Cereus hildmannianus and Pereskia aculeata. The first species produce edible, sweet, spineless fruits and the second species produces leaves with high protein content. Pollination studies were undertaken through fieldwork observations in three localities of Rio Grande do Sul, Brazil, over two flowering seasons, totaling over 130 observation hours. Breeding systems were elucidated utilizing controlled pollinations. Cereus hildmannianus is solely pollinated by nectar-gathering species of Sphingidae hawk moths. In contrast, the flowers of P. aculeata are pollinated by predominantly native Hymenoptera but also by Coleoptera and Diptera, which gather pollen and/or nectar. Both cacti species are pollinator-dependent; neither intact nor emasculated flowers turn into fruit, yet whereas C. hildmannianus is self-incompatible, P. aculeata is fully self-compatible. In sum, C. hildmannianus is more restrictive and specialized regarding its pollination and breeding system, whereas P. aculeata is more generalist. Understanding the pollination needs of these species is a necessary starting point towards their conservation but also for their proper management and eventual domestication.Brazil is one of the centers of diversity of Cactaceae, yet studies addressing both pollination biology and the breeding system in Brazilian cacti are scarce. We herein present a detailed analysis of two native species with economic relevance:  Rhipsalis baccifera (Sol.) Stearn, which occurs naturally in Africa and Sri Lanka, all remaining cacti are restricted to the Americas [Cactaceae is one of the most easily recognizable plant families, embracing about 2230 species distributed in about 176 genera [Americas . In contAmericas .The Pampa vegetation has always been neglected until recent research revealed remarkable records of plant diversity . Of 65 cCereus hildmannianus K. Schum. and Pereskia aculeata Mill. Cereus hildmannianus is popularly known as “Tuna” and its edible, sweet, spineless fruits make them valuable for horticultural purposes [Cereus peruvianus (Pfeiff.) K. Schum., has recently been established under cultivation for such purposes in Israel, where it is sold under the commercial name of “Koubo” [Pereskia aculeata is popularly known as “ora-pro-nobis” . Unlike most Cactaceae, this species presents well-developed, ovate leaves. In turn, these leaves present a high protein content and are highly appreciated in culinary usage [The present contribution aims to present a detailed study of the breeding system and natural pollination of two cactus species native to Rio Grande do Sul: (1) purposes . A very  “Koubo” . Further “Koubo” . (2) Perry usage ,14,15,16ry usage ,18.The questions behind this contribution are the following: (1) Are the study species pollinator-dependent? (2) If so, which are the pollinators? (3) Which is the breeding system in the study species? (4) How do pollinators contribute to cross-pollination?C. hildmannianus. According to the preceding bibliography [Cereus species [P. aculeata at the Porto Alegre Botanical Garden indicated that fruits are abundantly produced. According to Carneiro et al. (2016), the flowers are diurnal and strongly scented. Their color and shape strongly suggest bee pollination. In this context, we established the following hypotheses: (1) Both study species may be pollinator-dependent . (2) Owing to the apparent rarity of fruits, C. hildmannianus may be self-incompatible . (3) Owing to the commonness of fruits in P. aculeata, this species may be self-compatible. (4) Owing to its flower features and according to preceding literature in related species, C. hildmannianus may be pollinated by Sphingidae hawk moths. (5) Owing to its flower features, P. aculeata may be pollinated by bees.Our preliminary observations suggested that the natural fruit set is rare in iography , the flo species ,20. ConvCereus hildmannianus have a nocturnal anthesis, with the flowers starting to open around about 9:00 PM and fading between 6:00 AM and 9:00 AM, depending on climate conditions. The studied flowers showed a color variation on the external side of the tepals, varying from white to soft pink . The flowers of P. aculeata present diurnal anthesis, opening around 6:00 AM and closing around 1:00 PM. Perianth’s color is normally white .The flowers of oft pink a–c. Someoft pink c. The neoft pink c. The nely white d–f, excely white d–f. FlowC. hildmannianus aborted, indicating that this species is self-incompatible. Conversely, P. aculeata produced a high fruit set from either self-pollinated or cross-pollinated flowers and no statistically significant differences were observed between these treatments . Thus, P. aculeata can safely be considered fully self-compatible. The natural pollination success for both species was high: 70% in C. hildmannianus in São Francisco de Assis population (21/30 fruits produced) and 100% for Pereskia aculeata  as pollinators: Eumorpha satellitia (Linnaeus 1771) (Eumorpha vitis Linnaeus 1758 (Manduca sexta (Linnaeus 1763)  (Bombus pauloensis (Friese 1912)), and stingless bees (Trigona spinipes (Fabricius 1793)) were observed collecting pollen but in no instance did these bees touched the stigma and their passive behavior suggests that they are solely non-pollinating flower visitors. As a whole, we counted 44 hawk moth visits during the whole observation period. The photographic and film record indicates that all moth species uncoil their proboscises before landing into the flowers and that they use their forelegs to guide themselves into the floral tube (At the flowers of us 1771) e,f, Eumoeus 1758 c, and Madoptera) b,d (Suppdoptera) . At nighral tube a,b. Insiral tube c–f —B. pauloensis—Xylocopa frontalis (Olivier 1789)—Plebeia droryana (Friese 1900)—Scaptotrigona bipunctata (Lepeletier 1836)—T. spinipes), and wasps of Polybia sp. (Vespidae—Caryedes sp. (Chrysomelidae: Bruchini)  and Aellopos sp. (Shingidae)) were recorded only once gathering nectar at the flowers. During these brief visits, it was not possible to establish if they were, in fact, pollinators, but their infrequency indicates that is unlikely that they are important for the fruit set of P. aculeata; at least at the study site.Anthesis in abundant  and wereVespidae—g. The Coruchini) f and Dip surface . Based o surface , wasps fn pollen . Nectar-n pollen a,b,d,e. Apis mellifera (Apini) visited the flowers 33 times, which was more than bumblebees and carpenter bees but considerably less than native stingless bees , while B. pauloensis and B. morio spent about 3–4 s per visit. The native bees T. spinipes and S. bipunctata spent a mean of 35 and 32 s, respectively. It is an intermediate average time compared to the other bee species which spent less than 10 s or more than 50 s.When recording pollinator frequency, a total of 246 visits were documented. About 