AbstractOrthoceratium was collected in Greece that differs morphologically from the European species commonly presumed to be Orthoceratiumlacustre . Verification of the identity of the Greek species through comparison with 460 specimens of Orthoceratium from 17 West Palaearctic and one Afrotropical country, and examination of existing type material, revealed that the species recognized as O.lacustre in northwestern Europe for over 250 years is actually O.sabulosum , the other known species in the genus, which was originally described from Tunisia. Although the types of O.lacustre have been lost, a comparison of the distribution ranges of both species in Europe provided evidence that the species collected in Greece is conspecific with O.lacustre. Both species have distinct distributions in the West Palaearctic, with O.lacustre largely restricted to the northern border of the Mediterranean basin, and O.sabulosum more widespread, occurring in northwestern Europe, the western, southern, and eastern Mediterranean, the Middle East, and the Afrotropical Region (Tanzania). Both species are redescribed and fully illustrated, a neotype is designated for O.lacustre and a lectotype for O.sabulosum, and a key to males and females is provided. The misidentifications that lasted for over two centuries are explained by the omission by previous authors to study the type specimens, and inaccuracies in species descriptions and keys.Recently, a species of  Orthoceratium Schrank, 1803 is a nearly exclusively West Palaearctic dolichopodid genus in the subfamily Hydrophorinae  has also been reported from Tanzania  are given in the following key:phorinae , with onphorinae . OrthoceTanzania , though Tanzania . It seemO.lacustre in Flanders (northern Belgium) after an absence of nearly 40 years. The authors also provided a full account of the distribution records of this conspicuous species in the western Palaearctic realm , and remarked that another Orthoceratium species had been collected in a mountainous region in Greece (further referred to as ‘species B’). At present, this hydrophorine genus only includes one other species, O.sabulosum , thus far only recorded from Tunisia.O.lacustre had been lost , and that the status of O.sabulosum type specimens in Becker’s collection could be questioned. Moreover, series of Orthoceratium specimens from different European museums contained both O.sabulosum and ‘species B’. It thus appeared crucial to establish whether ‘species B’ actually corresponded with O.lacustre or represented a new, third species.In the process of verifying the identity of ‘species B’, the depository and availability of the type specimens of both described species was checked. This revealed that the Scopoli types of In the present paper, we present the results of this study, and give (re)descriptions of the species and information on their distribution and ecology. We finally discuss the plausible reasons for the continuous series of misidentifications, and the significance of type specimen examination.Orthoceratiumsabulosum from the Becker collection  with a lectotype and paralectotype label resp., were examined. Although the validity of their designations might be questioned (see Redescription of O.sabulosum), there is no doubt that these specimens are part of Becker’s original type series. Types (presumably syntypes) of both O.sabulosum  and O.lacustre (Scopoli collection) appear to be lost. All insects collected by Scopoli in Carniola were destroyed during fires in Scopoli’s house in Idria  in 1787  and that of Miroslav Barták  were also investigated.Two specimens of  in 1787 . None of(Re)descriptions are based on a large number of representative specimens of each species, both in alcohol and pin-mounted. A total of 173 character states was scored, with 35, 61, and 77 related to the head, thorax/abdomen/wing, and legs respectively. This allowed us to determine the most reliable and consistent decisive diagnostic features that were subsequently applied in the key.Relevant non-genitalic diagnostic characters in collected specimens were photographed by the junior author. The hypopygium and tergite V of each species were drawn using a camera lucida. The left lateral view of the hypopygium is illustrated here. In describing the hypopygium, ‘dorsal’ and ‘ventral’ refers to the morphological position prior to genitalic rotation and flexion. Thus, in the drawings showing a lateral view of the hypopygium, the top is morphologically ventral, while the bottom is dorsal.1, (vi) proximal versus apical section of vein CuA1, (vii) CuAx ratio  and (viii) relative lengths ratio of femur, tibia and tarsomeres of each leg. The latter relative lengths were recalculated so that the shortest leg part represents a value of “1”. Wing length was measured in both dry and wet specimens. All values given in this paper are average values, unless otherwise mentioned. Palp and proboscis size is compared to the eye size, measured as the vertical diameter (from about ocellar tubercle to the lower eye margin). Wing length was measured in 76 and 142 specimens of O.lacustre and O.sabulosum resp., to find out if differences occurred between both species and separate populations  of each gender in each of the two species unless otherwise mentioned, and include: (i) face width, (ii) body length, (iii) wing length (= distance between basis of basicosta and wing apex), (iv) relative wing width, (v) proximal versus apical section of vein MOrthoceratium specimens are given in Figure Capture locations of The general morphological terminology follows ac acrostichal bristles;ad anterodorsal;ant pprn anterior postpronotal  male secondary sexual character(s);npl notopleural;pal postalar;pd posterodorsal;psut ial presutural intra-alar  to apical (5) tarsomeres;T abdominal tergite;vtventral;I, II, III refers to fore, mid and hind leg;I–VI in the descriptions of abdominal segments (tergites/sternites) refers to basal (I) to caudal (VI) segments.Institutional, collection and other abbreviations:ANSC Andreas Stark private collection, Halle/S., Germany;NHMUKThe Natural History Museum, London, UK;HMNHHungarian Museum of Natural History, Budapest, Hungary;IBER Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria;MAPC Marc Pollet private collection, Welle, Belgium;MIBC Miroslav Barták private collection, Prague, Czech Republic;MNHNMuséum national de l’Histoire naturelle, Paris, France;MLUH Zentralmagazin naturwissenschaftlichen Sammlungen, Martin-Luther-Universität, Halle/S., Germany;NHMWNaturhistorisches Museum, Vienna, Austria;RBINSRoyal Belgian Institute of Natural Sciences, Brussels, Belgium;RMNHNaturalis Biodiversity Centre, Leiden, Netherlands;ZFMKZoologisches Forschunginstitut und Museum A. Koenig, Bonn, Germany;ZLKU Zoology Laboratory, Department of Biology, Faculty of Science, Muğla Sıtkı Koçman University, Muğla, Turkey;ZMHBMuseum für Naturkunde, Berlin, Germany;ZMUCZoological Museum, University of Copenhagen, Copenhagen, Denmark.MT: Malaise trap, SW: collected by sweepnet.Other abbreviations: Orthoceratium). The site code is also used in Figure Label information of mounted specimens is provided in full and with the original spelling. If not indicated otherwise, the label was white and rectangular, and information is from the top side. Label information is given from the top downward, with data from each label between quotation marks, and data from different lines on the same label separated by a slash (/). Information from different labels is separated by a semi-colon . The species record is followed by the repository of each specimen between square brackets . In addition to the label information, for non-type specimens, the most relevant label information is enriched, uniformly structured and given in the following format: “(site code) – COUNTRY: ♂, ♀, province , locality, location/area, latitude, longitude, altitude, sampling date (start) – sampling date (end), sampling method, collector [collection]”  also confirmed the identity of the specimens listed in Orthoceratium material: NMPC: National Museum , Prague, Czech Republic; MSNVE: Museo di Storia Naturale, Venice, Italy. The specimens originated from 17 different countries in the West Palaearctic, including 13 European, two North African and two Middle East ones, and one Afrotropical country.A total of 428 specimens of Orthoceratiumsabulosum revealed that the species widely known (and collected) as ‘O.lacustre’ in northwestern Europe was conspecific with this species. This held true for nearly all specimens of ‘O.lacustre’ examined from North Africa and the Middle East  as well. The question evidently raised if ‘species B’ then represented the true O.lacustre or not. As mentioned before, establishing the species concept of the latter species proved difficult due to the loss of the type material. Hence, the original description and other literature sources were studied carefully in search for information on significant diagnostic features that matched those of ‘species B’.To our surprise, the examination of the type material of Muscalacustris  like e.g., inland lakes, and did not seem to be confined to saltmarshes or brackish marshes like O.sabulosum in northwestern Europe.Unfortunately, all listed characters fit both O.lacustre were destroyed as early as 1787, it is unlikely that As the Scopoli type specimens of “Wings hyaline, usually tinged with ferruginous towards the fore edge, … Abdomen of the male … lamella oblong, compressed, broad at tip and truncated. … On waters, both fresh and brackish. (E[ngland]. I[reland]).”O.lacustre’ from England or Ireland, which now appears to be O.sabulosum.However, it can be assumed that Haliday based this description on ‘Liancaluslacustris):“Beine schwarz mit gelben Knieen und Gelenken. — Metallisch-grün. Untergesicht silberweiss schimmernd. Fühler schwarz. Rückenschild undeutlich gestriemt. Analanhänge länglich, zusammengedrückt, am Ende breit und abgestutzt. Schenkel oben grün, auch der hinterste Metatarsus. Flügel glashell, gegen den Vorderrand gewöhnlich bräunlich, gelblich tingirt, die vierte Längsader gebrochen. 