.7827 152.5247Allotype. NMV A 32247-002, 29.3 mm female (H1298), from type localityParatypes. LACM 60029-1, 34.5 mm male (H1295) & 31.4 mm female (H1299), from type locality.AMS I.47754-001, 32.8 mm male (H1296) & AMS I.47754-002, 31.3 mm female (H1303), from type localityDiagnosis.Hypseleotris moolooboolaensis distinguished among southeastern Hypseleotris except H. galii, H. acropinna by adult males possessing elongated rays in posterior portions of second dorsal, anal fins with fin tips extending along length of caudal peduncle. Mature males with median fins having distal bands of pale orange. Distinguished from H. galii by absence of dark pigment around genital papillae in females; H. galii females with prominent dark spot ventrally surrounding genital papilla . Morphologically very similar to H. acropinna, species additionally distinguished by their genotypes, geographical ranges. papilla . DistingDescription. Counts based on individuals of known SNP genotype HM; counts of holotype indicated with asterisk. Hypseleotris with first dorsal spines VII–VIII*, second dorsal fin elements I, 10–12*; anal fin elements I, 12*; pectoral fin rays 15*; lateral scales 33–34*; head scales 15–19 (16*) on dorsal midline. Head pores absent. Head pointed, with V-shaped snout, unlike rounder profiles of H. bucephala, H. gymnocephala. Coloration overall pale with dark-edged scales. Fins clear to dusky except in breeding males exhibiting bright bands on fins as described above. Urogenital papillae in males, females blunt, with lateral edges of papilla longer than incised centre, yielding w-shaped outline. No dark pigment around the genital papilla of females.Distribution. Found in the Mary River and its tributaries. Hemiclonal lineages involving this species and H. bucephala are present in the Mary, Maroochy, and Auburn (Burnett) rivers. A map showing the native distributions of southeastern Australian Hypseleotris is given in Remarks. The meristic counts agree with the higher range of counts previously tabulated for H. acropinna [Hypseleotris moolooboolaensis is very similar to H. galii and H. acropinna, and the three species form a clade as discussed above under the description of H. acropinna. The morphological differences between H. acropinna and H. moolooboolaensis are slight, but due to the additional genetic and geographic distinctions, we elect to formally describe the species.cropinna . HypseleEtymology. The species name is derived from the Aboriginal name for the Mary River, Moolooboola. The suffix -ensis indicates reference to a geographic locality.. 4Hypseleotris species in southeastern Australia can hybridize to form stable, hemiclonal lineages except for H. klunzingeri and H. compressa. Most of the possible hybrid combinations are known. These hemiclonal lineages are present at most locations and are often abundant. Hypseleotris bucephala and H. gymnocephala have extensively formed hybrid hemiclonal lineages that are found throughout the MDB, Cooper Creek and the Bulloo River. The closely related species H. acropinna, H. moolooboolaensis and H. galii do not hybridize with one another as far as we know, as each species is allopatric except for a couple of unusual occurrences of H. moolooboolaensis in the Maroochy and Auburn (Burnett) rivers (that require further investigation); however, each will form hybrid hemiclones with either H. bucephala or H. gymnocephala . In the cases of H. acropinna and H. bucephala, at least one of the parental sexual lineages coexist with their hemiclones. For H. gymnocephala, the pure parental sexual lineage is very rare, known only from two creeks that flow into the upper Lachlan River in New South Wales, thus any of its hemiclones do not co-occur with it today. In all other localities, the individuals previously identified as Lake's carp gudgeon are hemiclones. Hemiclones may be variable and difficult to distinguish from their parent taxa. H. acropinna, H. bucephala and H. gymnocephala.All of the sympatric Hypseleotris acropinna forms hybridogenic lineages with both H. bucephala  and H. gymnocephala . The H. acropinna-bucephala hemiclones are equivalent to the previously identified allozyme genotype HAxHB, and have the following meristic counts: dorsal spines VI–VII; second dorsal elements I, 11–12 (rarely 10); anal fin elements I, 11–12 (rarely 13); pectoral fin rays 14–15; lateral scales 31–34; head scales 10–16 at dorsal midline; head pores absent. These counts overlap broadly with both parental taxa. The sex ratio in these hemiclones is highly biased towards males; 98% of fish sampled in the Goulburn River system (Victoria) were males [H. acropinna-bucephala hemiclones is intermediate between the parental taxa with regard to head and snout shape, and the length/shapes of the median fins. However, because these fish do not generally have the rounded head typical of H. bucephala, they are generally identified as H. acropinna. At present precise identification as the pure H. acropinna species or a hemiclone is not easily possible without genotyping.ucephala a,d and H figures b,e. The Hypseleotris bucephala and H. galii have the following meristic counts: dorsal spines VIII; second dorsal elements I, 12; anal fin elements I, 12; pectoral fin rays 15; lateral scales 33; head scales 16 at dorsal midline; head pores absent. The similar hemiclone pairing between H. bucephala and H. moolooboolaensis exhibits counts of: dorsal spines VIII; second dorsal elements I, 11–12; anal fin elements I, 12; pectoral fin rays 15; lateral scales 33–34; head scales 16–18 at dorsal midline; head pores absent. These counts are within the ranges of counts for the more common H. acropinna × H. bucephala hemiclones, except that the H. bucephala × H. galii and H. bucephala × H. moolooboolaensis hemiclones have one more dorsal spine. The overall appearance of the individuals is likewise very similar, and they are generally identified as the non-H. bucephala parent. H. galii and an H. bucephala × H. galii hemiclone; they are very similar, but the H. bucephala × H. galii hemiclone is slightly more stocky, has slightly shorter dorsal and anal fins with more orange coloration, and a dusky tail (rather than red).Hemiclones between Hypseleotris acropinna and H. gymnocephala are more distinctive, generally resembling more slender H. gymnocephala. These hemiclones are equivalent to the previously identified allozyme genotype HA × HX, and have the following meristic counts: dorsal spines VII–VIII; second dorsal elements I, 10–11 (rarely 12); anal fin elements I, 10–12; pectoral fin rays 14–15; lateral scales 31–35; head scales sometimes absent, or if present, 10–16 at dorsal midline; head pores absent. The counts overlap broadly with both parental taxa, and the primary diagnostic character of H. gymnocephala, the absence of head and nape scales, is variably expressed. These individuals are also highly sex-biased, but in this case in favour of females, confirming the 96% predominance of females collected in the Goulburn River [H. acropinna if the head is scaled and more pointed.Hemiclones between Hypseleotris bucephala and H. gymnocephala correspond to the previously identified allozyme genotype HB × HX, and have the following meristic counts: dorsal spines VII–IX; second dorsal elements I, 10–12; anal fin elements I, 11–12; pectoral fin rays 14–16; lateral scales 33–37; head scales and head pores absent. These counts overlap with those of their parental taxa in most respects, except that the dorsal spine count may be as high as IX, and the lateral scales may be as high as 37; the body may be slightly more elongate. The sex ratio in these hemiclones is not skewed, with roughly equal numbers of females and males [c,f). They are most likely to be confused with H. acropinna × H. gymnocephala hemiclones which typically have a smaller scaleless patch on their head. They are unlikely to be confused with H. gymnocephala due to the extremely limited distribution of the pure parental lineage, thus, any bald-headed Hypseleotris found are almost certainly hemiclones.Finally, hemiclones between nd males . This he. 5A primary challenge of taxonomy is reconciling the names assigned