66% of these interactions were made by stingless bees (Meliponini) . All othess bees . The frelora sp.  were obslora sp. . CaryedeC. hildmannianus and P. aculeata. To the best of our knowledge, this is also the first pollination report for the whole cactus subfamily Pereskioideae, as it is currently defined [This is the first study coupling detailed pollination observations and breeding system experiments in  defined . Only a  defined ,8,9 and P. aculeata received a more diverse array of pollinators than the morphologically more complex flower of C. hildmannianus. An early divergent group, such as Pereskia, tends to show generalist morphology [C. hildmannianus present a set of features  that fit well in the syndrome of sphingophily (pollination by hawk moths) [C. peruvianus (ca. 27%) [Cereus fernambucensis Lem. (ca. 24%) [P. aculeata makes their flowers less restrictive. Overall flower features  likely fit well under the syndrome of mellitophily (bee pollination). However, insects other than bees were also observed as effective pollinators (see Results). The nectar concentration (ca. 10%) was lower than expected for “typical” melitophylous flowers (ca. 30%) [Denmoza rhodacantha  Britton & Rose (Cereeae: Trichocereinae) present features suggesting bird pollination, yet the actual pollinators are Halictidae bees [Trichocereus pasacana (F.A.C. Weber) Friederich & Rowley, whose flowers are nocturnal and apparently sphingophilous but are apparently pollinated by carpenter bees [Cipocereus laniflorus N.P. Taylor & Zappi [Echinopsis chiloensis subsp. chiloensis (Colla) Friederich & Rowley [Echinopsis ancistrophora Speg. [Echinopsis terscheckii (Parm. ex Pfeiff.) Friederich & Rowley [Echinopsis species are mainly pollinated by Sphingidae moths but also by other insects  [P. aculeata.As it is a rule in Cactaceae, both studied species presented ephemeral flowers that last less than one day. However, the two studied species present contrasting flower shapes  and phenologies . This is reflected in completely different sets of pollinators. As expected, the open-disk-like flowers of rphology . Thereforphology . The flok moths) , a fact ca. 27%) , Cereus ca. 24%) , and othca. 30%)  where fldae bees . A similter bees . Indeed,& Rowley , of Echira Speg. , and Echly bees) ,30. It hly bees) . The samly bees) . ExperimC. hildmannianus is self-incompatible; this is, unable to set fruit following pollination with the pollen of the same individual. Self-incompatibility has already been observed in C. peruvianus [C. repandus [C. fernambucensis with 45% of fruiting rate in manual self-pollination treatment [C. hildmannianus) usually have a long-life cycle and would tend towards self-incompatibility. In contrast, globose species that usually have short life cycles and produce few flowers need to be self-compatible to guarantee reproductive success [Cereus and related genera). In contrast, P. aculeata has proven to be self-compatible and we did not detect statistical differences between the percentages of fruits developed either through manual self-pollination or cross-pollination. Pereskia guamacho F.A.C. Weber, a species from the Caribbean region has proven to be self-incompatible [Leuenbergeria guamacho (F.A.C. Weber) Lodé), and, consequently, is not a “true” Pereskia. Unfortunately, the literature regarding the breeding system of Pereskia sensu stricto is scant and more species should be studied to obtain a more informative background. In this context, this contribution is the first report of the breeding system of a true Pereskia species.Both studied species are pollinator-dependent. Neither intact nor emasculated flowers set fruit, indicating that flowers do not undergo automatic self-pollination or develop fruit from apomixis. This points towards the necessity of pollinators’ agency for the plants to set fruit and viable seed. We herein report that ruvianus  and C. rrepandus . Conversreatment  is consireatment . Tree-li success . If thismpatible . HoweverC. hildmannianus show a clear sphingophilous syndrome; with nocturnal anthesis, long floral tube length, high nectar volume, and around 20% of sugar concentration [C. hildmannianus. This confirms our hypothesis for this species’ pollination and is also in line with precedent literature for the genus [C. peruvianus [C. fernambucensis [C. aethiops [Cereus spp. share the same set of flower features: nocturnal, nectariferous, tubulose, fragrant flowers with a perianth color varying from white to light pink. Pollinator behavior was also described similarly; with the Sphingidae uncoiling the proboscis still in flight and touching the stigma and stamens with the ventral part of the body. These preceding reports, however, didn’t give further details on insect permanency inside the floral tube [Cereus repandus Haw. but without giving details on pollinator behavior. Based on the prevalence of the observed flower features, we expect that all species of Cereus and Harrisia (Trichocereae) are pollinated by Sphingidae moths. One of us (OPB), has recently filmed unidentified Sphingidae pollinating flowers of Cereus hexagonus (L.) Mill. in the Colombian Andes. At least two other cactus species from Rio Grande do Sul present sphingophilous flower features: Epiphyllum phyllanthus (L.) Haw. and Echinopsis oxygona (Link.) Zucc. Globally, 24% of the published pollination reports in Cactaceae involve moths [Overall, the flowers of ntration . In agrehe genus ,20,25. Sruvianus , C. fernral tube ,20,25. Ive moths .P. aculeata reasonably matches with mellitophily, since the flowers present diurnal anthesis, short floral tube, low nectar volume, high pollen production, and a pleasant smell [P. aculeata, insects of other orders were also recorded as pollinators . Hence, we prefer to consider the flowers of P. aculeata as more generalistic in terms of pollination. It is important to stress that among the ten recorded pollinators, eight are bees and seven of these are native. Solitary native bees have already been documented as the main pollinators of sympatric yet unrelated Cactaceae [P. aculeata is self-compatible, this behavior clearly promotes some pollinator-mediated self-pollinations. The longer the insects stay at the flowers, the higher the chances of promoting self-pollination [Caryedes beetles  and Aellopus sp. (Sphingidae), were eventually recorded feeding on nectar. Only one visit was observed for each species. Thus, whereas they cannot be excluded as pollinators, their rareness makes it improbable that they consistently contribute to the fruit set of P. aculeata.Non-Apidae insects had a lower number of interactions compared with native bees and Cereus hildmannianus L. is a columnar (up to 15 m high) cactus  [) cactus a. This s) cactus a–c. The e leaves d,e. Thise leaves d–f. The e leaves d–f. Unlictaceae) .Cereus hildmannianus