2 2/3 ‘’’. Nach Scopoli in [Herzogtum] Krain; ich erhielt die Art durch Hrn. Micklitz aus dem Küstenlande.”O.lacustre. The former author did mention that O.lacustre  was considered much rarer than Liancalusvirens  and that its distribution ranged from England to Sicily. It seems like he actually saw specimens as he reports on the colour of teneral specimens.Neither Alloeoneurussabulosus on the basis of specimens from Tunis (HMHN); two years later this genus was listed as synonym of Orthoceratium by A.lacustris and featuring brownish wings. In his key to the species, he summarizes the most diagnostic features that separate both species as follows:O.lacustre in this key that matches ‘species B’ is the narrower face, compared to O.sabulosum. But specimens of both species show an equally large variation in the colour of the wings and the dusting of their pronotum. This is quite remarkable as we believe that Becker actually had specimens of both species at hand when he described O.sabulosum, as he repeatedly refers to O.lacustre. Why he used doubtful differences in his key or even mixed up features (see further) is unclear, but possibly he only had one single or a small number of O.lacustre specimens to compare with.The only feature of O.lacustre. The shape of the ventral process of the 5th tergite , and reported O.lacustre from France, and O.sabulosum from Tunisia. His description of O.lacustre, however, clearly points to O.sabulosum: face wide, about 2/5 of eye width, and a black bristle present amid coxa I. Some of his figures  confirm this conclusion.O.lacustre are included (O.sabulosum types (ZMHB), however, without giving details on the specimens. On the basis of the description and especially the drawings of the hypopygium , we can conclude that Negrobov’s O.lacustre matches ‘species B’ exactly. Next to the fact that it was unclear where the specimens – that he used for the description of O.lacustre – originated from , the question remained if his species (and ‘species B’) was the true O.lacustre. Indeed, Negrobov was not able to examine the type specimens of O.lacustre nor did the extant literature provided decisive information (as shown above).The key to both species by included  though tO.lacustre. Scopoli collected the species in Carniola (‘Krain’ in German), a historical region that corresponds mainly with inland parts of present-day Slovenia, including mountains. Since Scopoli stated that the species skated on backwaters [“ambulat super aquas stagnantes”] and occurred in or along lakes [“in lacubus”], it could further be assumed that he collected the species in inland wetland habitats .In a second stage of the verification process the type locality was considered to comprise a possible clue about the identity of O.sabulosum and ‘species B’, capture locations of both species were plotted on a map of the West Palaearctic , southwestern (Algeria and Tunisia in North Africa) and eastern borders of the Mediterranean basin , but also Iran , the locality closest to the type locality of this species, was selected as neotype Genus Taxon classificationAnimaliaDipteraDolichopodidaeMuscalacustrisOrthoceratium by Schrank (1803) Scopoli, 1763: 343. Type locality: Carniola (= present-day Slovenia) – presumably transferred to Muscaformosa Haliday, 1832 and Medeterusviridipes Macquart, 1834, previously listed as synonyms of O.lacustre, clearly refer to O.sabulosum (see further).1 with strong sinous bend at ½. Posterior border of wing indented at vein CuA1. Coxa I with strong white pubescence, and with three black bristles only at apex. Coxa II with only pale bristles at apex anteriorly. Pedicel with short apical bristles ; wing length: 5.2–6.2 mm (n = 44), 0.3 × as wide as long. Head ; crossvein dm-cu rather straight; posterior border of wing indented at vein CuA1. Proximal section of vein M1 1.9 × as long as apical section. Proximal section of vein CuA1 8.5 × as long as apical section. CuAx ratio: 1.7. Halter pale, calypteral fringe yellowish white. Legs ; with small ovoid brownish yellow pv tuft just beyond basal 1/4, about 1/8 of femur length (MSSC); with one rather small pv preapical bristle. Femur II with one strong ad preapical bristle, at less than apical 1/5, and with one small pv preapical bristle; with one row of very short white erect vt setae on basal 1/3 (MSSC), and with one row of short inclined pv setae along entire length, white on basal 2/3 and black on apical 1/3, longest at basis and apex. Femur III with one strong ad preapical bristle, at about apical 1/3, and one small pv preapical bristle; sometimes with some thin inclined (thus not erect!) ds bristles in basal 1/5. Tibiae I–III brilliant metallic green to violet, tibia I with basal 1/8, tibia II with basal 1/9, and tibia III with less than basal 1/10 pale yellow. Tibia I with two ds bristles, 2–3 × as long as tibia is deep; with two small ad bristles, 1–1.5 × as long as tibia is deep, and with two to three pv bristles, 2–3 × as long as tibia is deep; with white pilosity on av face along entire length, and with two small ap bristles. Tibia II with three ad bristles, about 3 × as long as tibia is deep, with basal bristle shorter; with two pd bristles, 2 × as long as tibia is deep, with basal bristle shorter, and with four ap bristles; with one av bristle at basal 2/3 and one pv bristle at basal 1/5, both 2 × as long as tibia is deep; and two small pv bristles in apical 1/2, not as long as tibia is deep. Tibia III with five ad bristles, about 2.5 × as long as tibia is deep, four strong and one small pd bristles, former about 2.5 x, latter not as long as tibia is deep, and four strong ap bristles; with distinct pd row on apical 1/2; with three-four av bristles, 1–1.5 × as long as tibia is deep, and with multiple shorter pv setae along entire length. Tarsi I–III dark brown, with taI1 mostly unmetallic and taII1 and taIII1 with metallic green to violet reflection. Tarsus I with taI1 with pale ventral pubescence (MSSC) with some darker short bristles; taI5 with long curved dorsal setae at apex, 0.7 × as long as taI5 length; only inner claw present (MSSC). Tarsus II with taII1 with multiple short vt bristles, nearly as long as taII1 is deep. Tarsus III with taIII1 with multiple inclined vt bristles, longest about as long as taIII1 is deep. Ratio of femur/tibia/tarsomeres 1–5 in leg I: 10.3/11.4/6.3/2.2/1.5/1/1.1, in leg II: 9.5/12.2/8.4/3.7/2.4/1.3/1, and in leg III: 10.6/15.3/6.7/4.6/2.8/1.4/1. Abdomen   at 1/2; crossvein dm-cu straight. Proximal section of M 1.7 × as long as apical section. Proximal section of CuA1 6.9 × as long as apical section. CuAx ratio: 1.6. Coxa I with two to three rather small, black ap bristles. Femur I bare ventrally. Femur III often with some thin inclined ds bristles in basal 1/5. Tibiae I–II with basal 1/10 pale yellow; tibia I with three pv bristles. Tibia II with two av bristles at basal 2/5 and 2/3, 2–2.5 × as long as tibia is deep, and four small pv bristles on entirely length, not as long as tibia is deep. Tibia III with ad bristles about 3 × as long as tibia is deep, and with four strong pd bristles, 2.5–4 × as long as tibia is deep. Tibia III with av bristles 1.5–2.0 × as long as tibia is deep. Tarsus I with taI1 with multiple short black ventral bristles, nearly as long as taI1 is deep; taI5 with both claws. Ratio of femur/tibia/tarsomeres 1–5 in leg I: 8.6/9.5/5.7/2.3/1.5/1/1.1, in leg II: 9.1/11.6/8.1/3.4/2/1.2/1, and in leg III: 10.6/15/6.4/4.3/2.6/1.4/1. Abdomen with five pubescent segments, 6th invisible; also STIV with strong whitish dusting.ead Fig. . Face si7 mm n = 5; wing lrax Figs . Mesonotg Figure . Smokey 7 mm n = 5; wing legs Figs , 6A–B. O7 mm n = 5; wing ln Figure . Basal f) Figure . ST withe Figure . Body leg Figure  with veiITALY: NEOTYPE (here designated to fix the identity of the species) ♂, [brownish rectangular] “Liancalus/ lacustris Scp”/ “Görz.” ; “Zool. Mus.”/ “Berlin”; “Orthoceratium lacustre”/ “”/ “det. Marc Pollet, 2017”; [red rectangular] “NEOTYPE” / “des. Marc Pollet, 2018” .Scopoli, 763”/ “deOrthoceratium.See Suppl. material O.lacustre was selected on the basis of the locality , and the preservation status of the specimen  one Trieste specimen featured a strong curved black bristle on the right fore coxa.O.lacustre in the literature – many of which refer to O.sabulosum in reality – must be considered unreliable. Our present study revealed that O.lacustre has been collected nearly exclusively along the northern border of the Mediterranean basin (incl. adjacent islands), both in coastal habitats and inland (montane) habitats , and on the islands of Sardinia  and Lesvos (Greece). In the latter site, both species have been collected (in different years by different collectors) in the same area.As a result of the taxonomic mix-up between both species in the past, previous distribution records of O.sabulosum  could not be verified due to a lack of specimens, but it is very likely that the Irish and Madeiran records refer to O.lacustre has been recorded from mostly lowland locations in a 25 km zone along the Mediterranean coast, where it seems to occur along inland lakes which also corresponds with the description of the habitat of the type specimens AlloeoneurussabulosusZMHB] – Alloeoneurus presumably synonymized with Orthoceratium by Kertész (1909) Becker, 1907: 112. Type locality: Tunis (Tunisia)  Medeterusviridipessyn. n. Macquart, 1834: 452. Type locality: Bordeaux (France) [unknown] Muscaformosa Haliday, 1832 and Medeterusviridipes Macquart, 1834 were previously listed as synonyms of O.lacustre by O.sabulosum.Diagnosis. Rather large, short-bodied, rather slender (but stouter than O.lacustre), brilliant green species with abdomen 1.3 × as long as thorax ; wing length: 5.2–6.1 mm (n = 77), 0.3 × as wide as long (n = 15) mm. Head ; crossvein dm-cu slightly concave; posterior border of wing indented at vein CuA1. Proximal section of vein M1 2.0 × as long as apical section. Proximal section of vein CuA1 7.1 × as long as apical section. CuAx ratio: 1.6. Halter pale, calypteral fringe yellowish white. Legs ; with one rather small pv preapical bristle. Femur II with one strong ad bristle, at less than apical 1/5, and one small pv preapical bristle; with one row of short inclined pv setae along entire length, white on basal 2/3 and black on apical 1/3, longest at basis and apex. Femur III with one strong ad bristle, at about apical 1/3, and with one small pv preapical bristle; with one thin erect black ds bristle at about basal 1/5, nearly 0.5 × as long as femur is deep. Tibia I brilliant metallic green, and tibia I with basal 1/8, tibia II with basal 1/9, and tibia III with basal 1/10 pale yellow. Tibia I with two ds bristles, 2–3 × as long as tibia is deep; with two small ad bristles, 1–1.5 × as long as tibia is deep, and with three pv bristles, 2 × as long as tibia is deep; with white pilosity on av face along entire length, and with two small ap bristles. Tibia II with two ad bristles, 4 × as long as tibia is deep, rarely with 3rd much shorter basal bristle; with two pd bristles, about 2.5 × as long as tibia is deep, with basal bristle shorter, and with four ap bristles; with two av bristles, at basal 1/3 and 2/3, 2 × and 1.5 × as long as tibia is deep resp.; with one pv bristle, at basal 1/6, 2 × as long as tibia is deep, and two small pv bristles in apical 1/2, not as long as tibia is deep. Tibia III with five ad bristles, about 3 × as long as tibia is deep, two strong and two small pd bristles, former about 3.5 ×, latter 2 × as long as tibia is deep, and four strong ap bristles; with distinct pd row on apical 1/2; with four to five av bristles, 1.5 × as long as tibia is deep, and multiple shorter pv setae along entire length. Tarsi I–III black, with taI–III1 with metallic green to bluish reflection and with multiple short black vt bristles, nearly as long as taI–III1 is deep; taI5 with long curved dorsal setae at apex, 0.8 × as long as taI5 is long; only inner claw present (MSSC). Ratio of femur/tibia/tarsomeres 1–5 in leg I: 9/10/5.4/2.1/1.5/1/1.1, in leg II: 9/11/8/3.5/2.3/1.2/1, and in leg III: 9.8/13.8/6/4.2/2.6/1.3/1. Abdomen  ; wing length: 5.2–6.5 mm (n = 65), 0.3 × as wide as long (n = 15). As male, except for: abdomen 1.4 × as long as thorax. Face 2.0–2.3 × (n = 5) as wide as postpedicel (length). Palp about 1/5–1/4 of eye, ovoid. Uppermost six to nine postocular bristles erect, black. One pair of