to taxa with the lineages that actually exist in nature. Species concepts are many and have engendered a long history of debate (reviewed in ) which wHypseleotris, the situation may be even more complicated in that it has been proposed that some hemiclones  may be generated from matings of two hemiclonal taxa  in addition to direct mating with a sexual species [Zoologists typically evaluate species boundaries with reference to sexually reproducing, bisexual lineages, which is why reproductive incompatibility, confirmed or inferred, is such a common species recognition criterion. Generally, animal species are also assumed to have arisen by divergence from other lineages rather than by reticulation among lineages. However, some organisms reproduce unisexually  and scientific names may also be applied to those lineages . Unisexu species .Menidia clarkhubbsi, a silverside from Texas that is all-female and a sperm parasite of its paternal parent taxon [Squalius alburnoides arose from hybridization involving a S. pyrenaicus female and an unknown, extinct male parent [Cnemidophorus lizards, some unisexual lineages have been given standard scientific names (Genus species or Genus species complex), even when their hybrid origin is known, a practice validated by the International Code of Zoological Nomenclature [The naming of unisexual lineages is rare enough that there is not a commonly accepted practice. Names of unisexual taxa may be expressed as Genus species A × Genus species B, or as Genus species A-species B ,41. In snt taxon . It is lnt taxon . The unie parent . This hye parent . Among Cnclature ,45.Ambystoma salamanders [Pelophylax water frogs [In hybridogenic amphibians, the male genotype is sometimes incorporated into the fertilized egg, resulting in polyploidy if the maternal diploid genome is maintained or diploidy if half of the maternal genetic contribution is discarded. This strategy is known as kleptogenesis, and the resulting lineage is a klepton. This pattern has been identified in amanders  and Peloer frogs . In caseHypseleotris (excluding H. compressa), five sexual species are known to form hybridogens and can be identified morphologically and genetically; four of those species are formally described here. Historical hybridization resulting in the formation of hemiclonal lineages is common among those five species and the resulting lineages, while identifiable genetically, are not 100% distinguishable from their parental taxa. Because several species are involved in the generation of hemiclones, it would be unwieldy to refer to this system as the H. acropinna/H. bucephala/H. galii/H. gymnocephala/H. moolooboolaensis complex. The precise mechanisms behind Hypseleotris hybridogenesis are still unclear but there is no evidence for kleptogenesis or polyploidy among the hemiclones, so we would not use the kl. designation in a species name. Although it would be possible to assign separate scientific names to the unisexual Hypseleotris lineages, we have elected not to do so. For each hemiclonal lineage we recommend using the combined scientific names from both parental species and/or their associated genotype codes, as per previous published work, for instance, H. acropinna × bucephala (HA × HB), H. acropinna × gymnocephala (HA × HX), H. bucephala × gymnocephala (HB × HX), H. bucephala × H. galii (HB × HG), or H. bucephala × H. moolooboolaensis (HB × HM). To avoid confusion with previous literature we recommend discontinuing previous common names used in this group  as they are now known to refer to multiple lineages. While many morphological characters of the parental species and hemiclones broadly overlap, hemiclonal combinations involving H. gymnocephala can be distinguished by their differing degree of baldness and to a lesser extent by their sex ratio and their current known distribution. Hemiclones involving H. acropinna, H. galii, or H. moolooboolaensis with H. bucephala are difficult to distinguish morphologically. Although subtle differences in body depth, fin length  and coloration  exist, it is not yet known if those differences are consistent among the hemiclone pairings.In the case of southeastern Australian . 6Hypseleotris individuals from six lineages in central and eastern Australia, we clarify the identity of five taxa that participate in the formation of hemiclonal hybrids and assign four of them new species names: H. acropinna, H. bucephala, H. gymnocephala and H. moolooboolaensis. We further designate a neotype and provide an updated description for the fifth hemiclone-forming species, H. galii. The discernment and identification of parental species and hemiclone hybrids in this complex is crucial to documenting and preserving these native Australian fishes and their habitats. We provide morphological counts, photographs and descriptions to facilitate these identifications, allow for better informed conservation planning and also to aid in unravelling the evolutionary history of this unique group.We use analysis of morphology correlated with SNP genotyping to untangle a difficult taxonomic problem: identification and description of pure parental taxa in a hemiclonal hybrid species complex of Australian freshwater fishes. After genotyping and examination of


Helicobacter pylori (H. pylori) and Mycoplasma hyorhinis (M. hyorhinis). Inflammation caused by H. pylori virulence factors, such as CagA, VacA, and oipA, disrupt epithelial integrity, which allows the primary tumor to progress through the metastatic process. Evidence supports the activation of aquaporin-5 by CagA-positive H. pylori infection, promoting epithelial–mesenchymal transition via the extracellular signal-regulated kinase/mitogen-activated protein kinase (MEK/ERK) pathway, thus laying the foundation for metastatic disease. M. hyorhinis has also been implicated in gastric neoplasia via β-catenin stabilization and subsequent activation of the WNT-signaling pathway, promoting gastric cancer cell motility and inciting cancer progression.Infectious bacteria influence primary gastric carcinogenesis, organotropism, and metastatic progression by altering the microenvironment at the primary and secondary tumors. Key species include Helicobacter pylori (H. pylori) infection is well-established as a contributor to gastric carcinoma. However, the role that these bacteria and others may play in gastric carcinoma metastasis is a current focus of study. A review of the literature was conducted to elucidate the process by which gastric adenocarcinoma metastasizes, including its ability to utilize both the lymphatic system and the venous system to disseminate. Studies that investigate the tumor microenvironment at both the primary and secondary sites were assessed in detail. H. pylori and Mycoplasma hyorhinis (M. hyorhinis) were found to be important drivers of the pathogenesis of gastric adenocarcinoma by modifying various steps in cell metastasis, including epithelial–mesenchymal transition, cell migration, and cell invasion. H. pylori is also a known driver of MALT lymphoma, which is often reversible simply with the eradication of infection. M. hyorhinis has been implicated in gastric neoplasia via β-catenin stabilization and subsequent activation of the WNT-signaling pathway, promoting gastric cancer cell motility and inciting cancer progression. Fusobacterium nucleatum (F. nucleatum) and its association with worse prognosis in diffuse-type gastric adenocarcinoma are also reviewed. Recognition of the roles that bacteria play within the metastatic cascade is vital in gastrointestinal adenocarcinoma treatment and potential reoccurrence. Further investigation is needed to establish potential treatment for metastatic gastric carcinoma by targeting the tumor microenvironment.Gastric cancer metastasis is a process in which the tumor microenvironment may carry significant influence.  