observations were conducted in the rural area of Caçapava do Sul municipality  and São Francisco de Assis . Pereskia aculeata was studied at the Porto Alegre Botanical Garden within the Porto Alegre municipality . While Caçapava do Sul and São Francisco de Assis are placed within the Pampa Biome, Porto Alegre is located in the transitional area between the Pampas and the Atlantic Rain Forest.The studies were conducted in three localities. All areas are natural occurrences for the respective species  and are P. aculeata and ten in C. hildmannianus) and bagged before anthesis. At the beginning of the anthesis, we measured the volume and sugar concentration  of nectar with a syringe and a manual refractometer.Nectar features were measured in situ from five flowers randomly chosen for each individual  Manual cross-pollination ; (2) manual self-pollination (flowers were pollinated with their own pollen); (3) apomixis (anthers were cut off); and (4) spontaneous self-pollination (intact flowers were bagged without any further manipulation). The number of individuals and flowers per treatment used in the experiments is summarized in eatments . All expividuals . After manus see . TreatmeC. hildmannianus and P. aculeata was measured by counting the number of fruits developed from 30 previously tagged flowers produced by five and four different individuals, respectively  in ectively .C. hildmannianus was studied during night hours (9:00 PM to 7:00 AM) and P. aculeata was studied during the day (6:00 AM to 1:00 PM). The pollination of C. hildmannianus in Caçapava do Sul was studied between January and February 2019, totaling 32 h of observation. The same species was studied in São Francisco de Assis in December 2019, totaling 25 h of observation. The natural pollination of P. aculeata was studied at the Porto Alegre Botanical Garden. Observations were made in March 2018 and March 2019, totaling 75 h of observation. It is important to stress that flower lifespan in both species is less than one day and that—unlike other cacti—the flowers do not open again the next day. The behavior of pollinators was documented through photos and videos. These files were used to analyze insect behavior and to determine if these animals were effective pollinators or non-pollinating visitors. For the purposes of this contribution, “pollinator” is defined as any animal pollen vector that, during foraging activities at the flowers, contacts both fertile flower whorls, thus promoting pollen transfer to the stigmas. Animals not fulfilling these requirements are thereafter considered non-pollinating flower visitors. Insects were collected for subsequent identification by specialists and vouchers were deposited at the entomological collection of the Fundação Estadual de Pesquisas Agropecuárias do Rio Grande do Sul (FEPAGRO). To ascertain pollinator frequency, in each individual of P. aculeata we selected five flowers to compute the duration and frequency of interactions during the anthesis. In C. hildmannianus, we selected one individual per night and observed all interactions. This operational difference between species is due to specific flowering characteristics of each species. As mentioned above, P. aculeata blooms abundantly during each blooming event whereas C. hildmannianus produces few flowers per night.Observations were performed during the flowering periods of both species in 2018 and 2019. Plant phenologies do not fully overlap. Anthesis also happens during different hour intervals (see Results); thus, pollination biology of C. hildmannianus should be self-incompatible ; (3) that, owing to the commonness of fruits in P. aculeata, this species may be self-compatible; (4) that owing to its flower features and according to preceding literature in related species, Cereus hildmannianus may be pollinated by Sphingidae moths; and (5) that owing to its flower features, P. aculeata may be pollinated by bees. Owing to our results, we can conclude that hypotheses 1, 2, 3, and 4 are fully confirmed. Hypothesis 5, however, is only partially confirmed. Native bees were indeed the main pollinators during our observations, but we recorded that P. aculeata also benefited from wasp, Diptera, and Coleoptera pollinators. Irrespective of these results, this contribution highlights the importance of native pollinators for the reproduction of cacti native to Southern Brazil, which is in line with the few preceding reports [The two studied species are clearly pollinator-dependent, yet presented contrasting breeding systems (self-compatibility and self-incompatibility) and pollinators (Sphingidae and mainly melitophilous). At the beginning of this contribution, we explicitly formulated the following hypotheses: (1) That both study species should be pollinator-dependent ; (2) that, owing to the rarity of fruits,  reports ,8,9. Thu reports ,36, meas


For Schistorchis Lühe, 1906, we report the type-species Sch. carneus Lühe, 1906 and Sch. skrjabini Parukhin, 1963. For Sphinteristomum Oshmarin, Mamaev & Parukhin, 1961 we report the type-species, Sph. acollum Oshmarin, Mamaev & Parukhin, 1961. We report and re-recognise Lobatotrema Manter, 1963, for the type and only species, L. aniferum Manter, 1963, previously a synonym of Sph. acollum. Lobatotrema aniferum is phylogenetically distant from, but morphologically similar to, Sph. acollum and Lobatotrema is recognised as a ‘cryptic genus’. We propose Blendiellan. gen. for B. trigintatestisn. sp. and B. tridecimtestisn. sp. These species are broadly consistent with the present morphological concept of Schistorchis but are phylogenetically distant from the type-species; a larger number of testes and some other subtle morphological characters in species of Blendiella serve to distinguish the two genera. We report three species of Paraschistorchis Blend, Karar & Dronen, 2017: P. stenosoma  Blend, Karar & Dronen, 2017 (type-species), P. seychellesiensis  Blend, Karar & Dronen, 2017, and P. zancli  Blend, Karar & Dronen, 2017. Lobatotrema aniferum, P. stenosoma, and Sch. carneus each have two distinct cox1 populations either over geographical range or in sympatry. Available evidence suggests that most of these species, but not all, are widespread in the tropical Indo-Pacific.We report nine species of the Schistorchiinae Yamaguti, 1942  from Indo-Pacific marine fishes. Molecular data (ITS2 and 28S rDNA and  Members of the Apocreadiidae Skrjabin, 1942 parasitise a wide range of marine and freshwater fishes, with the family comprising four subfamilies  and 23 genera  mitochondrial region. The complete ITS2 region was amplified and sequenced using the primers 3S . The cox1 alignment was transferred to Mesquite v.3.31  based on both morphological and molecular distinctions. Morphological OTUs were assigned to genera using the classification of Blend et