postocellar bristles, rarely with two pairs. Pedicel with some ventral bristles longer than pedicel is deep; postpedicel 0.8–1.0 × as long as deep; arista-like stylus 2.4–2.7 × as long as first three antennal segments combined. Thorax with five to eight ac, reaching between 4th and 5thdc. Vein R4+5 bended but straight near wing apex, there parallel with vein M1; apical section of vein M1 with weak bend (sinuous) at 1/2; crossvein dm-cu rather straight. Proximal section of M 1.7 × as long as apical section. Proximal section of CuA1 6.5 × as long as apical section. CuAx ratio: 1.5. Femur I bare ventrally. Femur III with two to four thin erect black bristles in basal 1/5, about 1/3 × as long as femur is deep. Tibia II with two large ad bristles, and often 3rd shorter basal bristle. Tarsus III black, unmetallic, taI1 with multiple short black ventral bristles ventrally, nearly as long as taI1 is deep; taI5 with both claws. Ratio of femur/tibia/tarsomeres 1–5 in leg I: 8.5/9/5.3/2/1.5/1/1, in leg II: 9/10.5/7.8/3.2/1.9/1/1, and in leg III: 10.1/14.5/6.2/4.2/2.4/1.2/1. Abdomen with five pubescent segments, 6th invisible; also STIV with strong whitish dusting.ead Fig. . Face six Figure . Mesonotg Figure . Slightlegs Figs , 6C. Oven Figure . Basal f) Figure . ST withLECTOTYPE (here designated to fix the identity of the species) ♂, TUNISIA: [Tunis governate] “Tunis904” / “Ujhelyi”, [bottom side] “X 26”; “Alloeoneurus” / “sabulosus Beck.” / “det. Becker”; [red rectangular] “Lectotypus”; “Zool. Mus.” / “Berlin” [ZMHB] (TN-01). PARALECTOTYPE ♀, TUNISIA: “Tunis904” / “Ujhelyi”, [bottom side] “XI 2”; [red rectangular] “Typus”; “Zool. Mus.” / “Berlin” [ZMHB] (TN-01).HMNH at the time of the description. A holotype was not formally designated in ZMHB were part of the type series. Fact is that both specimens in the ZMHB were collected by Ujhelyi in 1904 in Tunis, where Biró (HMNH) also collected Diptera in 1903 . The Becker catalogues at the ZMHB list a few specimens that he received as a gift from Biró, but these specimens always carry according labels . However, no such labels were found on the current lectotype and paralectotype.The original description of this species by  in 1903 . And as Orthoceratium.See Suppl. material O.lacustre, O.sabulosum is much more widespread in the West Palaearctic and currently known with certainty from 14 countries, although it has not (yet) been collected in the northern part of the Mediterranean basin , it is confined to humid coastal habitats, with a strong preference for salt marshes and brackish marshes. In Belgium, the species has only been collected in sea-aster (Astertripolium) vegetations, bordering shallow brackish to saltwater ponds . Also in Greece (Lesvos), Portugal , Spain (Alicante) and north Africa , records originate from locations close to the sea. In sharp contrast, the species is also known from locations between 700 m and 1,907 m in Portugal, Spain, Turkey and Iran, mostly in (coniferous) forest habitat and often with or close to small streams or open water (lakes).Based on the current records, the distribution range of er ponds  and in wO.lacustre and O.sabulosum, resp. On average, wings in males (+ 0.1 mm) and females (+ 0.2 mm) of O.lacustre are only very slightly longer than in O.sabulosum. Indifferently, both overall and in separate populations or datasets per country, wings in males of both species were approximately 0.5 mm shorter than in females. Size variations within the same sex in separate populations mostly proved higher in the females, with a maximum of 0.9 mm in males of Greece and even of 1.5 mm in females of France, both in O.lacustre. Size differences between both species in the keys by Table Orthoceratium – compared to other dolichopodid lineages, it remains surprising that key features of this genus have been overlooked by previous authors. The ventral process of the 5th tergite was only mentioned by th sternite! – and the posteroventral spot on the fore femur only by O.sabulosumO.lacustre, i.e. the strong black bristle on the fore coxa. All authors ignored the presence of one single claw of the fore tarsus in the male and Considering the fair size of O.sabulosum. The description of the ventral process of the 5th sternite “… aber der vierte Bauchring ist anders und hier [in O.sabulosum] ganz einfach gebildet, während er bei All.lacustris spitz dreieckig endigt und vorsteht …” clearly points towards O.lacustre. This erroneous interpretation was copied by O.lacustre.Even more worrisome is the fact that O.sabulosum in his description of O.lacustre, which must have been simple misfortune. In fact, Parent listed records of his ‘O.lacustre’ from localities in the north of France  and the south , two regions where either O.sabulosum or O.lacustre have been recorded, resp. . Indeed, only when the type locality of O.lacustre proved to be situated within the distribution range of ‘species B’, it became apparent that both species were conspecific. If this would not have been the case, and assuming that the face width would have been unreliable like the other four features used in the extant identification keys, O.lacustre might as well have rendered nomen dubium, and ‘species B’ might have been described as new. In this respect, the use of alternatives to physical type specimens deposited in a museum (and photos thereof) like e.g., field images of uncollected specimens as promoted by Here lies the importance of type material that cannot be underestimated. Only after the examination of the types of Orthoceratium species that occurs in northwestern Europe has indifferently been considered as O.lacustre, while it was, in reality, O.sabulosum. This is all the more surprising since Orthoceratium species are among the larger and more conspicuous dolichopodid species in Europe. The main reasons for this continuous series of misinterpretations seemed to be the mere copying of keys by successive authors that contained misleading information, the omission of examining type specimens of O.sabulosum, and the loss of the type specimens of O.lacustre. The importance of type specimens and the examination thereof is stressed, as well as that of unequivocal, detailed, and well-illustrated descriptions to avoid this kind of taxonomic confusion.For more than 2.5 centuries the


Anaplasma phagocytophilum is an obligate parasitic intracellular bacterium. It is the causative agent of granulocytic anaplasmosis, with effects on human and animal health. In Europe, the pathogen is mainly transmitted among a wide range of vertebrate hosts by blood-sucking arthropods. The aim of this study was to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dogs (Nyctereutes procyonoides), badgers (Meles meles), foxes (Vulpes vulpes), martens (Martes sp.) and European polecats (Mustela putorius), using molecular methods.16S ribosomal RNA gene partial sequence was used as a marker to identify A. phagocytophilum in spleen samples collected from carnivores using nested PCR.In the present study, 174 spleen samples were collected from adult, wild carnivores hunted in the years 2013–2016. A short fragment (383 bp) of the A. phagocytophilum in wild carnivores was 31.61% (55/174). Seven sequences of A. phagocytophilum were generated from two raccoon dogs, two badgers, one marten, one red fox and one European polecat. Six identical nucleotide sequences were obtained from one raccoon dog, two badgers, one marten, one red fox and one European polecat , and these were identical to many A. phagocytophilum sequences in the GenBank database (100% similarity). The second sequence (A. phagocytophilum sequence 2: MH328210) obtained from the raccoon dog shared 99.74% identity with A. phagocytophilum sequence 1.The prevalence of A. phagocytophilum in wild carnivores, viz raccoon dog, badger, marten and European polecat, in Poland. The detected A. phagocytophilum sequences (1 and 2) were closely related with those of A. phagocytophilum occurring in a wide range of wild and domestic animals and vectors.To our knowledge, this is the first study to use molecular methods to determine the presence of  Anaplasma phagocytophilum is a Gram-negative bacterium belonging to the family Anaplasmataceae  hunted in Poland around the Głęboki Bród Forest District  during the years 2013–2016. These were stored at −20 °C until further processing. DNA was isolated from the middle part of the spleen using a Genomic Mini AX Tissue kit  and according to the manufacturer’s instructions.A. phagocytophilum was based on semi-nested PCR amplification of the partial 16S rRNA gene according to Werszko et al. [A. phagocytophilum: A480F, A520F and A900R. The first PCR reaction was performed with primers A480F and A900R. For the second reaction (semi nested PCR), 1 μl of the first reaction product and primers A520F and A900R were used. PCR reactions were conducted in 50 µl of reaction mixture containing the following: 40 μl of deionized water in first reaction (41 μl in second), 1 μl of a 25 mM solution of MgCl2, 0.5 μl of Allegro Taq DNA Polymerase , 0.5 μl of dNTPmix (10 mM), 5 μl of 10× Taq DNA Polymerase Buffer (with 25 mM MgCl2), 0.5 μl of each primer (20 pmol/μl), 2 μl of template DNA in the first reaction and 1 μl of first reaction PCR product in the second reaction. DNA isolated from red deer (Cervus elaphus) infected with A. phagocytophilum (GenBank: GQ450278) was used as the positive control. Nuclease-free water was added to the PCR mix as a negative control. The PCR reaction was performed in an automated DNA Engine PTC-200 Peltier Thermal Cycler . The thermocycling profile was as follows: denaturation at 92 °C for 2 min, followed by 35 cycles with 30 s denaturation at 94 °C, 10 s primer annealing at 60 °C, and 1 min at 72 °C for primer extension, with a final extension step of 5 min at 72 °C.Molecular detection of o et al. . Three phttps://www.graphpad.com).The PCR products were subjected to electrophoresis  on a 1.0% agarose gel stained with ethidium bromide and visualized using ChemiDoc, MP Lab software ; a Nova 100 bp DNA Ladder  was used for comparison. The PCR amplicons were purified using a QIAEX II Gel Extraction Kit , sequenced by Genomed  and assembled into contigs using ContigExpress, Vector NTI Advance v.11.0 . The obtained sequences were compared with sequences available on GenBank using the Basic Local Alignment Search Tool (BLAST). Statistical analyses were performed using the Chi-square test in GraphPad online (A. phagocytophilum infection was 31.60% (55/174); however, among all tested groups, the prevalence of infection varied according to the host species. The highest prevalence was observed in martens , followed by raccoon dogs  and foxes , with the lowest prevalence observed in badgers . Anaplasma phagocytophilum was detected in two of four investigated European polecats. The prevalence of A. phagocytophilum was significantly higher in martens than in badgers . The prevalence in raccoon dogs was significantly higher than in badgers . During the tests, a large difference was observed between the first and the second reaction in nested PCR. After the first reaction only 4 samples were positive in all tested animals (2.30%). After the second amplification step, 55 samples out of 174 were positive (31.60%).The overall prevalence of A. phagocytophilum PCR-positive samples and submitted to the GenBank database . Six sequences  were found to be identical (100% similarity) with many A. phagocytophilum sequences obtained from Europe, Asia, Africa and North America. Anaplasma phagocytophilum sequences were obtained from a wide range of hosts: human, dog, raccoon dog, fox, sheep, black-striped field mouse, northern red-backed vole, tick, cow, goat and horse. More detailed data are shown in Table Anaplasma phagocytophilum sequence 2 was highly similar to all examples of A. phagocytophilum sequence 1 obtained in this study (99.74% identity) and differed only by one nucleotide from sequence 1.Seven sequences were obtained from A. phagocytophilum, and that the composition of this reservoir varies according to geographical regions [A. phagocytophilum infection in the tested animals provides compelling evidence for the involvement of wild carnivores in the enzootic cycle. So far in Europe, A. phagocytophilum has been detected in red foxes in Germany (8.20%), Italy (16.60%) and Hungary (12.50%) [Anaplasma phagocytophilum can be detected in many various organs such as spleen, lung and liver tissue [Anaplasma in spleen samples, and that it is more sensitive than standard PCR: while the detected prevalence was only 4/174 (2.30%) in all tested animals after the first reaction, the detection level was increased to 55/174 (31.60%) after the second stage.Studies performed in Europe confirm that a wide range of mammals can serve as competent animal reservoirs for  regions , 28. The12.50%) , 13, 16.