This literature review is the second of a two-part in-depth literature review on bacterial involvement in gastrointestinal neoplasia metastasis. The first publication focused on bacterial involvement in colorectal carcinomas . The curHelicobacter pylori (H. pylori) infection . H.. H.H. py species . Histoloukocytes . AdditiogA) gene , vacuolagA) gene , and outgA) gene  gene aregA) gene . CagA-exH. pylori [H. pylori infection does not cause ulcers in every infected person is unknown. The relationship between H. pylori virulence factors and host immunity is one element that determines the fraction of infected individuals who will develop serious illnesses [beta (IL-1β) [H. pylori isolated in populations with an increased prevalence of gastric adenocarcinoma [Over half of the world’s population carries . pylori ; howeverllnesses . Geneticllnesses  and inte (IL-1β) , or decr (IL-1β) , are assarcinoma . H. pylori are detected in the gastroduodenal mucosa in the majority of patients with duodenal ulcers, but only a minority (10 to 15%) of H. pylori-infected patients develop peptic ulcer disease [Other factors that determine the severity of disease progression in some individuals and not in others are ongoing areas of research. For example,  disease ,80. It i disease . It is lH. pylori virulence factors has been identified as a common mutation that drives this progression [H. pylori infection, which promotes epithelial–mesenchymal transition via the extracellular signal-regulated kinase/mitogen-activated protein kinase (MEK/ERK) pathway, thus laying the foundation for metastatic disease [Human gastric carcinogenesis is a multi-step and multifactorial process. This process begins as superficial gastritis and progresses into atrophic gastritis, metaplasia, dysplasia, and then gastric adenocarcinoma, in a manner first described by Corea et al. in 1988 ,83. Consgression ,85. Evid disease . H. pylori are also associated with extra nodal marginal zone B-cell lymphoma, also known as mucosa-associated lymphoid tissue (MALT) lymphoma [H. pylori infection [H. pylori can evidently stimulate NF-κB activation through the MALT1/BCL-10 pathway in MALT lymphomas that lack inciting translocations [H. pylori eradication, but tumors with translocations that involve MALT1 or BCL-10 persist after eradication [H. pylori eradication [H. pylori focuses on its epidemiology [H. pylori and lifestyle modifications, including reducing salt intake, smoking, obesity, and alcohol, remains key in gastric CA control [H. pylori is thought to be inhibited by glycans found in glandular mucus, and a high-salt diet  is thought to increase superficial mucus cell mucus, while decreasing glandular mucus cell mucus [lymphoma . MALT isnfection . Three tnfection . H. pyloocations . This hydication . MALT lydication . Currentemiology ,73, impremiology , treatmeemiology , and a bemiology , and itsemiology . Primaryll mucus .Mycoplasma hyorhinis (M. hyorhinis) are Gram variable cell wall-deficient bacteria. This small Gram-negative pleomorphic coccobacilli is known to infect the respiratory tract of pigs. Numerous swine diseases have been linked to M. hyorhinis, with severe pathogenicity, high mortality rates, and ensuing financial losses. Antibiotic interventions to lessen these effects encourage the growth of drug-resistant M. hyorhinis strains which have the potential to infect human stomachs through pork consumption [M. hyorhinis in 56% of gastric cancer [sumption . A previc cancer .M. hyorhinis dysbiosis via the NLRP3 inflammasome [M. hyorhinis infection results in pathology in human studies. Serology studies have confirmed M. hyorhinis in gastric carcinoma, colon carcinoma, and prostate and lung carcinoma biopsies [Gastric and prostate neoplasms have been linked to ammasome . NLRP3 ibiopsies ,95.M. hyorhinis involvement in gastric neoplasia via β-catenin stabilization and subsequent activation of the WNT-signaling pathway, promoting gastric cancer cell motility and inciting cancer progression [M. hyorhinis infection and metastatic disease.A 2019 study by Liu et al. revealed gression . They fogression . In vitrgression , veritabFusobacterium nucleatum (F. nucleatum) is an opportunistic, Gram-negative obligate anaerobic bacterium which is commonly located in the oral cavity of humans [F. nucleatum is frequently detected in primary colorectal cancer (CRC) and its metastases, and has been linked to a worse prognosis in gastrointestinal cancers in general. Recently, studies have shown increased loads of F. nucleatum in gastric cancer tumor samples [F. nucleatum positivity showed no association with chronic gastritis or preneoplastic conditions, such as intestinal metaplasia [f humans ,99. F. n samples , althougtaplasia .F. nucleatum is associated with worse prognosis in Lauren’s diffuse-type gastric cancer patients, but not in the intestinal type, in contrast to H. pylori [F. nucleatum increases cell proliferation and tumor-promoting inflammation, while avoiding immune destruction, ultimately promoting a pro-inflammatory state and tumorigenic environment [F. nucleatum is thought to stimulate cell proliferation via two primary signaling mechanisms, including (1) FadA binding to E-Cadherin to activate the WNT/B catenin pathway [. pylori . F. nuclironment ,101. F.  pathway  and (2)  pathway ,104. HowInvestigations into the complex interactions of microorganisms and tumor behavior are uncovering a variety of potential mechanisms by which infection/colonization contributes to more aggressive tumors. Factors such as increased acute and chronic inflammation, induction of various enzymes, stimulation of cytokines, changes in the microbiome, and even potential alterations in local oxygen tension may modulate the growth, invasiveness, and spread of neoplastic cells.H. pylori and M. hyorhinis have been shown to induce metalloproteinase activity [Tumor cells may degrade interstitial connective tissue and the basement membrane by secreting proteolytic enzymes or by inducing stromal cell proteolysis ,106. Ulcactivity ,109. MMPactivity . They alactivity .H. pylori has been repeatedly shown to decrease the risk of neoplasia, especially when treated before pathologic site changes have occurred and even in those with prior gastric cancer history [It is important to note that the regions within the stomach have varying microflora compositions . Eradica history . Prognos history , but tha history . For exa history .Proteobacteria flourished after H. pylori eradication, which may warrant probiotic consideration in the treatment plan [Propionibacterium acnes, Streptococcus anginosus, and Prevotella melaninogenica in the gastric tumoral microenvironment, which are associated with gastric cancer tumorigenesis through their specific mechanisms that increase local inflammation [P. acnes releases short-chain fatty acids, which can cause lymphocytic gastritis. S. anginosus induces cytokine release via sulfur metabolism. P. melaninogenica contributes to excess gastric acidity in non-atrophic sites [However, bacterial consideration does not end there. A study showed detrimental gut bacteria such as ent plan . Bik et ammation . P. acneic sites ,120,121.For early-stage tumors, gastrectomy is the most successful definitive treatment. A landmark study in 1982 recommended excision margins of at least six centimeters to properly excise serosa infiltrations and constituted the standard of treatment for nearly 30 years. However, increasing the amounts of gastric tissue removed leads to poorer patient quality of life and nutrition ,123. ManRalstonia and Helicobacter predominating before and Streptococcus and Prevotella predominating after tumor resection [Anaerosinus, Butyrivibrio, Campylobacter, Clostridium, Coprococcus, Desulfovibrio, Oscillospira, Oxalobacter, Slackia, Sporobacter, Veillonella, and Victivallis after a BII or RYGJ, compared to controls without these surgeries [Many studies have analyzed the alteration in the microbiome post-gastrectomy. The Roux-en-Y gastrojejunal anastomosis (RYGJ) and Billroth II anastomosis (BII) are two types of partial gastrectomy that may be performed for neoplasm resection and are shown to alter gut microbiota. One study showed esection . Anotherurgeries . Althougurgeries . E. coli, K. pneumoniae, E. faecalis, and Streptococcus species [Streptococcus spp. and a few Veillonella spp., which are metabolically dependent on Streptococcus spp. in oral biofilms, to overcome the previously inhibitory gastric barrier [Higher concentrations of dissolved oxygen in the hindgut after an RYGJ are thought to lead to an increase in facultative anaerobes, such as  species . Both th species . As a re barrier . As with barrier ,132. TheWe have gained insight by comparing alterations in the microbiome after partial gastrectomy for cancer and for morbid obesity. The majority of partial gastrectomies are performed to resect tumors. So, the question remains whether tumors found post-gastrectomy can truly be attributed to bacteria-induced inflammatory changes versus recurrence.Yokenella regensburgei and Fusobacterium varium were found after bariatric surgery; these two, in particular, have already been associated with colonic inflammation, which can be theorized to occur in the stomach as well [Furthermore, partial gastrectomy to treat morbid obesity yields different alterations. In one study,  as well . Evidenc as well ,135,136. as well ; therefoH. pylori can halt gastric MALT lymphoma progression and reverse early metaplasia. Inflammation and disruption of epithelial integrity, brought on by virulence factors such as CagA, VacA, and oipA, enable the primary tumor to go through the critical stages of the metastatic process. The evidence supports the activation of aquaporin-5 by CagA-positive H. pylori infection, which promotes epithelial–mesenchymal transition via the extracellular signal-regulated kinase/mitogen-activated protein kinase (MEK/ERK) pathway, thus laying the foundation for metastatic disease. M. hyorhinis has been implicated in gastric neoplasia via β-catenin stabilization and subsequent activation of the WNT-signaling pathway, promoting gastric cancer cell motility and inciting cancer progression. F. nucleatum is a driver of gastrointestinal cancers in general, and further investigation into its involvement in gastric cancer is needed. F. nucleatum is associated with worse prognosis in diffuse-type gastric adenocarcinoma. Recognition of the roles that bacteria play within the metastatic cascade is vital in gastrointestinal adenocarcinoma treatment and potential reoccurrence. At this time, further investigation is needed to establish potential treatment for metastatic gastric carcinoma by targeting the microbial contribution to the tumor microenvironment.Bacterial involvement in gastric neoplasia and metastasis is significant. Primary tumors are frequently curable with gastrectomy, when detected early. However, metastatic disease, which accounts for the majority of cancer-related deaths, remains a deadly clinical scenario. Growing evidence indicates that bacterial infection influences organotropism and metastatic progression by altering the microenvironment at the primary and secondary tumors, in addition to promoting carcinogenesis in primary gastric CA. Eradication of


Patients with hepatocellular carcinoma (HCC) have poor prognosis, especially in advanced stages. Targeted therapy is the main treatment for advanced HCC patients, but the optimal targets for HCC remain poorly understood. The main purpose of this study was to identify potential novel prognostic markers and therapeutic targets.Firstly, differentially expressed genes (DEGs) in HCC were identified from the Gene Expression Omnibus (GEO) database. The expression, significance in prognosis, and potential mechanisms of DEGs were analyzed using GEPIA, TIMER, HPA, Kaplan Meier Plotter, CBioPortal, miRWalk, TargetScan, and ENCORI databases. Immunohistochemical staining was used to determine the protein expression levels of potential candidate genes.MND1, STXBP6, and CLGN were significantly increased in HCC (p< 0.01). HCC patients with elevated CLGN mRNA levels had poorer overall survival (OS), disease-free survival (DFS), progression-free survival (PFS), and disease-specific survival (DSS) (p < 0.05). Higher MND1 mRNA levels significantly correlated with poorer DFS in HCC patients (p< 0.05). However, there was no significant correlation between STXBP6 expression and prognosis of HCC (p> 0.05). Further analysis revealed that patients with elevated CLGN mRNA expression in advanced pathology stages had poorer prognosis (p< 0.01). In addition, CLGN protein levels were elevated in HCC compared to their levels in normal tissues. The mRNA levels of CLGN had no significant correlation with the abundance of six common tumor infiltrating lymphocytes in HCC (COR < 0.5). Moreover, the mutation rate of CLGN was less than 1% in HCC patients (10/1089). Finally, the expression level of hsa-miR-194-3p in HCC was significantly lower than that in normal tissues (p < 0.05), and prognosis of HCC with low expression of hsa-miR-194 was poor (p < 0.05).The mRNA levels of CLGN in HCC is significantly associated with poor patient prognosis, especially in the advanced stages, and may be regulated by hsa-miR-194-3p. These findings suggest that CLGN may be closely related to the progression of HCC, and is a potential therapeutic target and prognostic indicator for patients with advanced HCC.The upregulation of  Primary liver cancer remains the sixth most common cancer and the third leading cause of cancer-related deaths worldwide in 2020, according to the latest statistics . HepatocIn recent years, bioinformatic databases have provided abundant transcriptomic and clinical data , 9, whichttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE54236) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE121248) were downloaded from Gene Expression Omnibus  (http://gepia.cancer-pku.cn/) (https://cistrome.shinyapps.io/timer/)   and GSE1ov/geo/) . D ifferpku.cn/)  and Tumo/timer/)  were usehttp://kmplot.com/analysis/index.php?p=service&cancer=liver_rnaseq)  and disease-free survival (DFS) in HCC patients. Kaplan Meier Plotter (_rnaseq)  was usedEthics Statement: The study was conducted in accordance with the principles of the Declaration of Helsinki . The colImmunohistochemical Staining: Samples from 24 HCC patients who underwent surgical resection at Liuzhou Workers’ Hospital were collected. The acquisition of specimens was approved by the Ethics Committee of Liuzhou Workers’ Hospital.CLGN  was performed using the avidin-biotin- peroxidase complex method. The primary antibody (1:200 dilution for CLGN) was applied to the sections and allowed to bind for 1 h at room temperature. The sections were then incubated with biotinylated anti-mouse/rabbit antibody for 30 min and an avidin-biotin-peroxidase reagent for 10 min. After color development with diaminobenzidine, the sections were counterstained with hematoxylin.Five-micrometer sections were obtained from paraffin-embedded tumor and non-tumor specimens. All sections were dewaxed in xylene and rehydrated in alcohol, followed by wet autoclave pretreatment in citrate buffer for antigen retrieval  and then rinsed with phosphate buffer saline. Immunohistochemical staining with antibody to https://www.proteinatlas.org/) database was used to verify the expression of CLGN protein in HCC patient specimens and normal liver tissue.In addition, PATHOLOGY module in HPA   in HCC were estimated using the TIMER algorithm. Statistical significance was set at p values of <0.05 and correlation coefficients (COR) of ≥ 0.5.The CBioportal database   was usedhttp://mirwalk.umm.uni-heidelberg.de/) (http://www.targetscan.org/vert_72)    and Targvert_72)  databaseays.php)  databaseCLGN mRNA expression.Gene expression data were cleaned and visualized using R (Version 3.6.3) and the R packages ggplot2 and VennDiagram. The cleaning algorithm for the gene expression data included removal of non-coding genes, miRNA, and blank probes; FDR ≤0.05 and FC ≥2; retaining one level from one gene that had several high or, consistently, low expression levels in different specimens. We removed genes that had both high expression and low expression in different specimens. Cytoscape (Version: 3.7.2) and cytoHubba plugin were used to analyze miRNAs with the potential to regulate MND1, STXBP6, and CLGN in HCC were significantly higher than those in normal tissues.The DEG analysis revealed 290 genes with significantly high expression and 518 genes with significantly low expression in the GSE54236 (G1) dataset  had poorer OS   of cases  of patients  