al. . Moleculy et al. , incorpoSpecimens consistent with the concept of the Schistorchiinae were collected from four families of the Tetraodontiformes  and one of the Acanthuriformes (Zanclidae). Our analyses of further samples from scarids and siganids are inconclusive and those specimens, together with those of several rare species, are reserved for future publication dependent on further collections. Here we report on nine species, including two proposed as new.Schistorchis Lühe, Schistorchis carneus Lühe, Schistorchis skrjabini Parukhin, Sphinteristomum Oshmarin, Mamaev & Parukhin, Sphinteristomum acollum Oshmarin, Mamaev & Parukhin, Lobatotrema Manter, Lobatotrema aniferum Manter, Sphinteristomum and Sph. acollum. A new genus, Blendiellan. gen., is proposed for two new species consistent with the present diagnosis for Schistorchis Lühe, Paraschistorchis Blend, Karar & Dronen, Paraschistorchis stenosoma  and, for five of these, intraspecific variation was insignificant, ranging from only 0–4 bp. In contrast, three morphotypes incorporated deep divisions in cox1 sequence data. Sequences from specimens consistent with L. aniferum from Ningaloo Reef and from off Lizard Island differed at 22 bp. Sequences from specimens consistent with P. stenosoma collected from C. pardalis from off both Heron and Lizard Islands differed at 48 bp. Sequences from specimens consistent with Sch. carneus from Arothron stellatus and A. manilensis, all from off the Queensland coast, differed at 48–49 bp.Neighbour joining analysis of the Blendiella differed at only 3 bp in the ITS2 analysis whereas they differed at 56–57 bp in the cox1 analysis. The next most similar combination of putative species differed at 12 bp (Sph. acollum v. the two Blendiella species). The six ITS2 sequences relating to P. stenosoma were all identical, in contrast to the cox1 analysis in which they differed at 48 bp. The five sequences of L. aniferum from Ningaloo Reef differed from the six from off Lizard Island by just 1 bp in the ITS2 dataset, whereas they differed at 22 bp in the cox1 dataset. The four sequences relating to Sch. carneus from A. stellatus differed from the two from A. manilensis by 3 bp (the same as for the two species of Blendiella), whereas they differed by 48 bp in the cox1 analysis.Neighbour joining analysis of the ITS2 dataset distinguished the nine morphotypes by 3–51 bp  were used principally to explore phylogenetic relationships (see below). In terms of species delineation, combinations of the nine morphospecies differed at 8–116 bp; the lowest distinction of 8 bp was between the two new species of Blendiella. The three morphospecies for which cox1 data demonstrated deep divisions  all had identical 28S sequences for corresponding specimens.Twelve newly generated 28S rDNA sequences  specimens consistent with P. stenosoma was not replicated in ITS2 or 28S rDNA sequences. We recognise both forms as a single species; comparable sympatric and morphologically indistinguishable populations have been reported for two species of Preptetos Pritchard, 1960 on the GBR  ………...Lobatotrema Manter, 3a. Parasites of species of Abalistes and Rhinecanthus  and Sparidae…………………………....Sphincteristomum Oshmarin, Mamaev & Parukhin, 3b. Parasites of species of 4a. Oral sucker highly glandular……………………………………………...……5.4b. Oral sucker normally muscular, or not strongly glandular………………………………..6.Schistorchis Lühe, 5a. Testes 8–11, normally in two distinct columns; body robust……. Blendiella n. gen.5b. Testes ≥11, normally mainly in single column; body slender…………..via uroproct.……….….…Neomegacreadium Machida & Kuramochi, 6a. Caeca open 6b. Caeca terminate blindly or at separate ani……………………………….……………….7.Plesioschistorchis Blend, Karar & Dronen, 7a. Caeca end blindly……………………....Paraschistorchis Blend, Karar & Dronen, 7b. Caeca open at marginal or submarginal ani………..……………………………… ……………………………………………….Apocreadiidae Skrjabin, 1942Schistorchiinae Yamaguti, 1942Schistorchis  Lühe, 1906Type-species:Schistorchis carneus Lühe, 1906, by original designationsensu Blend et al.  Blend, Karar & Dronen, 2017Schistorchis carneus Lühe, 1906Pleorchis oligorchis Johnston, 1913Syn. Type-host: Arothron stellatus (Bloch & Schneider), Stellate puffer (Tetraodontiformes: Tetraodontidae).Type-locality: South Modragam Paar, Sri Lanka.New materialHosts: Arothron manilensis (Marion de Procé), Narrowlined puffer; A. stellatus (Tetraodontiformes: Tetraodontidae).Localities: off Lizard Island , northern GBR; Mackay Harbour , north Queensland; Moreton Bay , south-east Queensland, Australia.Site in host: Intestine.Prevalence: off Lizard Island: 1 of 3 (33%) A. stellatus. Mackay Harbour: 1 of 1 A. manilensis. Moreton Bay: 3 of 5 (60%) A. stellatus.Deposition of specimens: QM G240397–406.Representative DNA sequences: Partial cox1 mtDNA, seven sequences ; ITS2 rDNA, six sequences ; partial 28S rDNA, three sequences .Measurements: Table Arothron stellatus off Sri Lanka. It was subsequently reported from Arothron hispidus (Linnaeus) by Johnston  (Tetraodontidae) from the Bay of Bengal, India.This species (the type-species of the type-genus of the Schistorchiinae) was described by Lühe  from AroJohnston  as Pleor. Odhner  recogniseezullah  from A. i et al.  from LagA. manilensis from off Mackay are morphologically consistent with previous descriptions of this species and are immediately distinct from all other described species of Schistorchis in their massive bodies. We conclude that the sequences reported here for specimens from the type-host  serve to establish the molecular identity of the type-species of the type-genus for the Schistorchiinae.New specimens reported from the type-host, from the northern GBR and Moreton Bay, and from Sch. carneus from A. stellatus from off Lizard Island and from Moreton Bay and from A. manilensis from Mackay Harbour, almost midway between the two other sites. cox1 sequence data for samples from the two fish species differ at 48 bp, ITS2 sequence data differ at 3 bp, and 28S sequence data are identical. The 48 bp cox1 distinction, although substantial, is less than that between the most similar combination of species recognised here (56–57 bp), the two new species of Blendiella, and identical to the 48 bp difference between what is below interpreted as two sympatric populations of P. stenosoma. The ITS2 distinction of 3 bp is identical to that between the two species of Blendiella, and greater than any distinction interpreted as intraspecific . The absence of distinction between 28S sequences contrasts with a difference of 8 bp between the two species of Blendiella which are otherwise by far the most similar combination of species recognised in this data set for that marker. These data present a host-associated distinction in the molecular characterisation of these forms, but we have been unable to