(12.50%) . The hig(12.50%) , or the (12.50%)  which isr tissue , 12, 13..50% , 13A. phagocytophilum observed in raccoon dogs in Poland (35.30%) is higher than in Germany (23.00%) [A. phagocytophilum infection in this host to be 1.00% in Korea, Hildebrandt et al. [Foxes are well adapted to the urban environment and are accustomed to the presence of humans. In addition, their frequent exposure to tick bites and their potential to act as reservoirs or maintenance hosts for pathogens infecting humans and domestic animals highlights their importance in public health . The pre(23.00%) . Interes(23.00%)  and Kangoland 35.% is hight et al.  reportedA. phagocytophilum infection; however, Garcia-Pérez et al. [Anaplasma infection was found in two of the four tested European polecats in the present study, previous studies have found the prevalence to be 4.30% in Germany and 4.90% in the Netherlands [A. phagocytophilum was found to be more common in pine martens (Martes martes) in the present study (41.70%) than in Belgium (22.00%) [According to the present findings, 18.70% of the tested badgers in north-eastern Poland displayed z et al.  reportedz et al.  found itherlands , 23. SimAnaplasma phagocytophilum in the natural environment. Most isolates obtained in this study were identical to A. phagocytophilum sequences deposited in GenBank. Our findings represent the first detection of A. phagocytophilum in badgers, raccoon dogs, martens and polecats from Poland. The nested PCR used in the present study was more sensitive to detection of A. phagocytophilum than standard PCR for spleen tissue samples.All five tested species of carnivore may serve as suitable reservoir hosts in the ecology of


Various biological materials unrelated to humans are found at crime scenes and it is often important to elucidate the origin of these materials. A genetic locus common to several species is conventionally PCR-amplified with universal primers to identify species. However, not all species can be identified using a single locus.In this study, DNA from 13 commonly handled taxa was analyzed to identify species by a genome profiling (GP) method, which involves random PCR and temperature gradient gel electrophoresis.In a clustering analysis, we successfully obtained a single cluster for each species.The GP method is cost-effective and does not require advanced techniques and knowledge in molecular biology. The random sampling of the whole genome using multiple primers provides substantial genomic information. Therefore, the method is effective for classifying a wide range of species, including animals, plants, and insects, and is useful for crime scene investigations.The online version of this article (10.1186/s12863-019-0753-9) contains supplementary material, which is available to authorized users. Several methods to detect human-specific DNA have been developed to identify victims or suspects, using biological materials such as blood stains, hair, small tissue particles, and body fluids found at crime scenes –3. It isFor non-human biological materials, such as meat pieces, blood, hair, and bone fragments, the amplification of a species-specific locus by PCR is commonly performed –8. Howevspiddos) are obtained on the temperature gradient gel. These spiddos are corrected using internal standards. Then, the spiddos pattern is compared between samples and references and a pattern similarity score (PaSS) is obtained. Using this PaSS, a cluster analysis is performed to identify species.In this study, we focused on the genome profiling (GP) method. The GP method was developed by Nishigaki et al. in the bioindustry field in 1971 . In the This method corresponds to random sampling in statistics. It is possible to analyze information for the entire genome at a very low cost in a short period of time, without requiring any special knowledge or techniques. Using this GP method, we have identified human body fluid , 23 and The GP method is highly sensitive and accordingly it can potentially be used like the Ames test for mutagen analyses . Additiospiddos were obtained for all samples, regardless of species (Table spiddos obtained from each sample was 11.7 ± 3.1 (range 5–21) for SP-1, 12.7 ± 3.8 (range 5–22) for SP-2, and 7.0 ± 3.0 (range 4–16) for SP-3.Four or more es Table . The avespiddos obtained using SP-1 to − 3 were 27.8 for cattle, 33.0 for pigs, 26.8 for sheep, 36.0 for chickens, 40 for greater amberjack, 32.5 for bigeye tuna, 35.8 for silver salmon, 30.8 for Japanese horse mackerel, 30.0 for Japanese halibut, 33.0 for dog, 36.8 for cat, 16.3 for rat, and 31.0 for human.The average numbers of spiddos.Phylogenetic trees were generated using PaSS values for each primer. Using the SP-1 primer, the same species were classified into a single cluster for cattle, sheep, Japanese halibut, greater amberjack, Japanese horse mackerel, and rat. However, mixed clusters were obtained for other species  analysis is generally accepted for the personal identification of biological materials such as blood, semen, and saliva stains collected at a crime scene , 29. HowThough there is a report that simple sequence repeat (SSR) is very effective for determining the differences among species , many respiddos obtained by random PCR. A spiddos shows not only the cleaved temperature of the double-stranded DNA fragment amplified by random PCR, but also specific DNA sequences. Therefore, analyzing spiddos is the same as performing random sampling for large-scale genome information. For this reason, primers that can amplify a larger number of spiddos are useful to maximize the genome information that is obtained.For the GP method, the accuracy of species determination depends on the number of spiddos that can be obtained using a single primer. Accordingly, the number of spiddos can be increased by increasing the number of random primers, resulting in a more accurate cluster analysis [However, there is a limit to the number of analysis . In our spiddos [spiddos in various species. Further studies are required to evaluate this species specificity. A second problem is related to the reference samples. Although there are international databases for DNA sequences, a database of TGGE images does not exist. Therefore, it is necessary to prepare DNA for the suspected species once to obtain images for comparison.Classification based on a very small part of the whole genome is sometimes difficult for closely related species. Moreover, the reliability of the results may be insufficient when the whole genome is not analyzed. The GP method, in which the whole genome is analyzed by random sampling, addresses these limitations and is sufficient for species classification. However, the GP method has various limitations. First, it is difficult to apply to mixed samples. Although mixed samples were not examined in this report, both genomes are expected to be randomly amplified, and it may be difficult to determine their species. In previous studies of virus detection using body fluids, we found species-specific spiddos , 24. TheDespite the issues described above, the GP method can save time, labor, and costs, and although it is a random sampling approach, it can be used to obtain whole genome information. Therefore, the GP method is also an effective approach for classifying species and can be used for criminal investigations.(Bos taurus), pig (Sus scrofa domesticus), sheep (Ovis aries), chicken , greater amberjack (Seriola dumerili), bigeye tuna (Thunnus obesus), silver salmon (Oncorhynchus kisutch), Japanese horse mackerel (Trachurus japonicus), Japanese halibut , dog (Canis lupus familiaris), cat (Felis catus), and rat (Rattus norvegicus). The meats of cattle, pig, sheep, chicken, greater amberjack, bigeye tuna, silver salmon, Japanese horse mackerel, and Japanese halibut were purchased at a grocery store in the city market. Samples were collected by scrubbing the surface of each raw meat with a cotton swab. The dog, cat, and rat samples were obtained at pet shops by scrubbing the buccal mucosa of each species with a cotton swab. Human samples were obtained from healthy adult volunteers who provided written informed consent. The buccal mucosa of each volunteer was scrubbed with a cotton swab. A total of 49 samples  were collected.Human and 12 species that are often found in the house or household kitchens were used in this study, i.e., cattle Cotton swabs were digested with Proteinase K overnight at 56 °C. DNA was extracted using QIAamp® DNA Mini Kits . The DNA concentration was adjusted to 5 ng/μL.Three random primers, SP-1 (pfm12) (5′-AGAACGCGCCTG-3′), SP-2 (pfm19) (5′-CAGGGCGCGCGTAC-3′), and SP-3 (hunt) (5′-TGCTGCTGCTGC-3′) were used . PCR ampTwo types of reference DNA were prepared as TGGE internal standards . For refFor reference 2 (Ref2), PCR amplification was performed using a 50-μL reaction solution containing 2.0 μL of M13 phage DNA , 3.5 μL each of 10 μM Ref6F (5′-GCCGGCATCACCGGCGCCACAGGTGCGGTTG-3′) and Ref6R (5′-TAGCGAGGTGCCGCCGGCTTCCATTCAGGTC-3′), 4.0 μL of dNTP (2.5 mM each), 5.0 μL of 10× Buffer, 0.25 μL of Ex Taq Polymerase (Takara Bio Inc.), 10 mM primers, and 1.0 μL of extracted DNA. PCR conditions were 30 cycles of 94 °C for 15 s, 44 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 30 s.A total of 1.0 μL of reference DNA solution was obtained by mixing the reaction solutions of Ref1 and Ref2 at a ratio of 1:1 and 1.0 μL of the PCR solution, followed by electrophoresis. The mixed sample was applied to a 6% polyacrylamide gel and electrophoresed at 100 V for 10 min with a temperature gradient of 15 °C to 65 °C , 37. Aftspiddos) were manually plotted. The spiddos were corrected by two reference DNA spiddos. Representative images of the electrophoresed gels and corrected figures are shown in Fig. From the images of the electrophoresed gels, the melting points of amplified double-stranded DNA . (CSV 18 kb)Additional file 2:PaSS values (SP-2). (CSV 16 kb)Additional file 3:PaSS values (SP-3). (CSV 18 kb)Additional file 4:PaSS values (average of SP-1-3). (CSV 25 kb)