is a testis-specific endoplasmic reticulum chaperone protein , 24. Preosterone . HoweverCLGN were highly expressed in HCC compared with normal liver tissues. Furthermore, patients with higher CLGN mRNA levels had poorer prognoses, and CLGN mRNA levels were valuable in predicting the prognosis of patients with advanced pathology. These results suggest that CLGN is associated with malignant progression in HCC patients. Additionally, CLGN was found to be significantly upregulated in invasive breast cancer, chromophobe renal cell carcinoma, papillary renal cell carcinoma lung adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, and uterine corpus endometrial carcinoma. To the best of our knowledge, no studies have investigated the role of CLGN in these tumors, which is worth of further investigation.In this study, it was found that the mRNA and protein of MND1 and STXBP6 in HCC remains unknown. Notably, Zierhut et al. found that MND1 was necessary for meiosis homologue repair  are small non-coding RNAs composed of approximately 20 nucleotides that regulate gene expression by binding to the 3’-UTR of the target mRNA . Recent CLGN is a potential prognostic marker for HCC and is associated with HCC progression, however, several limitations are associated with our study. Specifically, the relationship between CLGN expression and patient prognosis has not yet been verified at the center. Additionally, laboratory research on the cancer-promoting mechanisms of CLGN were not available, and the sample size used for immunohistochemistry in this study was small. Therefore, a large-scale verification study is required in the future.Our findings suggest that CLGN is upregulated in HCC and significantly correlates with patient prognosis, especially in the advanced stages. In addition, the mRNA levels of CLGN were found to be potentially regulated by hsa-miR-194-3p. These results suggest that CLGN may have important therapeutic and prognostic value in HCC patients.Gene Expression Omnibus (GEO) , Gene Expression Profiling Interactive Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), Kaplan Meier Plotter, The Human Protein Atlas (HPA), cBioPortal, miRWalk, TargetScan, and The Encyclopedia of RNA Interactomes (ENCORI).The datasets analyzed during the current study are available on The studies involving human participants were reviewed and approved by Ethics Committee of Liuzhou Workers’ Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.ZC: Data analysis and writing of the first draft. BC, YL and ZW: Data analysis, manuscript editing, review, and approval. JW, ZC, and GC: Data analysis, data collection and experiments. DL: Data analysis, manuscript editing and review. All authors have read and approved the final manuscript.


Borrelia burgdorferi that regulates the expression of surface lipoproteins with roles in virulence. Further studies indicated that this protein binds specifically and non-specifically to DNA and colocalises with nucleoids in this bacterium. The data showed that this protein binds to DNA as a homodimer, although it can form other organised structures. Crystallography analysis indicated that the protein possesses domains responsible for protein–protein interactions and forms a “tweezer” structure probably involved in DNA binding. Moreover, sequence analysis revealed conserved motifs that may be associated with dimerisation. Structural analysis also showed that the tridimensional structure of EbfC/YbaB is highly conserved within the bacterial domain. The DNA-binding activity was observed in different bacterial species, suggesting that this protein can protect DNA during stress conditions. These findings indicate that EbfC/YbaB is a broadly distributed NAP. Here, we present a review of the existing data on this NAP.Genomic compaction is an essential characteristic of living organisms. Nucleoid-associated proteins (NAPs) are a group of small proteins that play crucial roles in chromosome architecture and affect DNA replication, transcription, and recombination by imposing topological alterations in genomic DNA, thereby modulating global gene expression. EbfC/YbaB was first described as a DNA-binding protein of  Escherichia coli and other species [The genome of a living organism must be compacted in an organised process to fit into the cell. Therefore, genomic compaction is an essential characteristic for all organisms. In prokaryotes, the chromosome is compacted by a combination of factors such as molecular crowding, DNA supercoiling, DNA transactions, and association with different DNA-binding proteins . Nucleoi species .Salmonella enterica, Fis is characteristically expressed during exponential growth [NAPs show variations in expression level and the DNA sequences they bind to, and some exhibit preferences for sequences and/or DNA topological features located along the bacterial genome. For instance, in l growth ,5, wherel growth . In addil growth . For insl growth . FurtherIn addition to their topological effects on DNA, NAPs modulate many biological functions ,7,8. ForIn contrast, NAPs can allow contact between enhancer and promoter sequences by bending the DNA, thereby promoting transcription. Direct contact between NAPs and RNApol has also been reported, where the former guides the latter in the transcription process . Howevererp-binding factor, chromosomal), also known as YbaB, is a less-studied and widely distributed NAP among prokaryotes [Borrelia burgdorferi sensu lato (referred here as B. burgdorferi), which causes Lyme disease [Haemophilus influenzae [E. coli , and other bacterial species have been determined. Structural analyses did not reveal a characteristic DNA-binding domain; however, experimental studies have demonstrated that these proteins bind to DNA with distinct sequence preferences [E. coli, but this does not seem to be the case for Streptomyces [Salmonella enterica is not essential for pathogenesis in a mouse model of systemic infection (unpublished results).EbfC  [dnaX, ybaB, and recR in the chromosome of E. coli has also been observed in other bacteria. The colocalisation of ybaB with dnaX and recR suggests a possible role for ybaB in DNA recombination, repair, and/or replication. The involvement of NAPs in the modulation of DNA transactions corroborates this suggestion. However, synteny among dnaX-ebfC/ybaB-recR has not been observed in many other bacterial species, which contradicts this hypothesis.RecR is a component of the RecFOR recombination system found in different bacterial species and is involved in DNA recombination and repair . dnaX en(DNApol) . The orgybaB belonged to the sigma E (σE) regulon in E. coli [E. coli by an unknown mechanism [ebfC/ybaB are discussed further in this paper.Using a two-plasmid system to identify promoters recognised by RNA polymerase containing the sigma E factor,  E. coli . The σE  E. coli . Interesechanism . This obhttps://www.ebi.ac.uk/interpro/entry/InterPro/IPR004401/protein/UniProt/#table, accessed on 10 June 2022). The majority (99.19%) of putative YbaB/EbfC proteins contain 94–133 amino acids. The largest sequences comprising 180 and 182 amino acids also belonged to the YbaB/EbfC family  [E. coli (PDB accession 1pug), Clostridium thermocellum (PDB accession 1ybx), Helicobacter pylori (PDB accession 3f42), and Mycobacterium tuberculosis (PDB accession 5yrx) [So far, the investigations have revealed that YbaB forms homodimers composed of α-helices and β-sheets with two α-helices protruding from a globular region, forming a tweezer-like structure ,34. To don 1j8b) , E. colion 5yrx) . The canon 5yrx) ,33,48. Ton 5yrx) ,49. The on 5yrx) .B. burgdorferi, H. influenzae, and E. coli binds to DNA [This structure has no characteristic DNA-binding domain; however, experimental evidence showed that the EbfC (YbaB) of s to DNA ,34. Addis to DNA . This tolta1) that controls the tiller angle and gravity response in rice and has homologs in other plants presents a conserved YbaB DNA-binding domain [Thus far, all the data indicate that EbfC/YbaB could be grouped into a family of DNA-binding proteins. The members of this family are encoded by a broad range of bacteria ,53,54,55g domain . HoweverB. burgdorferi, H. influenzae, E. coli, Deinococcus radiodurans, and C. crescentus [B. burgdorferi, EbfC has been shown to bind to DNA as a homodimer, but other organised structures can also be formed [B. burgdorferi EbfC were demonstrated using an electrophoretic mobility shift assay (EMSA). Experimental data indicated that this protein binds to DNA through its α-helical domains. As discussed above, the two α-domains form a protruding tweezer, which is hypothesised to form a DNA-binding domain. Indeed, EMSA analyses showed that EbfC protein variants carrying mutations in nine amino acids located in the α-helical regions  were unable to bind to the promoter region of erp genes, even at a higher concentration of mutant proteins than that of the wild-type. Protein variants were constructed by mutating wild-type amino acids with alanine [B. burgdorferi [H. influenza. Although mutations at these residues lead to a loss of the DNA-binding capacity of the B. burgdorferi EbfC protein, the mutants still formed higher-order structures such as tetramers and octamers in solution, except for N77. The location of the N77 residue near the β-sheet region probably affects oligomerisation. These results indicated that EbfC multimerisation is independent of its ability to bind DNA [The DNA binding ability of EbfC/YbaB has been demonstrated in escentus ,34,47,55e formed ,29. The  alanine . These r alanine . Interesgdorferi  are not bind DNA .B. burgdorferi indicated that both the N- and C-termini are involved in the formation of the α-domains and participate in DNA-binding activity [C. crescentus lacking the putative C-terminal DNA-binding domain was constructed and tested for its binding activity to the operator sequences of erpAB operon. The truncated YbaB protein could not form stable protein–DNA complexes, indicating that this domain is essential for DNA-binding activity. Its removal leads to the loss of double-stranded DNA-binding activity in C. crescentus [The data obtained for the EbfC protein of activity . To deteescentus .B. burgdorferi [GTnAC-3′ with high affinity, which is spread throughout the genome of B. burgdorferi [B. burgdorferi cells containing a fusion GFP-tagged EbfC protein indicated that EbfC colocalises with bacterial nucleoids in multiple centres [H. influenzae and E. coli exhibited preferences for specific DNA sequences, they did not show high-affinity binding for the palindromic sequence 5′-GtnAC-3′, as observed for B. burgdorferi [H. influenzae and E. coli are unknown, but they are probably different from those of B. burgdorferi [D. radiodurans and C. crescentus [C. crescentus suggested that the binding of the YbaB protein to DNA is not sequence-dependent; however, further studies are needed to clarify this question.Available evidence suggests that EbfC/YbaB binds to DNA in sequence-specific and non-sequence-specific manners ,30,33,34gdorferi . EMSA, dgdorferi ,29,30. I centres ,29. Alth centres . Althougescentus ,55. BothB. burgdorferi, EbfC regulates the expression of pathogenic traits [erp operons by binding to operator sequences in the promoter region of erp genes. Both EbfC and BpaB  bind to the regulatory region of the erp operons. BpaB is a repressor that inhibits the transcription of erp. This protein binds cooperatively to the erp operator, thereby blocking the binding of RNApol to the promoter. This repression is enhanced by the binding of BpuR , another DNA-binding protein [erp operons and competes with BpaB to bind to the regulatory region, thus stimulating the transcription of erp genes [ebfC exhibited a higher transcription level during the exponential growth phase than during the stationary phase, where transcripts were not detected. The expression of bpaB and bpuR was higher in unfed ticks in the insect’s midgut; however, ebfC expression was higher during the transmission of the bacteria from the feeding tick to the host, which explains the activation of erp transcription [erp operons is inhibited by BpaB in the non-fed tick vector, but it is highly induced during the infection of mammals [erp-binding factor, chromosomal (EbfC) [bpaB is a gene located in extra-chromosomal elements, whereas bpuR and ebfC are chromosomal genes [The functions of EbfC/YbaB have been described for several bacterial species . For exac traits . This bac traits . Cp32s, c traits . Among oc traits . EbfC wa protein ,58. EbfCrp genes ,38. ebfCcription ,31,38. T mammals ,31,38,59l (EbfC) ,29,30,31dnaX-ebfC expression. DnaA is necessary for DNA replication and functions as a regulatory protein [dnaX encodes DNA polymerase III subunits, these findings suggest a link between DNA replication and ebfC expression in B. burgdorferi [As discussed previously, Stevenson et al.  recently protein . Howevergdorferi .B. burgdorferi by ebfC deletion have been unsuccessful, suggesting that this is an essential protein in this species [E. coli, Streptomyces lividans, and Deinococcus radiodurans [Attempts to construct mutants of  species ,31. Howeiodurans ,55,61.B. burgdorferi, and the transcriptome was analysed using RNA-seq [ebfC transcripts was 28-fold in the bacteria overexpressing EbfC than in the uninduced control. This overexpression did not measurably affect the growth rate; however, transcriptome analyses indicated that EbfC affects gene expression globally in B. burgdorferi. In the EbfC-overexpressing strain, the expression levels of 52 genes were affected positively or negatively, representing approximately 4.5% of the gene content of the bacterial strain studied [B. burgdorferi may explain this observation. The authors specified that smaller replicons carry most infection-related genes, whereas the chromosome is rich in housekeeping genes [dbpB, involved in adherence to host tissues, enzymes such as alanine racemase, and the gamma subunit of exodeoxyribonuclease V. The genes repressed by EbfC included flhB, which encodes a flagellar assembly/export protein, and bbb07, an outer membrane protein involved in tick colonisation [As an alternative method for analysing the effects of EbfC on global gene expression, the EbfC protein was overexpressed in  RNA-seq . Upon co studied . The genng genes . The funng genes . For insnisation .ebfC/ybaB has been suggested to play a role in DNA repair by forming an operon with recR. Indeed, results indicate that YbaB plays a role in DNA repair because a null-mutant E. coli exhibited higher sensitivity to radiation than the wild-type strain [Streptomyces is a genus of bacteria frequently used to study DNA repair because genetic instability is a common characteristic in this group. For example, a study to characterise the recR gene of Streptomyces demonstrated that mutants for recR or orf1recR of S. lividans presented an increased susceptibility to DNA-damaging agents [recR gene alone. Considering that orf1 is orthologous to ebfC/ybaB, orf1 does not seem essential for the repair function in S. lividans [orf107recR from Bacillus subtilis; however, attempts to complete them with B. subtilis recR alone or with the ybaBrecR region of E. coli failed [orf107 is also orthologous to the ebfC/ybaB gene. In addition, dr0199, a gene orthologous to ebfC in Deinococcus radiodurans, has been suggested to play a role in DNA repair [ybaB mutants, the occurrence of polar effects on recR should be considered. This is particularly important in species in which ybaB and recR translations are coupled, such as E. coli. Based on all these results, further studies are necessary to characterise better the role of EbfC/YbaB protein in DNA repair.In some bacterial species, e strain . Howeverg agents . Both mulividans . Interesi failed . orf107 A repair . HoweverybaB appears to belong to the σE regulon of E. coli, as demonstrated by a two-plasmid system [S. enterica σE regulon [Enterobacteriaceae family. The σE regulon is activated by a cascade of events in response to misfolded and/or mis-translocated outer membrane proteins or LPS in Gram-negative bacteria as a consequence of exposure to different stress conditions [E coli, σE is an essential gene; however, compensatory mutations can generate variants that can survive.Studies have also indicated that EbfC/YbaB may