detect a consistent morphological difference between specimens from A. manilensis and A. stellatus. These data are problematic. Strictly, based on the species recognition criteria that we employ, combined molecular and host distinction creates a basis for the recognition of separate species. However, we choose not to propose a new species because of three qualitative weaknesses in the case. First, the molecular distinction is generally lower than that typically seen between combinations of species in this family. Secondly, the host distinction is marginal in that other records interpreted as S. carneus incorporate further host richness  which either casts doubt on the importance of host distinction or might suggest that even more unrecognised richness is present in the genus. Finally, there is only a single collection of two specimens from a single A. manilensis, so that there is negligible replication of the basis of the key distinction in the data set. We conclude that the conservative option is to identify all these specimens as S. carneus pending the gathering of further evidence.Sequence data are available for specimens here identified as Schistorchis skrjabini Parukhin, 1963Type-host: Triacanthus biaculeatus (Bloch), Short-nosed tripodfish (Tetraodontiformes: Triacanthidae) [as Triacanthus brevirostris Temminck & Schlegel].Type-locality: South China Sea.New materialHosts: Tripodichthys angustifrons (Hollard), Black-flag tripodfish (Tetraodontiformes: Triacanthidae).Locality: Moreton Bay , south-east Queensland, Australia.Site in host: Intestine.Prevalence: 13 of 57 (23%).Deposition of specimens: QM G240407–22.Representative DNA sequences: Partial cox1 mtDNA, five sequences ; ITS2 rDNA, four sequences ; partial 28S rDNA, four sequences .Measurements: Table  Body robust, with maximum breadth in mid-hindbody and posterior end more sharply tapered than anterior end. Tegument spinous to level of ventral sucker. Pre-oral lobe strongly developed. Eye-spot pigment dispersed in forebody. Oral sucker round in outline, highly glandular; U-shaped muscular sphincter at oral aperture prominent. Prepharynx short. Pharynx unspecialised, rounded, typically partly dorsal to posterior margin of oral sucker. Oesophagus absent. Intestinal bifurcation immediately posterior to pharynx, close to level of anterior margin of ventral sucker. Caeca open at separate ani close to posterior extremity. Ventral sucker round in outline, unspecialised, far smaller than oral sucker. Testes 11 (10 in one specimen), entire, in compact group up to three testes wide and five testes long, close to ventral sucker; post-testicular region occupying approximately two-fifths to half body length. Seminal vesicle large, saccular, dextrally antero-dorsal to ventral sucker. Genital pore ventro-submedian, immediately anterior to ventral sucker. Ovary entire, ovoid, median, immediately anterior to most anterior median testis, usually partly overlaps ventral sucker dorsally. Canalicular seminal receptacle not detected. Vitelline follicles extend from level of ventral sucker or just entering forebody to close to posterior extremity, restricted to lateral margins anteriorly, occupying most available space posterior to testes, dorsally confluent immediately posterior to testes then separated into four variably distinct columns by intestinal caeca and excretory vesicle. Uterus short, passes from level of ovary to just anterior to ventral sucker. Excretory vesicle I-shaped, terminates at level of posterior margin of most posterior testis. Excretory arms extensive, to anterior margin of oral sucker. Excretory pore terminal.T. biaculeatus (as T. brevirostris) and from the balistid Abalistes stellatus (Bloch & Schneider) [as Abalistes stellaris (Bloch & Schneider)]; T. brevirostris was the first-named host and is interpreted as the type-host here. The species was subsequently reported by Hafeezullah (T. biaculeatus (again as T. brevirostris) from the Bay of Bengal.This species was described by Parukhin  from theeezullah  in T. biSch. skrjabini. The specimens also resemble Sch. paruchini Kurochkin, Meuschenia australis (Donovan) [as Navodon australis (Donovan)], from the Great Australian Bight . The report of this species in a balistid is probably exceptional or erroneous. In Moreton Bay, Sch. skrjabini occurs commonly in T. angustifrons but in none of multiple monacanthids and tetraodontids examined there. However, we have examined no balistids from this location.The specimens reported here are strongly consistent with the two previous descriptions of Sphincteristomum Oshmarin, Mamaev & Parukhin, 1961Type-species: Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, 1961by original designation.sensu Blend et al.  in the Indo-Pacific and sparid fishes in the Mediterranean Sea.With characters of Schistorchiinae d et al. . Genus pType-species: Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, Other species: Sphincteristomum mediterraneae Abid-Kachour, Mouffok & Boutiba, Sphincteristomum nikolevi Parukhin, Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, 1961Type-host: Abalistes stellatus (Bloch & Schneider), Starry triggerfish .Type-locality: Tonkin Bay, Vietnam.New materialHost: A. stellatus.Locality: off Lizard Island , northern GBR, Australia.Site in host: Intestine.Prevalence: 1 of 1 (100%).Deposition of specimens: Hologenophore QM G240423.Representative DNA sequences: Partial cox1 mtDNA, one sequence ; ITS2 rDNA, one sequence ; partial 28S rDNA, one sequence .Measurements: Table A. stellatus from off Lizard Island.] Body broad, with maximum breadth in mid-hindbody; posterior margin damaged. Tegument spinous to mid-hindbody. Pre-oral lobe distinct. Eye-spot pigment dispersed in forebody. Oral sucker roughly pyriform, highly glandular; U-shaped muscular sphincter embedded at aperture. Prepharynx short. Pharynx large, rounded, with anterior portion dorsal to oral sucker. Oesophagus absent. Intestinal bifurcation just anterior to anterior margin of ventral sucker. One caecum opening at anus on damaged posterior margin; end of second caecum removed in portion used for sequencing. Ventral sucker round in outline, unspecialised, far smaller than oral sucker. Testes two, tandem, contiguous, in anterior hindbody, distinctly lobed; anterior testis significantly wider than long; posterior testis triangular; post-testicular region occupying approximately two-fifths body length (based on estimated full body length). Seminal vesicle saccular, prominent, dextral to ventral sucker. Genital pore ventro-submedian, slightly sinistral, immediately anterior to ventral sucker. Ovary ovoid, median, contiguous with anterior testis, mostly posterior to ventral sucker. Canalicular seminal receptacle not detected. Vitelline follicles extend from level of posterior margin of oral sucker to close to posterior