Chlamys farreri using an infrared-based method, and evaluated the effects of several biotic  and environmental factors (circadian rhythm and temperature) on scallop heart rate (HR), amplitude (HA), and rate-amplitude product (RAP). Results revealed that size has a significant effect on both HR (negative) and HA (positive), but RAP values are similar in different sized scallops. Age also affects scallop cardiac performance, significantly for HR, but not for HA or RAP. Circadian rhythm affects cardiac activity, with significant elevation of HR, HA and RAP during 1:00–8:00 and 17:00–19:00. With seawater temperature elevation, HR peaks at 30.03 ± 0.23°C, HA at 15.08 ± 0.02°C, and RAP at 15.10 ± 0.19 and 30.12 ± 0.28°C. This suggests HR is a good indicator for thermal limit, whereas HA may indicate optimal growth temperature, and RAP could be an index of myocardial oxygen consumption to indicate myocardium stress. Our study provides basic information on the factors that may affect scallop cardiac performance. It also elucidates the feasibility of HA and RAP as cardiac indices in marine molluscs.Cardiac activity has been widely used in marine molluscs as an indicator for their physiological status in response to environmental changes, which is, however, largely less studied in scallops. Here, we monitored cardiac performance of Zhikong scallop  Gadus morhua, HR is also an important indicator for evaluating influences of short-term and prolonged exposure to hypoxia  is widely used in vertebrates for evaluating physiological status. For example, HR is an independent predictor of cardiovascular and overall mortality in human . In Atla hypoxia . Similar hypoxia  and Moll hypoxia , HR is w hypoxia , suggestlinities . The imps edulis , and braand zinc . Fluctuaand zinc . All theCarcinus maenas, HR is dependent on body size, with small crabs having a faster HR than the large ones , there are other parameters besides HR in the ECG which can also provide valuable information on cardiac activity. For example, amplitudes of the P-QRS-T waves represent the variation in membrane potential during the depolarization or repolarization of different pumping chambers. There are also PR interval which reflects conduction through the atrioventricular node, and QT interval that represents the time taken for ventricular depolarization and repolarization. Most importantly, these parameters have been widely used in the clinical diagnosis of various cardiovascular diseases. In contrast, previous studies on cardiac performance in molluscs generally focus on HR, which may provide incomplete information on their cardiac activity.Crassostrea gigas, Zhikong scallop Chlamys farreri, and Yesso scallop Patinopecten yessoensis. Considering their ecological and economic importance, study on the cardiac activity in these animals could benefit ecological surveys as well as aquacultural industry. In this study, we measured the cardiac performance of Zhikong scallop and examined the effects of several biotic factors  and environmental factors (temperature and circadian rhythm) on cardiac parameters – heart rate (HR), amplitude (HA), and rate-amplitude product (RAP). It should facilitate a better understanding on the scallops’ physiological status under different conditions.Bivalves, including clams, oysters, mussels, and scallops, are a large group of molluscs consisting of ∼14,000 species worldwide . They caAll Zhikong scallops used in present study were collected from artificial scallop-rearing substrates installed in Xunshan Fishery Group Co., Rongcheng .For the study of size effect, 96 24-month-old healthy scallops with shell height varying from 40.25 to 67.02 mm and total weight ranging from 42.39 to 67.81 g, were collected, respectively. For the study of age effect, scallops with similar shell height (40.53 ± 2.87 mm) were collected for each age group  to avoid the interference of size factor. For the study of temperature effect, 24 scallops with similar shell height (48.69 ± 1.91 mm) were collected. For the study of circadian rhythm effect, 48 24-month-old scallops (24 as control group and 24 as treatment group) with similar shell height (52.53 ± 2.62 mm) were collected. To prevent potential genetic effect on the experiments, all individuals were sampled from a pool of various geographical populations including Yantai , Weihai , and Qingdao .After sample collection, encrusted organisms on scallop shells were removed. The scallops were placed in plastic tanks with filtered and aerated seawater at 15°C for 7 days for acclimation . To avoiCardiac performance was monitored by the non-invasive method improved from the technique of To detect the effects of size and age on cardiac performance, scallops were maintained at 15°C which is within the optimum temperature range for their growth (14–22°C) . To inve∗V). For examining the effects of size on cardiac performance, a regression analysis was performed. To detect the effect of age on cardiac performance, one-way ANOVA was conducted followed by Duncan’s test. The HR/HA/RAP increment rate with temperature was obtained for each individual by regression analysis. To compare the differences in HR/HA/RAP between control and experimental groups under circadian rhythm influence, independent sample t-tests were performed. Differences and correlations were considered significant when P < 0.05. All data were analyzed using SPSS 21.0 .The heart rate (HR) was counted in beats per min (bpm) , and ther = −0.85, P < 0.001), but increases with HA . The effects of total weight on cardiac performance are similar to that of shell height, with the correlation coefficient of −0.80 (P < 0.001) and 0.80 (P < 0.001) with HR and HA, respectively  = 20.229, P < 0.001], suggesting older scallops have relatively faster HR. But age has no significant effect on HA   = 1.986, P = 0.145]  higher HA was found from 12 to 16°C, with the maximal HA (1.70 ± 0.02 V) at 15.08 ± 0.02°C. Before the peak, HA increased gradually with an average rate of 0.04 V/°C. Afterward, HA decreased abruptly at the rate of 0.10–17 V/°C, and then declined moderately at 0.01 V/°C until 37°C. ∗V) at 15.10 ± 0.19°C with an average increasing rate of 2.42 ± 0.07 bpm∗V/°C, and the second peak (76.38 ± 4.27 bpm∗V) at 30.12 ± 0.28°C with the rate of 2.42 ± 0.13 bpm∗V/°C. Afterward, RAP decreased sharply down to less than 10 bpm∗V at 34°C, with the rate of 16.36 ± 0.52 bpm∗V/°C.We then examined the changes in cardiac performance when temperature elevated from 5 to 37°C. As shown in During temperature elevation, we observed various plethysmogram patterns . In cont∗V in RAP were recorded in the experimental group that constantly kept in the dark, and there is no significant difference in either HR, HA, or RAP during the 24 h period. In contrast, HR, HA, and RAP showed marked changes when scallops were exposed to natural light. Significantly higher HR was recorded during 1:00–8:00, with HR ranging from 27.16 ± 0.96 to 30.54 ± 0.80 bpm. Correspondingly, we found HA and RAP were significantly higher during this period, with the values ranging from 1.98 ± 0.03 to 2.37 ± 0.03 V, and 53.77 ± 3.52 to 72.37 ± 2.86 bpm∗V, respectively. There is another small peak during 17:00–19:00 in HR, HA and RAP curves, with the maximal value of 29.35 ± 1.65 bpm in HR, 2.30 ± 0.05 V in HA, and 67.51 ± 6.14 bpm∗V in RAP at 18:00. The above results suggest circadian rhythm has a significant effect on scallop cardiac activity, and HR and HA exhibit coordinate changes under natural light.The effect of circadian rhythm on cardiac performance was also evaluated. As displayed in Research on cardiac performance has been widely conducted in marine molluscs, such as limpets  mussels M. edulis  because RPP is a marker of cardiac function which is relatively stable in healthy people irrespective of height or age (The independence of RAP with scallop size and age inspires us to consider the possible similarity between scallop RAP and human rate-pressure product (HRt or age . That bet or age . This inTemperature has been found to be a major environmental factor that affects HR of various marine molluscs . In ZhikCoelomactra antiquate (Although the effect of circadian rhythm on mollusc cardiac performance has never been reported before, we found that in scallops, circadian rhythm has a similar effect on HR, HA, and RAP. The same two peaks were found in HR, HA, and RAP curves, with the first one spanning across an 8 h-period from 1:00 to 8:00, and the other across a 3 h-period from 17:00 to 19:00, indicating Zhikong scallops are primarily active at night. This fluctuation pattern is very similar to the daily rhythm of oxygen consumption rate of Zhikong scallop  and anotntiquate . Since 8ntiquate , and/or ntiquate , thus inTo sum up, our study revealed that all the investigated factors including size, age, environmental temperature and circadian rhythm have significant effects on scallop cardiac performance, therefore, these factors should be taken into account in the experimental design. But genetic background could potentially affect cardiac performance  and shouAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed.SW, LZ, and ZB conceived and designed the experiments. YaL and HG collected the samples. QX, YuL, and XZ performed the experiments. QX and LZ analyzed the data. QX, LZ, and SW wrote the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Success of drying was evaluated with several quality criteria: shrinkage (change of volume), color change, shape, content of L-ascorbic acid, total phenolic content, flavonoid content, anthocyanin content, and antioxidant activity. A considerable influence of convective drying on color changes was not observed, as ΔE was low for all samples. It was obvious that fresh raspberries had less physical changes than frozen ones. On average, convective drying reduced L–ascorbic acid content by 80.00–99.99%, but less than 60% for other biologically active compounds as compared to fresh raspberries. Convective dried Polana raspberry may be considered as a viable replacement for freeze-dried raspberries.Raspberries are one of Serbia’s best-known and most widely exported fruits. Due to market fluctuation, producers are looking for ways to preserve this fresh product. Drying is a widely accepted method for preserving berries, as is the case with freeze-drying. Hence, the aim was to evaluate convective drying as an alternative to freeze-drying due to better accessibility, simplicity, and cost-effectiveness of Polana raspberries and compare it to a freeze-drying. Three factors were in experimental design: air temperature , air velocity (0,5 and 1,5 m Raspberries (Rubus idaeus) are one of the most important fruits in Serbian agriculture. It has been recently reported that raspberry production in 2017 was 109,742 t, which positions Serbia as one from the three leading countries in raspberry