participate in the stress response ,55,62. Ad system . However regulon ,65, a phnditions . In E coS. enterica, and the mutants are viable [E. coli, in some cases by a factor of 10-fold [E. coli [In contrast, this gene is not essential in e viable . Interes 10-fold . Therefo[E. coli . These rD. radiodurans is a model organism for studying DNA repair based on its high capacity to repair DNA damage caused by mutagenic agents. This bacterium contains a gene (dr0199) orthologous to EbfC. A strain carrying a deletion mutation in dr0199 exhibited high sensitivity to hydrogen peroxide and UV and gamma radiation, which was complemented by the reintroduction of the wild-type gene [D. radiodurans. In Paenibacillus riograndensis, a plant growth-promoting bacterium, the ebfC/ybaB ortholog (0116.0006 0.0231) was highly expressed during growth in an iron-limiting medium when compared to growth under sufficient iron conditions [C. crescentus demonstrated that this protein binds to DNA in a non-specific manner, can compact DNA, and protects it against enzymatic degradation [Studies with different bacteria have suggested a possible role for EbfC/YbaB in DNA protection. ype gene . The autnditions . The mecnditions . Furtherradation . The rolradation .E. coli, ClpYQ is an ATP-dependent protease, and YbaB/EbfC was demonstrated to be one of the targets of this protease [E. coli, reinforcing the need for further studies to better characterise the roles of this NAP in prokaryotes.Protein turnover is an essential characteristic of any organism and is necessary for modulating peptide and protein levels, including misfolding and regulatory proteins. In protease . These dB. burgdorferi, EbfC binds specifically to DNA sequences with high affinity. However, this protein can also bind to DNA sequences in a lower affinity non-specific manner. Data from the literature suggest that EbfC/YbaB orthologs may be involved in the bacterial envelope stress response, DNA protection, and repair. However, further studies are required to clarify the role of EbfC/YbaB orthologs in these processes. EbfC of B. burgdorferi is undoubtedly the best-characterised EbfC/YbaB ortholog in terms of DNA binding, regulation of pathogenic traits, and as a global regulator. Taken together, these data suggest that EbfC/YbaB is a NAP, but its biological function has not yet been characterised in most prokaryotic species. Therefore, further studies are necessary to better describe the biological roles of this NAP in prokaryotes.EbfC/YbaB is a widely distributed family of prokaryote proteins capable of binding to DNA, as demonstrated in different studies. Furthermore, the structure of these proteins is conserved, exhibiting a general topology of N-α1/β1/β2/β3/α2-C, with regions involved in dimerisation and DNA-binding. The DNA-binding region, structured as a pair of tweezers, probably represents a DNA-binding domain that is not well-characterised yet. Despite its wide distribution, the roles of EbfC/YbaB have been described in a few bacterial species. In


Tumor multifocality is frequently seen in Papillary thyroid carcinoma (PTC). However, few studies have analysed the impact of bilateral multifocality in PTC. The incidence of bilateral multifocality, its clinico-pathological associations and prognostic impact in PTC from Middle Eastern ethnicity remains unestablished.We retrospectively evaluated 1283 patients who underwent total thyroidectomy for PTC. Bilateral and unilateral multifocality were decided based on the final pathology result. Primary outcome was recurrence free survival (RFS). Risk factors for bilateral multifocality were analyzed by multivariate logistic regression analysis.Multifocal PTC was found in 54.3% (697/1283) of patients. Among the 697 multifocal PTCs, 210 patients (30.1%) had unilateral multifocal PTC and 487 patients (69.9%) had bilateral multifocality. Bilateral multifocality was significantly associated with older age at diagnosis (p = 0.0263), male gender (p = 0.0201), gross extrathyroidal extension (p = 0.0332), larger primary tumor size , lateral lymph node metastasis (p = 0.0008), distant metastasis at diagnosis (p = 0.0195) and recurrence (p = 0.0001). Bilateral multifocality was also found to be an independent predictor of RFS . Multivariate logistic regression analysis demonstrated tumor diameter >4cm to be the only independent risk factors for bilaterality in multifocal PTC .Incidence of bilateral multifocality is high in Middle Eastern PTC. Tumor diameter >4cm can be considered as a predictive factor for bilateral multifocal PTC. Bilateral multifocality appears to be an important prognostic factor for PTC and an independent predictor of RFS. Therefore, patients with bilateral multifocal PTC may benefit from more frequent follow-up to identify recurrences earlier. Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy . Its incPTC with multifocality frequently arise in thyroid gland and has been considered as prognostic marker in PTC –17. SeveIdentifying predictive markers of bilateral multifocality and its clinico-pathological associations as well as clinical impact on patients’ prognosis could guide surgeons and oncologists to determine the appropriate treatment for PTC patients. In this retrospective study we investigated the incidence of multifocality in PTC from Middle Eastern ethnicity and its clinico-pathological associations. Moreover, we aimed to determine whether bilateral multifocality can predict patients’ prognosis in this ethnicity.One thousand five-hundred and fifteen consecutive unselected PTC patients diagnosed between 1988 and 2018 at King Faisal Specialist Hospital and Research Centre  were available to be included in the study. Only those patients with information on tumor focality who underwent total thyroidectomy were included in the study. A total of 1283 PTC cases were available to be included in the study. Of these, 697 patients had multifocal PTCs and were included for final analysis. Cases were identified based on clinical history followed by fine needle aspiration cytology for confirmation. The Institutional Review Board of the hospital approved this study and the Research Advisory Council (RAC) provided waiver of consent under project RAC # 221 1168 and # 2110 031.Baseline clinico-pathological data were collected from case records and have been summarized in BRAF mutation data was assessed in our laboratory by Sanger sequencing and has been published by us previously (eviously .Radioactive iodine (I-131) therapy was given to 83.2% (1068/1283) of PTC patients in our cohort. All ATA high- and intermediate-risk patients were offered I-131 therapy, whereas low-risk patients with lymph node metastasis also received I-131 therapy. Post-operative serum thyroglobulin levels were analyzed at 6 months and 1 year after surgery. 6.5% (84/1283) of patients showed an elevated post-operative serum thyroglobulin level (> 2ng/ml). Tumor recurrences were detected by serum thyroglobulin levels, whole body scan, ultrasound or CT scans performed during regular follow-up visits. Recurrence-free survival (RFS) was defined as the time (in months) from date of initial surgery to the occurrence of any tumor recurrence . In case of no recurrence, date of last follow-up was the study endpoint for RFS.The associations between clinico-pathological variables and tumor focality was performed using contingency table analysis and Chi square tests. Mantel-Cox log-rank test was used to evaluate RFS. Survival curves were generated using the Kaplan-Meier method. Cox proportional hazards model and logistic regression were used for multivariate analysis. Two-sided tests were used for statistical analyses with a limit of significance defined as p value < 0.05. Data analyses were performed using the JMP14.0  software package.BRAF mutation data was available in 576 cases, with mutation being present in 62.7% (361/576) of multifocal PTCs. 20.5% (143/697) of patients developed tumor recurrence. (Median age of the entire cohort was 38.2 years (range: 6 – 89 years), with a male: female ratio of 1:3. Majority of the tumors were of classical variant . 