extremity, mainly restricted to lateral margins anteriorly, occupying most available space around and posterior to testes, dorsally confluent immediately posterior to testes then broadly separated into columns by intestinal caeca and excretory vesicle. Uterus short, between level of ovary and genital pore. Excretory vesicle I-shaped, reaches to posterior testis. Excretory pore missing from damaged posterior extremity. Body elongate linguiform, with maximum breadth at level of testes; posterior end slightly tapered, bluntly rounded. Tegument spinous in forebody. Pre-oral lobe distinct. Eye-spot pigment dispersed in forebody. Oral sucker ovoid to elongate pyriform, highly glandular; U-shaped muscular sphincter embedded at aperture. Prepharynx short. Pharynx subquadrate, immediately posterior to or slightly overlapping oral sucker. Oesophagus insignificant to absent. Intestinal bifurcation close to level of anterior margin of ventral sucker. Caeca open separately at posterior extremity at marginal ani. Ventral sucker round in outline, unspecialised, far smaller than oral sucker. Testes two, tandem, in midbody, contiguous, usually entire, sometimes distinctly lobed; post-testicular region occupying approximately two-fifths body length. Seminal vesicle saccular, small, dextral, mainly dorsal to ventral sucker. Genital pore immediately anterior to ventral sucker, slightly sinistrally submedian. Ovary ovoid, slightly dextral, contiguous with anterior testis, usually immediately posterior to ventral sucker. Canalicular seminal receptacle saccular, dorsal to ovary. Vitelline follicles extend from just anterior to ventral sucker, usually paralleling intestinal caeca, to close to posterior extremity, mainly restricted to lateral margins anteriorly, becoming more extensive posteriorly and occupying most available space around and posterior to testes, dorsally confluent immediately posterior to testes then broadly separated into four poorly defined columns by intestinal caeca and excretory vesicle. Uterus extends anteriorly from level of posterior margin of ovary. Excretory vesicle I-shaped, reaches to posterior testis. Excretory pore terminal., which has the testes at the posterior end of the body  fell in Clade 3. These results indicated that neither Balistoides nor Pseudobalistes as presently constituted is monophyletic. The proposed solution included recognition of P. fuscus as a species of Balistes and B. viridescens as a species of Pseudobalistes. These changes do not appear to have been adopted in the fish literature, although we detect no literature presenting a dissenting view. Regardless, the balistid relationships reported by McCord & Westneat (L. aniferum in two (non-sister) clades and those of Sph. acollum in the third. Unfortunately, these insights do not materially improve understanding of genus-level-distinction between Lobatotrema and Sphincteristomum.The evidence from New Caledonia, Ningaloo Reef, the GBR and Japan, suggests that Westneat  inferredWestneat  have theBlendiella n. gen.sensu Blend et al.  Blend and his extended contribution to this field, including specifically to the understanding of the Schistorchiinae.ZooBank LSID: urn:lsid:zoobank.org:act:3F9CF5AA-DCB0-4F70-AD47-51E865EEF228.Schistorchis on the basis of their highly glandular oral suckers and large numbers of testes. However, the molecular phylogenetic analyses presented here show that the two new species form a clade distant from the type-species of Schistorchis, Sch. carneus, which forms a strongly supported clade with one other sequenced species consistent with the present concept for the genus, Sch. skrjabini. If schistorchiine genera are to be maintained as monophyletic, several solutions to this problem are possible. Schistorchis and Sphincteristomum could be synonymised, recognising Schistorchis as a genus in which the testis number ranges from 2–33. We find that idea unsatisfactory for these parasites, although we note that Miller & Cribb  (B. trigintatestis) and 10–16 (13) (B. tridecimtestis) testes whereas species of Schistorchis almost always have fewer . Just two of 19 B. tridecimtestis had fewer than 12 testes. The two new species are also significantly smaller and narrower than the four Schistorchis species. The largest specimen of the new genus is 1,631 µm long in comparison with recorded maxima of 14,400 µm for Sch. carneus, 2,900 µm for S. kurochkini, 3,600 µm for S. skrjabini and 13,500 µm for Sch. tetraodontis. The oral suckers of the two new species have a distinctive shape, constricted anteriorly, whereas those of species of Schistorchis are evenly round in outline. Finally, the two new species both infect balistids whereas the four recognised species of Schistorchis are reported from monacanthids, tetraodontids and triacanthids. We acknowledge that these distinctions are relatively subtle, but in combination they allow recognition of the new genus.In the recent classification proposed by Blend et al. , the two & Cribb  took thaBlendiella trigintatestis n. sp.Type-host: Sufflamen chrysopterum (Bloch & Schneider), Halfmoon triggerfish .Other hosts: Rhinecanthus aculeatus (Linnaeus), White-banded triggerfish; Balistapus undulatus (Park), Orange-lined triggerfish .Type-locality: off Lizard Island , northern GBR, Australia.Site in host: Intestine.Prevalence: 37 of 78 (47%) S. chrysopterum; 7 of 39 (18%) R. aculeatus; 13 of 19 (68%) B. undulatus.Deposition of specimens: QM G240439–72.Representative DNA sequences: Partial cox1 mtDNA, six sequences ; ITS2 rDNA, nine sequences ; partial 28S rDNA, two sequences .Measurements: Table ZooBank LSID: urn:lsid:zoobank.org:act:86F228BA-4F4F-4FEF-B082-3CD8A82540E8.Etymology: the specific name is composed from the Latin for 30 (triginta), and testis, in reference to the exceptional number of testes in this species.S. chrysopterum from off Lizard Island.] Body small, elongate with nearly parallel margins, maximum breadth anywhere from level of ventral sucker to mid-hindbody; posterior end tapered and bluntly rounded. Tegument spinous in anterior forebody. Pre-oral lobe distinct. Eye-spot pigment dispersed in forebody. Oral sucker pyriform, with broad posterior margin, highly glandular; small U-shaped muscular sphincter embedded at aperture. Prepharynx short. Pharynx subquadrate, muscular and immediately posterior to oral sucker, sometimes dorsally overlapping oral sucker slightly. Oesophagus absent. Intestinal bifurcation in mid-forebody. Caeca open at separate marginal ani close to posterior extremity. Ventral sucker round in outline, unspecialised, far smaller than oral sucker. Testes 25–33, entire, contiguous, arranged in broad column from just posterior to ventral sucker to one-third of hindbody length from posterior extremity; column initially three or four testes wide, but narrows progressively, eventually to one