world production ,4,5,6,7;In Serbia, raspberries are often processed to jelly, diary, or confectionery products able to be stored for a longer time ,15,16,17Kowalski et al.  tested dRubus idaeus var. Autumn Bliss) and boysenberries (Rubus ursinus × Rubus idaeus var. Black Satin). Berries were applied to different convective drying conditions: 50 °C for 48 h, 65 °C for 20 h or 130 °C for 2 h until a moisture content was below 15%. Obtained results indicated that drying regime at 65 °C during 20 h was optimum for the best preservation of color, polyphenol content and antioxidant activity in dried berries. Moreover, authors argued that conventional drying is more economical than freeze-drying, and with considerable increase of total polyphenolic content due to depolymerization of native polyphenols which lead to improved antioxidant activity. Different raspberry cultivars, have different sensitivity to convective drying mostly evident by changes in their physical , mechanical , nutritive , and sensory properties . Furthermore, Pavkov et al. [Bustos et al.  studied v et al.  found thTherefore, the aim of this research was to investigate the influence of convective drying on physiochemical properties and quality parameters of red Polana raspberry for fresh and frozen samples. Evaluated quality parameters included changes in volume and shape, color, L-ascorbic acid, total phenolic content, flavonoid content, anthocyanin content, and antioxidant activity. All results were controlled against freeze-dried raspberries.n = 500) that were as following; (i) moisture content Xd.b = 5.45 ± 0.45 kgw/kgd.b.; (ii) mass m = 3.30 ± 0.24 g; (iii) length a = 21.26 ± 0.74 mm, width b = 20.08 ± 0.56 mm, and thickness c = 18.63 ± 0.62 mm; (iv) volume V = 3.17 ± 0.22 cm3; and (iv) water activity aw = 0.979 ± 0.001, at the temperature of 20 °C, total soluble solids of 6.6% and pH = 3.26 ± 0.02.Polana variety samples of red raspberries at full maturity were taken from the local farmers during August and October, 2017, at area of Novi Sad, Republic of Serbia. The raspberry fruits were harvested few hours prior to each experiment. The selected samples were approximately equal in size, volume, color, mass, and humidity. Average values of these properties were obtained from raspberry fruits subsample  with lab-scale convective dryer, designed and constructed to control for air flow , air drying temperature, and continuous monitoring of the sample mass in processing . The dry−1). The absolute air humidity was approximately constant in the experiments with average value 0.011 ± 0.002 kgw/kgd.air. For each experimental run, the initial mass of the raspberries was approximately 500 g. Raspberry samples were set on perforated sieve in a thin stagnant layer and placed in a drying chamber. Air flew along or across the surface of the material in the dryer, and the samples were dried until the same value of moisture content of approximately Xd.b = 0.152 kgw/kgd.b. The experiment was conducted with three repetitions for each experimental run.Convective raspberry drying was three-factor experiment with one qualitative and two quantitative factors. The qualitative factor represented the initial state of the raspberry fruit before drying with two levels (fresh and frozen raspberries). Quantitative factors of the experiment were drying temperatures  and air velocity (0.5 to 1.5 m·ssh) is one of the most common physical and quality indicators of the final product. Shrinkage is expressed by the ratio between the change of volume after drying and volume of sample before drying. The samples volume (n = 45) were measured by immersing the raspberries into 96% concentration of ethanol [0 is the mass of liquid and the immersed sample (kg), ml is the mass of liquid, and ρt is the liquid density (kg/m3). The volumetric shrinkage of raspberries (Vsh) was based on the following equation [0 is initial average volume and Vi is volume of each raspberry after drying.When drying some biomaterial, volume shrinkage , able to detect the changes after drying ,30,31. The fruit .n = 45) by colorimeter Konica Minolta CR400(C-light source and the observer angle of 2°). Where L* was whiteness/brightness, a* was redness/greenness, and b* represented yellowness/blueness. The total color difference (ΔE), hue angle (ho) and chromaticity (C*) were calculated by Equations (4)–(6) [0, a0, and b0 are the color values before drying, while L*, a*, and b* are the color values after drying.Before and after each drying regime, CIELab color parameters were assessed for raspberry samples ( (4)–(6) ,34:ΔE = Methanol extract was prepared from the fresh/dried raspberry samples to determine the contents of total phenols, flavonoids, and radical scavenging capacity. Briefly, 50 mL of an extraction solvent   was poured over the raspberry sample into an Erlenmeyer flask. The flasks were covered and placed on a laboratory stirrer for 24 h (in a dark place). After the extraction, the samples were transferred to volumetric flasks of determined volume, filtered, and stored in a dark and cool place until the analysis was carried out.2CO3 (m/v). After 3 min of stirring, distilled water was added and made up to volume of 50 mL. Reaction mixture was left to stand at room temperature for 2h and absorbencies were measured at 765 nm by UV–Vis spectrophotometer. Based on the measured absorbance, the concentration (mg/mL) of TPC was calculated from the calibration curve of the standard solution of gallic acid. The results are expressed in g of gallic acid equivalents (GAE) per 100 g of fruit dried basis (gGAE/100gd.b).The TPC in methanol extracts of fresh and dried raspberries was determined by Folin–Ciocalteu spectrophotometric method . In a 502 solution (m/v). Then mixture was incubated at room temperature for five minutes, and then 0.3 mL of 10% AlCl3 (m/v) was added. After six minutes, when the solution became very yellow, 2 mL of NaOH was added. Distilled water was added to the reaction mixture and made up the volume to 10 mL in a volumetric flask. The absorbance was measured at 510 nm. The TF were calculated according to the catechin standard calibration curve and expressed in mg of catechin equivalents (CAE) per 100 g of fruit dried basis (mgCAE/100gd.b).The TFL was determined by previously described colorimetric method . In shorblank is the absorbance of the blank and Asample is the absorbance of the sample. The obtained results were presented as a mass of dry sample material that is necessary for inhibition of 50% of DPPH (IC50 (mgd.b/mL)). The free radical scavenging capacity (RSC) of raspberry extracts was determined using a simple and fast spectrophotometric method described by Espin et al. . Briefly510 = sample absorbance at λ = 510nm; A700 = sample absorbance at λ = 700nm; Mw = molecular weight of cyanidin-3-glucoside (449.2), Df = dilution factor = original solution volume; ɛ = molar extraction coefficient of cyanidin-3-glucoside (26900); and m = sample weight (g). The content of AC was expressed in mg per 100 g of fruit dried basis (mg/100gd.b).The sample preparation for the content of total AC was conducted by previously described method . Here an18, 5 µm (250 × 4.6 mm I.D.) column . Separation was performed with standard method BS EN 14130:2003 (Foodstuffs. Determination of vitamin C by HPLC). The content of AC was expressed in mg ascorbic acid (the sum of ascorbic acid and its oxidative form of dehydroascorbic acid) per 100 g of fruit dried basis (mg/100gd.b).Separations and quantifications of vitamin C were performed by HPLC equipment (Thermo Scientific™ UltiMate 3000) on Nucleosil 100-5Cp < 0.05.For the purposes of statistical tests, analysis of variance was performed (ANOVA) with Statistica13 . In order to define homogenous groups of samples an additional Duncan test was performed with statistical significance at sh = 16.49 ± 2.75%). For convective drying, the least changes in volume had fresh raspberry samples dried at T = 60 °C and air velocity of 1.5 m·s−1 (Vsh = 35.74 ± 6.78%). Pavkov et al. 2017 [−1. Judging by the volume shrinkage during convective drying, air temperature of 50 °C will lead to a total collapse and loss of the product shape. Interestingly, the least volume shrinkage (23.17%) was achieved with air temperature at 70 °C. Drying with air temperature at 60 °C provoked shrinkage of 28.74%, what is still lower than it was obtained in the current study. Samples dried with air temperature of 80 °C reached volume shrinkage of 43.13%. Results obtained from these two experiments revealed that higher air temperatures do not necessarily lead to higher changes in volume shrinkage. This may be explained by the fact that higher air temperature have tendency to lean towards mechanical stabilization of the raspberry surface, thus limiting the degree of shrinkage.Comparison of convective and freeze-drying technique for fresh vs. frozen samples revealed that the least changes in volume had freeze-drying (V−1 (Vsh = 79.07 ± 4.07%). As previously reported, drying temperature of 80 °C had similar trend on volume shrinkage [Air temperature and initial state of the raspberry, prior to convective drying significantly changed the volume of dried raspberry. On the other hand, the air velocity did not have any impact on the change in raspberry volume . The moshrinkage .−1. Here they found higher volume shrinkage (Vsh = 81 ± 3%) than what was reported in the current study.Additional for convective drying, some reports indicated variations in volume shrinkages with regards to raspberry varieties. Sette et al.  dehydrat−1. On the contrary, Duncan’s test revealed that there are no significant differences in the volume shrinkage of the samples which were dried at the same temperature and with velocity of1.5 m·s−1. The reason for this may be the faster drying rate in the first drying period, which can lead to a faster mechanical stabilization of the surface, hence the preservation of the volume. As expected, initial raspberry state had an effect on volume shrinkage, and the results after drying are presented in −1.Initial raspberry state effected the shrinkage, which was not surprising as creation of the ice crystals tends to destabilize cellular structures and this is particularly emphasized with drying air velocity of 0.5 m·s−1 (k = 0.2694 ± 0.003). Results showed that under the same experimental conditions, the convective dried frozen raspberries had greater deviation of size as compared to the fresh raspberries. Hence, Heywood shape factor corresponded with the results for volume shrinkage.The referent Heywood shape factor before drying of fresh raspberry was k = 0.3323 , and allCIE Lab color parameters L*, a*, b*, C*, h*, and ΔE* measured on fresh and dried raspberries at different drying conditions are shown in p < 0.05) . The largest color change for this drying type was at T = 80 °C and air velocity of 0.5 m·s−1 for both frozen and fresh raspberry when ΔE was 11.17 and 10.12, respectively. Unexpectedly, freeze-dried raspberries had the largest color changes (ΔE = 19.60) that were caused by increase in all of the three-color parameters, and especially for a* (Δa = 18.04). Bustos et al. [Cassia alata from 40 °C to 60 °C reduced drying time from 180 min to 120 min [Air temperature, air