24.0% (167/697) of multifocal PTCs were larger than 4cm in largest diameter. Regional LN metastasis was noted in 58.1% (405/697) of cases and distant metastasis was present at diagnosis in 6.5% (45/697). urrence. Table 1.Of the 697 multifocal PTCs included in our study, 69.9% (487/697) were bilateral and 30.1% (210/697) were unilateral. Bilateral multifocality was significantly associated with older age at diagnosis (p = 0.0263), male gender (p = 0.0201), gross extrathyroidal extension (p = 0.0332), larger primary tumor size , lateral lymph node metastasis (p = 0.0008) and distant metastasis at diagnosis (p = 0.0195) Table 2.Since we found bilateral multifocal PTCs to be associated with tumor recurrence, we analysed the prognostic significance of bilateral multifocality with regards to RFS. Using Mantel Cox log rank test, we found that bilateral multifocality was associated with shorter RFS (p = 0.0006) Figure 1Next, we sought to determine the clinico-pathological features that could predict bilateral multifocality in PTCs. On univariate analysis using logistic regression model, we found older at diagnosis, male gender, gross extrathyroidal extension, larger tumor size (>4cm), regional lymph node metastasis and distant metastasis at diagnosis to be significantly associated with bilateral multifocality. We included these significant clinico-pathological parameters in a multivariate logistic regression model and found that only tumor size >4cm  was an independent predictor for bilateral multifocality in PTCs Table 4.The optimal clinical management of PTC depends on appropriate clinico-pathological risk stratification. Therefore, accurately evaluating the clinico-pathological risk factors is important in achieving the balance between higher therapy benefit and lower adverse complications. Multifocality as prognostic marker remains a highly controversial subject , 29–33. In our cohort, multifocality was observed in 54.3% of patients, which is comparable with what has been reported in literature , 23, 35.Interestingly, bilateral multifocality was significantly associated with unfavorable clinical outcomes, being an independent predictor of RFS. The prognostic value of multifocality was significant in bilateral multifocality subgroup compared to unilateral multifocal PTC subgroup, showing an approximately two-fold increased risk of recurrence. Therefore, this study has identified that bilateral multifocality is a risk factor for recurrence after total thyroidectomy in Middle Eastern PTC patients. This is consistent with a previous report which showed that bilateral multifocality was an independent risk factor for neck recurrence as well as distant metastasis .BRAF mutation as predictors of bilateral multifocal disease (BRAF mutation was not associated with bilateral multifocal disease in this study.Given the strong prognostic impact of bilateral multifocality, we further evaluated if there are any predictive markers of bilateral multifocal PTCs. We found that tumor diameter of >4cm was the only independent predictor for bilaterality in multifocal PTCs. Previous studies have identified number of tumor foci, TSH level, and  disease , 43. HowThe present study supported the finding of previous studies that multifocality is associated with aggressive disease , 42, 43.Our study has a few limitations. Firstly, this is a retrospective single institute study which includes only PTC patients from Saudi Arabia and hence selection bias is inevitable. The results of this study should be interpreted with caution and generalizability of our results to other ethnicities needs to be validated in large studies from different ethnicities. Secondly, data on the number of tumor foci are not routinely recorded in our histopathological reports and hence we were unable to incorporate this interesting data in our current study.In conclusion, bilateral multifocality is high in Middle Eastern PTC and is associated with aggressive clinico-pathological markers. Tumor diameter >4cm was predictive of bilateral multifocality in PTC. Bilateral multifocality appears to be an important prognostic factor for PTC and an independent predictor of RFS. Therefore, patient with bilateral multifocal PTC may benefit from more frequent follow-up to identify recurrences earlier.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Research Advisory Council, King Faisal Specialist Hospital and Research Centre. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.Study concept and design: KA-K, SP, AS. Executed the study: SP, AS, PA, NS, SA-S, FA-D. Statistical analysis: SP. Drafting the article: KA-K, AS, SP. Critical revision of the article for important intellectual content, writing of the article, and approval of the final version: KA-K, SP, AS, PA, NS, SA-S, FA-D. All authors contributed to the article and approved the submitted version.


Leishmania parasites, which are the causative agents of leishmaniasis. Herein, we developed an amplicon-based next-generation sequencing (Amp-NGS) to characterize sand flies and Leishmania parasites simultaneously targeting partial fragments of 18S rDNA and ITS1 genes, respectively.Phlebotomine sand flies are vectors of n = 8) and Leishmania spp. (n = 9) samples and validated using wild-caught sand flies from Palestine. The assay was highly specific, and all DNA references were successfully identified to the species level.Our assay was optimized using reference sand fly (n = 187), Phlebotomus spp. represented 95% of the collected samples (177/187), including Ph. sergenti , Ph. papatasi , Ph. perfiliewi , Ph. tobbi  and Ph. syriacus . Sergentomyia spp. represented only 5% (10/187) of the collected samples and included S. dentata (n = 6), S. fallax (n = 2), S. schwetzi (n = 1) and S. ghesquiere (n = 1). The study observed strong positive correlation between sand fly identification results of the Amp-NGS and morphological identification method . Some discrepancies between the two methods in the identification of closely related species  were observed. Leishmania DNA was detected and identified as L. tropica in 14 samples .Among the wild-caught sand flies  and identify The online version contains supplementary material available at 10.1186/s13071-022-05388-3. Leishmania spp. described worldwide, about 20 cause disease in humans, including cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis (MCL)  with read length < 100 bp were removed from the analysis [Leishmania parasite identification.Amp-NGS for both analysis . Moreovek) = 0.976], indicating high reliability of the Amp-NGS . Similarly, some samples of Sergentomyia fallax, S. dentata and S. theodori species were interchangeably identified by the morphological method. The separation of these species is mainly based on differences in shape and armature of the structures of mouthparts and the pharynx. It is reported that some sand flies belonging to the same genus do not comply with all known morphological features, leading to misidentifications [The sand fly species identified by Amp-NGS were in perfect agreement with the morphological identification as the reference method [kappa index . The genus Phlebotomus was further divided into two sub-clusters, CL and VL , the main dominant genus of the sand fly in Palestine was Phlebotomus spp. (95%) compared to the low prevalent Sergentomyia (5%). Phlebotomus spp. are the main incriminated vectors of Leishmania parasite [Phlebotomus spp., 83% were Ph. sergenti, the L. tropica vector, and 11% were Ph. papatasi, the L. major vector, which concords with a previous study [Here, despite a relatively small size of samples tested (parasite . Out of Leishmania species. This assay allows the identification of large numbers of sand flies, which may be useful for large-scale field studies.Our newly developed Amp-NGS assay was optimized and validated as a powerful tool for the simultaneous identification of sand flies and Additional file 1: Table S1. Reference DNA samples for Leishmania and sand fly species used in the study. Table S2. Sand flies and Leishmania GenBank accession numbers used in this study for primer designing. Table S3. The virtual probe sequences used for detection and identification of sand flies and Leishmania parasites.