posteriorly. Seminal vesicle small, saccular, dorso-dextral to ventral sucker. Genital pore immediately anterior to ventral sucker, slightly sinistro-submedian. Ovary roughly ovoid, posterior, and slightly dextral to ventral sucker, contiguous with anteriormost dextral testes. Canalicular seminal receptacle saccular, antero-dorsal to ovary. Vitelline follicles extend from just anterior to ventral sucker to close to posterior extremity, mainly paralleling intestinal caeca anteriorly, becoming more extensive posteriorly and occupying most available space around and posterior to testes, confluent immediately posterior to testes then broadly separated into four poorly defined columns by intestinal caeca and excretory vesicle. Uterus short, restricted to area between ovary and pharynx. Excretory vesicle I-shaped, relatively short, terminates well short of posterior testis. Excretory pore terminal.. Body relatively small, elongate with nearly parallel margins, with maximum breadth anywhere from level of ventral sucker to mid-hindbody. Posterior end tapered and bluntly rounded. Tegument spinous in anterior forebody. Pre-oral lobe distinct. Eye-spot pigment dispersed in forebody. Oral sucker pyriform, with broad posterior margin, highly glandular; small U-shaped muscular sphincter embedded at aperture. Prepharynx short. Pharynx subquadrate, muscular and immediately posterior to oral sucker, sometimes dorsally overlapping oral sucker slightly. Oesophagus absent. Intestinal bifurcation in mid-forebody. Caeca open at separate marginal ani close to posterior extremity. Ventral sucker round in outline, unspecialised, far smaller than oral sucker. Testes 11–16, entire, contiguous anteriorly, slightly separated posteriorly, arranged in broad column from distinctly posterior to ventral sucker to mid-hindbody; column initially two or three testes wide but rapidly narrows progressively to one for posterior half. Seminal vesicle small, saccular, dorso-dextral or dorso-posterior to ventral sucker. Genital pore immediately anterior to ventral sucker, sightly sinistro-submedian. Ovary roughly ovoid, posterior to  and slightly dextral to ventral sucker, contiguous with anteriormost testes. Canalicular seminal receptacle saccular, antero-dorsal to ovary. Vitelline follicles extend from about level of pharynx to close to posterior extremity, mainly paralleling intestinal caeca anteriorly, becoming more extensive posteriorly and occupying most available space around and posterior to testes, confluent immediately posterior to testes then broadly separated into four columns by intestinal caeca and excretory vesicle. Uterus short, restricted to area between ovary and pharynx. Excretory vesicle I-shaped, relatively short, terminates well short of posterior testis. Excretory pore terminal. (Tetraodontiformes: Monacanthidae).Type-locality: Hawaii, United States of America (USA).New materialHosts: Cantherhines pardalis; Cantheschenia grandisquamis Hutchins, Large-scaled leatherjacket (Tetraodontiformes: Monacanthidae).Localities: off Lizard Island , northern GBR; off Heron Island , southern GBR, Australia.Site in host: Intestine.Prevalence: off Lizard Island: 3 of 9 (33%) C. pardalis. Off Heron Island: 17 of 35 (49%) Cantherhines pardalis; 26 of 43 (60%) Cantheschenia grandisquamis.Deposition of specimens: Hologenophores Mitochondrial Genotype A QM G240498–500; vouchers not differentiated by mitochondrial sequences QM G240501–27.Representative DNA sequences: Partial cox1 mtDNA, six sequences ; ITS2 rDNA, seven sequences ; partial 28S rDNA, four sequences .Measurements: Table C. pardalis from off Heron Island.] Body narrow, with almost parallel margins and maximum breadth usually slightly anterior to ventral sucker. Posterior margin rounded, roughly symmetrical relative to anterior margin. Tegument spinous to level of ventral sucker. Pre-oral lobe distinct. Eye-spot pigment dispersed in forebody. Oral sucker rounded in outline, muscular but with clear glandular components; U-shaped muscular sphincter embedded at aperture discernible, but not conspicuous. Prepharynx short. Pharynx subquadrate, typically partly dorsal to oral sucker. Oesophagus distinct. Intestinal bifurcation immediately anterior to ventral sucker. Caeca broad, terminate at separate ani at posterior extremity. Ventral sucker round in outline, unspecialised, only marginally smaller than oral sucker. Testes usually 11, , usually contiguous, commencing well posterior to ventral sucker, arrangement dependent on extension of body; in well-extended specimens testes in single column; in more contracted specimens, testes may be opposite anteriorly and zigzag posteriorly; post-testicular region occupying approximately one-quarter body length. Seminal vesicle saccular, small, postero-dorsal to ventral sucker. Genital pore ventro-submedian, immediately anterior to ventral sucker, slightly sinistral. Ovary rounded in outline, well-separated from and slightly dextral to ventral sucker. Canalicular seminal receptacle not detected. Vitelline follicles extend from about level of posterior margin of ovary to close to posterior extremity, occupying most available space around and posterior to testes, unevenly ventrally confluent posterior to testes, only weakly separated into columns by intestinal caeca and excretory vesicle. Uterus relatively long, extends slightly posterior to ovary, fills space between ovary and ventral sucker. Excretory vesicle I-shaped, reaches to posterior testis. Excretory pore terminal. (Tetraodontiformes: Monacanthidae).Type-locality: Seychelles Islands, Indian Ocean.New materialHost: C. pardalis.Localities: off Heron Island , southern GBR; off Lizard Island , northern GBR, Australia.Site in host: Intestine.Prevalence: off Lizard Island 8 of 9 (89%). Off Heron Island 14 of 35 (40%).Deposition of specimens: QM G240528–41.Representative DNA sequences: Partial cox1 mtDNA, seven sequences ; ITS2 rDNA, seven sequences ; partial 28S rDNA, two sequences .Measurements: Measurements in Table C. pardalis from off Heron Island.] Body broad, linguiform, with maximum breadth at mid-hindbody; posterior margin sometimes indented slightly at each anus; anterior margin rounded. Tegument spinous to mid-forebody. Pre-oral lobe well developed. Eye-spot pigment dispersed in forebody. Oral sucker rounded in outline, muscular but with clear glandular components; U-shaped muscular sphincter embedded at aperture, well-developed and prominent. Prepharynx short. Pharynx small, subquadrate, typically partly dorsal to oral sucker. Oesophagus short but distinct in well-extended specimens. Intestinal bifurcation immediately anterior to ventral sucker. Caeca broad, terminate at separate ani at posterior extremity. Ventral sucker round in outline, unspecialised, noticeably smaller than oral sucker. Testes 9–11, roughly ovoid, in broad