velocity, and initial state of raspberry before drying had statistically significant effect on color change ( < 0.05) . The leas et al.  reporteds et al.  reporteds et al. ,41. Duris et al. . Increas 120 min . Consequd.b. (18.92 mg/100gw.b.) (Table 4), which was similar to quantities reported by Bobinaite et al. [−1. This was regardless of the fact that the raspberry temperature reached T = 80 °C, and equaled to 27.46 ± 1.12 mg/100gd.b. and 22.54 ± 1.28 mg/100d.b., after drying of frozen and fresh raspberry, respectively (Table 4). Conversely, the degradation of 99% L-ascorbic acid was detected for longest drying with T = 60 °C and air velocity of 0.5 m·s−1. Accordingly, this might mean that L-ascorbic acid degradation is more induced by longer exposure to higher oxygen levels during the convective drying than to the drying temperature itself.The average amount of L-ascorbic acid in fresh samples before drying was 118.27 mg/100ge et al. . This coe et al. ,45,46,47d.b.), since there was neither thermal nor oxygen degradation involved.This reasoning is in accordance with Verbeyst et al.  researchd.b. All three individual experimental factors had influence on the content of total phenols . On the contrary, they were least preserved at 60 °C and air velocity of 0.5 m·s−1(0.92 gGAE/100gd.b.). Freeze-drying preserved 1.10 g GAE/100gd.b. of total phenols, and, as expected, convective drying reduced polyphenolic content in the samples. The exceptions were the samples freshly dried at air velocity of 1.5 m·s−1 and drying temperatures of 70 °C; and 80 °C in which higher total phenolic content was observed in comparison to freeze-dried samples. Similar results were recently reported where higher phenolic content was found in convectively hot air-dried Cassia alata in comparison to freeze-dried samples [When these samples were dried convectively the best preserved polyphenolic content was at T = 70 °C and air velocity of 1.5 m·s samples . Hossain samples .w.b.), one with medium level (0.2–0.5 gGAE/100gw.b.), and the third with high levels (>1.0 gGAE/g100gw.b.). This classification was accepted by others [Vasco et al.  made clay others ,51, and d.b. After convective drying, anthocyanin content was preserved from 40–56%. Anthocyanin content (287.0 mg/100d.b.) was best preserved with drying of fresh raspberries at T = 70 °C and air velocity of 0.5 m·s−1. Their least retention occurred after convective drying of frozen samples at T = 70 °C and air velocity of 0.5 m·s−1 (205.3 mg/100d.b.). Thermal degradation of anthocyanins and complementary oxidization is the origin of the maroon color that was detected with the CIELab analysis. After freeze-drying, the content of anthocyanin in raspberry was 410.4 mg/100gd.b. which is expected due to minimized considerable influence of temperature and oxygen. Temperature had significant influence on the content of anthocyanin , howeverd.b./mL. Freeze-dried samples had highly preserved radical scavenging capacity that was equal to 0.0641 mgd.b./mL, likely due to high preservation of all bioactive compounds. The lowest IC50 value  had convective drying for frozen samples, of IC50 = 0.0845 mgd.b./mL which was obtained at T = 80 °C and air velocity of 0.5 m·s−1. The main reason for this may be the high preservation of total flavonoid content (0.97%) in dried samples with same convective drying regime. Raspberry belongs to a group of biomaterial with high radical scavenging capacity [As expected, all experimental factors influenced the radical scavenging capacity . As a smcapacity ,50. It icapacity . As prev−1. Convective drying of raspberry had influenced all measured biologically active compounds. In comparison to fresh samples, in convectively dried raspberries 60–78% of total phenols was preserved as well as 75–97% of flavonoids and 40–56% of anthocyanins. Consequently, lower radical scavenging capacity was found in convectively dries samples as compared to fresh or freeze-dried. The largest shortcoming for convective drying was observed in difference between freeze-dried for preservation levels of L-ascorbic acid. Freeze-drying preserved more than 97% of L–ascorbic acid, while convective drying samples had degradation of over 80% of this compound. This might not be as relevant where L–ascorbic acid is added in processing of raspberries . In conclusion, Polana raspberry dried convectively with air temperature of 60 °C and air velocity of 1.5 m·s−1, may be considered as sufficient alternative to freeze-drying.Using physical properties, contents of various biologically active compounds and radical scavenging capacity proved to be useful in selecting alternatives for preservation of raspberries as in the case of convective and freeze-drying. For Polana variety, the most desirable results against freeze-drying as standard in terms of color, volume shrinkage, and Heywood shape factor change was achieved with convective drying of fresh raspberry at T = 60 °C with air velocity of 1.5 m·s


N = 133 children; 86 male; mean age = 9.42, SD = 1.715; age range = 5.92–13.75y) with a broad range of abilities, and uses whole‐brain structural connectomics based on diffusion‐weighted MRI data. The results indicate that academic attainment is associated with differences in structural brain organization, something not seen when focusing on the integrity of specific regions. Furthermore, simulated disruption of highly‐connected brain regions known as hubs suggests that the role of these regions for maintaining the architecture of the network may be more important than specific aspects of processing. Our findings indicate that distributed brain systems contribute to the etiology of difficulties with academic learning, which cannot be captured using a more traditional voxel‐wise statistical approach.Literacy and numeracy are important skills that are typically learned during childhood, a time that coincides with considerable shifts in large‐scale brain organization. However, most studies emphasize focal brain contributions to literacy and numeracy development by employing case‐control designs and voxel‐by‐voxel statistical comparisons. This approach has been valuable, but may underestimate the contribution of overall brain network organization. The current study includes children ( First investigation of structural connectome associations of academic attainment in children.First direct comparison of voxel‐wise and connectome associations approaches in a developmental sample.1The literature on structural brain mechanisms supporting reading and math abilities strongly emphasizes the contribution of particular anatomical substrates  who were referred for problems in attention, learning and/or memory, by educational and clinical professionals working in various specialist children's services. The sample is therefore not already restricted to children who have met narrowly defined criteria, for example, particular cut‐off scores on cognitive assessments. Instead, the sample consists of children who are struggling educationally and display a broad range of abilities from deficits to age‐expected performance. This also includes deficits in both reading and math, which are known to often co‐occur —the inverse of the distance from any node to any other node; and average clustering coefficient (CG)—the extent to which node's neighbours are also neighbours. These metrics can be calculated at a whole‐brain level, or at the level of networks' nodes (brain regions). To investigate how the organization of the structural brain network may relate to academic attainment, we constructed a network that represents white matter connections throughout the brain based on diffusion‐weighted MRI data. We then explored how the organization of this network varied according to children's literacy and numeracy abilities, and whether age‐related differences in structural organization mediate the relationship between academic attainment and age.A network science approach was adopted to obtain a comprehensive account of the neuroimaging data. In this, brain regions are described as nodes and their connections as edges. Nodes typically correspond to regions of interest  research clinic. At the clinic, children were recruited on the basis of ongoing problems in attention, learning, and/or memory in school and were identified by professionals working in schools or specialist children's community services. Families were invited for an assessment that lasted approximately three hours. The assessment included the academic attainment measures reported here. Exclusion criteria for referrals were significant or severe known problems in vision or hearing that were uncorrected, conditions of known genetic origin, and having a native language other than English. This study was approved by the local NHS research ethics committee (Reference: 13/EE/0157). Written parental consent was obtained and children provided verbal assent.SD = 1.715, range: 5.92–13.75y). Sample average cognitive scores in the sample were below age expectation with verbal IQ, short‐term memory, and working memory scores about half a standard deviation below the population mean . The most common was Attention Deficit Hyperactivity Disorder , followed by Dyslexia , and autism spectrum disorder . Seven children had other diagnoses .The initial sample included 206 children. Of this sample, children were selected who had good‐quality MRI data , and completed assessments of math and reading. We further restricted the age range to focus on the age range with most available participants . The final sample consisted of 133 children  , using a 32‐channel quadrature head coil. The imaging protocol consisted of two sequences: T1‐weighted MRI and a diffusion‐weighted sequence.T1‐weighted volume scans were acquired using a whole‐brain coverage 3D Magnetization Prepared Rapid Acquisition Gradient Echo (MP RAGE) sequence acquired using 1 mm isometric image resolution. Echo time was 2.98 ms, and repetition time was 2250 ms.Diffusion scans were acquired using echo‐planar diffusion‐weighted images with an isotropic set of 60 non‐collinear directions, using a weighting factor of b = 1000s*mm‐2, interleaved with a T2‐weighted (b = 0) volume. Whole brain coverage was obtained with 60 contiguous axial slices and isometric image resolution of 2 mm. Echo time was 90 ms and repetition time was 8400 ms.2.4The white matter connectome reconstruction followed the general procedure of estimating the most probable white matter connections for each individual, and then obtaining measures of fractional anisotropy (FA) between regions . Regions of interests (ROIs) were based on the Desikan‐Killiany parcellation of the MNI template  v1.9 , and local and global efficiency  were calculated using the python implementation of the Brain Connectivity Toolbox as described in Rubinov and Sporns  keeping age separate meant that we could specifically test to the role of age in our model, and use both quadratic and linear terms; and (ii) we could subsequently conduct a mediation analysis using age. Global graph metrics were entered into the model as predictors alongside control variables for brain volume and movement during the diffusion sequence (see below). Model:G, EG) leading to an adjusted significance criterion of p < .0125.Bonferroni correction was used to adjust for multiple comparisons associated with the two outcomes  and the two predictors  = 0.36, β = −0.05, p = .550; Reading: F = 3.00, β = −0.15, p = .086). There was a significant effect of total brain volume on the CG  = 9.97, β = −0.27, p = .002) and on EG  = 7.64, β = −0.24, p = .007). There was an effect of participant motion on math scores  = 4.78, β = −0.19, p = .031), indicating that participants who moved more during the sequences scored lower on the math attainment task. Participant motion was not related to reading attainment, the CG, or the EG  = 0.84, β = −0.08, p = .360, CG: F = 0.63, β = 0.07, p = .429; EG: F = 0.16, β = 0.04, p = .690). Total brain volume and participant motion were included as control variables in all analyses that used the GLM approach.Influence of extraneous variables: We investigated the influence of total brain volume estimated through FSL SIENA .2.7G and EG were calculated in the targeted network. This process was repeated 100 times at each step and the results were averaged to remove effects associated with any particular node. Nodes were not removed completely to keep the number of nodes in the network constant. Different knock‐down values  produced similar results to the reported findings. Node‐level metrics  were calculated and averaged for hub nodes or peripheral nodes to investigate their association with academic attainment measures.In order to assess the role of highly connected nodes for the relationship between global graph measures and ability scores, we carried out a simulated attack on highly connected nodes (rich club), peripheral nodes, and randomly chosen nodes. To this end, between one and 20 nodes from each category were selected at random and their existing edge weights were knocked‐down to the lowest observed value in the group‐average network, that is, 0.01. Then, C2.8In order to contrast the structural connectome approach with more commonly used voxel‐wise statistical analysis, FA maps were processed using tract‐based spatial statistics (TBSS) as implemented in FSL v5.0.9  were compared to participants who scored in the typical range expected for their age (age‐standardized attainment score 90 < score < 120). See Table 3SE = 1.459, reading: mean = 85.41 SE = 1.538, see Figure At the group level, ability scores in math and reading were in the low range  = 34.81, p < .001) and higher EG  = 8.12, p < .001) compared to scrambled networks with the same degree of distribution and connection strength indicating small‐world organization in the observed structural networks . The relationship between global graph metrics and reading remained when regressing the linear and quadratic effect of age  but not for math .Regression analysis indicated a significant relationship between learning attainment measures and global graph metrics  = 76.653, p < .001, mediation effect: p = .026, see Figure F = 74.556, p < .001, mediation effect: p = .131). Similarly, EG significantly mediated age‐related improvements in reading  = 70.765, p < .001, mediation effect: p = .012) but not math  = 76.653, p < .001, mediation effect: p = .076). No significant mediation effects were observed for the squared age term for reading  = 4.641, p = .2, mediation effect: p = .976; EG: F = 11.411, p = .01, mediation effect: p = .977) or math  = 2.101, p = .552, mediation effect: p = .976; EG: F = 5.148, p = .161, mediation effect: p = .977)Next, a mediation analysis was carried out to investigate the relationship between age, global graph metrics, and academic attainment scores. Ceading , left precuneus , and right middle frontal cortex  was most closely associated with reading scores. Math scores were most closely associated with Cj of the right superior frontal cortex (βC = 9.37), the left cerebellum (βC = 2.23), and the left lingual cortex (βC = 2.19), and the Ej of the right middle frontal cortex (βE = 0.38), the right superior frontal cortex (βE = 0.34), and the right accumbens area (βE = 0.32), and Ej of the right middle frontal cortex (βE = 0.38), the right superior frontal cortex (βE = 0.34), and the right accumbens area (βE = 0.32).Next, the relationship between node‐level graph metrics and academic attainment scores was investigated. Overall, stronger relationships were observed between C3.4G and EG compared to peripheral nodes , but not hub nodes . Regarding the relationship with academic attainment, reading scores were associated with the Cj and Ej of hub nodes , and, to a lesser extent, Ej of peripheral nodes  but not their Cj . Math was closely related to Cj and Ej of hub nodes , but not to peripheral nodes .In order to assess the importance of hub and peripheral nodes on brain organization, simulated attacks were carried out. Knock‐down of hub nodes led to a larger decrement in C3.5pcorrected < .05  research clinic is based at and supported by funding from the MRC Cognition and Brain Sciences Unit, University of Cambridge. The Principal Investigators are Joni Holmes (Head of CALM), Susan Gathercole (Chair of CALM Management Committee), Duncan Astle, Tom Manly and Rogier Kievit. Data collection is assisted by a team of researchers and PhD students at the CBSU. This currently includes: Sarah Bishop, Annie Bryant, Sally Butterfield, Fanchea Daily, Laura Forde, Erin Hawkins, Sinead O'Brien, Cliodhna O'Leary, Joseph Rennie, and Mengya Zhang. The authors wish to thank the many professionals working in children's services in the South‐East and East of England for their support, and the children and their families for giving up their time to visit the clinic.


Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modification is common.m/z 94.9362) derived from PS moiety. PS is not derived from endogenous substances in animal body, and the product ion is a suitable marker for the detection of PSOs. With our strategy, reproducible target analyses were achieved for identifying the specific substances, with a LOD of 0.1 ng/mL and a quantification rage of 0.1–200 ng/mL in deproteinated plasma. Non-target analyses could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix. These results suggested that the detection of PSOs in horse blood is possible by targeting the product ion using LC/MS/MS.We demonstrated the specific detection of phosphorothioated oligonucleotides (PSOs) using LC/MS/MS. PSOs produce the specific product ion (The online version of this article (10.1186/s13104-018-3885-5) contains supplementary material, which is available to authorized users. Horseracing originated in the early 18th century in Britain with the production of Thoroughbreds by breeding Arabian stallions with British native mares, and it then spread to the rest of the world as a sport  and 10 mM triethylamine [TEA]) and solvent B (methanol), with gradient elution as follows: 5 to 50 to 90% B in 2 and 0.01 min and hold for 0.49 min, then back to 5% B in 0.01 min, and hold for 1.49 min after pre-equilibrium in 5% solvent B for 0.5 min at a flow rate of 0.4 mL/min. Sample injection volume was 2 μL.The samples were analyzed on a Prominence UFLC XR HPLC system  using an ACQUITY UPLC BEH C® 6600 System, SCIEX, Framingham, MA, U.S.A.) was used.A quadrupole-time-of-flight (Q-TOF) tandem mass spectrometer equipped with a DuoSpray™ ion source  in high sensitive mode and enhance ion mode. The MS parameters for the scan range, gas 1 (nebulizer gas), gas 2 (heater gas), curtain gas, temperature, ion spray voltage, declustering potential, and collision energy (CE) were set as follows: m/z 70–300, 60 psi, 80 psi, 25 psi, 600 °C, − 4000 V, − 120 V, and − 92 V, respectively. Mass spectrometer was calibrated with external standard solution before acquisition.For target analyses, a product ion scan was carried out using the precursor ion of m/z 450–2800 for full scan and m/z 70–2800 for product ion scan, respectively.For non-target analyses, Data Dependent Acquisition (DDA) was used to search for the PSOs that could produce the specific product ion of negatively charged PS. It was possible to acquire product ion scan data by triggering the 10 most intense precursor ions within the DDA criteria every 0.2 s cycle. The MS parameters were the same as in the target analyses except for the scan ranges: ®TF 1.7.1 Software (SCIEX), MultiQuant™ 3.0.2 Software (SCIEX), and PeakView® 2.2 Software (SCIEX) were used for data acquisition, target analysis, and non-target analysis, respectively. The peak areas of analyte vs. nominal analyte concentrations were fitted by quadratic regression using 1/x2 weighting factors.Analyst9−, theoretical m/z 785.18) was selected as a precursor ion for target analysis  was performed based on the peak height, peak shape, and signal to noise ratio. MS conditions for oligo 1 (PSO) were examined by flow injection analysis under the LC conditions described above. Multiply charged ions with charge numbers of 3–10 were detected in negative ion mode, and the most-intense-charged ion  of 0.1 ng/mL. We also evaluated the quantitative performance using curve fitting, and reproducibility, assuming that quantitative analysis would be required for pharmacokinetic studies in drug development. For quantitative analyses, good curve fittings were obtained in the range of 0.1–200 ng/mL with a correlation coefficient r ≥ 0.990 and 82–118% accuracy, and within-day and day-to-day reproducibilities were confirmed  and the samples spiked at concentrations of 1–20,000 ng/mL in the same matrix were analyzed with DDA using the specific product ion of Synthetic oligonucleotides containing natural nucleic acids, such as DNA and RNA, are quickly degraded by nucleases when administered into the body . Therefom/z 94.9362) would be a good marker to detect and monitor PSOs in horse blood for gene doping and medicine development.For target analysis, we successfully detected PSO in deproteinated horse plasma with an LOD of 0.1 ng/mL. Furthermore, good curve fittings and reproducibility were confirmed in the range of 0.1 to 200 ng/mL, indicating that the product ion  could be detected with 100 ng/mL. PSOs were selectively detected in the complex biological matrix, including natural nucleic acids, without any sequence information. The results suggested that DDA of a Q-TOF tandem mass spectrometer is a useful method for the detection of unknown PSOs used for doping in the same manner as the model samples used in this study.For non-target analysis, exhaustive scanning of PSOs in deproteinated plasma using the DDA function revealed that the product ions (m/z 94.9362) is an effective marker for the detection of non-approved PSOs used for doping. The product ion is also a good marker for the pharmacokinetics of PSOs because of its quantitative sensitivity.In conclusion, the product ion (The limitation of this study is a model case report that used phosphorothioated (PS) oligonucleotides spiked in deproteinated plasma. Therefore, studying with animal models is recommended.Additional file 1: Figure S1. Mass spectra of full scan and product ion scan for phosphorothioated oligonucleotides (PSOs). Figure S2. Quadratic calibration curves of phosphorothioated oligonucleotides (PSOs) in deproteined plasma. Table S1. Accuracy and reproducibility of phosphorothioated oligonucleotides (PSOs) detection.