contiguous column, initially 2 testes wide but narrowing to one testis posteriorly; post-testicular region occupying approximately one-third body length. Seminal vesicle saccular, small, postero-dorsal to ventral sucker. Genital pore ventro-submedian, immediately anterior to ventral sucker, slightly sinistral. Ovary rounded in outline, dextral, typically slightly separated from ventral sucker anteriorly and anteriormost testis posteriorly. Canalicular seminal receptacle saccular, antero-dorsal to ovary. Vitelline follicles extend from about level of anterior margin of ventral sucker to close to posterior extremity, anteriorly principally lateral to intestinal caeca, posteriorly occupying most available space around and posterior to testes, unevenly ventrally confluent posterior to testes, separated into relatively distinct columns by intestinal caeca, testes, and excretory vesicle. Uterus short, restricted to area lateral and anterior to ovary to ventral sucker. Excretory vesicle I-shaped, reaches close to level of posterior testis. Excretory pore terminal.[Based on 13 wholemount specimens, including three hologenophores from C. pardalis from off the Seychelles Islands in the Indian Ocean based on just one adult and one immature specimen. The present material is from the same host, broadly agrees with the original description of P. seychellesiensis, and is distinguished from the other three species by the breadth of the body. Notably, the original description shows the testes as forming a single column in contrast to the present specimens in which the column commences as two testes wide and narrows to one for the last few testes. This distinction translates into the post-testicular region in the original description occupying a considerably smaller proportion of total body length (16.3%) than in the present specimens, 21.0–38.4 (30.5)%. Whether this difference should be considered as intra-specific or inter-specific is debateable. Here we take the view that the similarities between these samples outweigh the differences and thus identify them as P. seychellesiensis. Clearly, further sampling and, ideally, sequencing from the type-location is necessary to improve confidence in this identification.This species was described by Toman  from C. P. stenosoma and P. seychellesiensis as indicated by the two figures presented here, the two are not always as easily distinguished as might be expected. Inter-specific differences are most pronounced in the largest specimens. In these, the two species differ clearly in shape (P. seychellesiensis is much broader), oral sucker width , the pharynx width tends to be larger in P. seychellesiensis, and the vitelline follicles commence further from the anterior end than do those of P. seychellesiensis. However, in specimens <2 mm long, all these distinctions converge. We recognised just one character distinction present in large easily distinguished specimens (including the hologenophores) that was also present in smaller specimens. The partial oral sucker sphincter of P. seychellesiensis is always discernible as a strongly and clearly recognisable structure whereas in P. stenosoma it is present, but far less easy to distinguish. Although the sphincters are here figured as comparable, that of P. seychellesiensis is consistently far better developed.Despite the seemingly obvious differences between cox1, ITS2 and 28S datasets  Blend, Karar & Dronen, 2017Hanson,  Blend, KSchistorchis zancli Hanson, 1953Syn. Type-host: Zanclus cornutus (Linnaeus), Moorish idol .Type-locality: Hawaii, USA.New materialHost: Z. cornutus.Localities: off Heron Island , southern GBR, Australia. Off Moorea , Society Archipelago, French Polynesia. Off New Caledonia . Off Palau .Site in host: Intestine.Prevalence: off Heron Island 3 of 30 (10%). Off Moorea 9 of 15 (60%). Off New Caledonia 1 of 2 (50%). Off Palau 1 of 1 (100%).Deposition of specimens: QM G240542–71.Representative DNA sequences: Partial cox1 mtDNA, four sequences ; ITS2 rDNA, two sequences ; partial 28S rDNA, one sequence .Measurements: Table Paraschistorchis zancli was originally described from off Hawaii by Hanson  off Hawaii. Lo et al. (Z. cornutus from off Moorea (French Polynesia) and Heron Island (southern GBR) and reported identical 28S and ITS2 sequences and only inconsequential differences in morphology.y Hanson  from Z. ritchard . Yamagutritchard  reportedo et al.  reported. Careful examination of multiple specimens from three localities  leads us to conclude that a sphincter is probably present but that, unlike in its congeners, it is exceptionally difficult to discern. We further note that, unlike in the congeners P. stenosoma and P. seychellesiensis, there is little if any development of gland cells in the oral sucker of this species.The new specimens are consistent with the previous morphological descriptions of the species with minor differences in appearance attributed to geographical or intraspecific variation. A novel observation here is that the supposedly key schistorchiine characteristic of a partial sphincter embedded in the oral sucker is exceptionally hard to detect. We note that the first two descriptions of the species Hanson,  made no cox1 from French Polynesia and Palau and for ITS2 from French Polynesia, Heron Island and Palau , although not nearly as pronounced as those of the species of Blendiella, Lobatotrema, Schistorchis and Sphincteristomum. In addition, we do not detect clear gland cells at all in the oral sucker of P. zancli. Strikingly, the sphincter in the oral sucker of P. zancli is reduced to the point that we have found it difficult to detect, although our view is that a weak structure is present. We hypothesise that glandular oral suckers and the oral sphincter are linked characters of the Schistorchiinae as a whole. We suggest a functionality whereby the sphincter allows host tissue to be securely captured in the cavity of the oral sucker where secretions of the gland cells begin some aspect of the digestion of the host tissue. If broadly correct, we would predict that the strongest gland cell development would be associated with strongly developed sphincters, and vice versa (as appears to be the case for P. zancli). This idea suggests that some level of glandular development may be expected in almost all schistorchiines although it need not obviate its careful use in the distinction of genera.One of the key characters used by Blend et al.  to distiL. aniferum and P. zancli) suggest that widespread distributions are plausible. In addition, the species under consideration are relatively morphologically distinctive. In this context there is evidence for the distribution of Sch. carneus in Sri Lanka, the Red Sea and eastern Australia, for P. zancli from Hawaii, French Polynesia, Palau and the GBR, for L. aniferum from Fiji, Japan, New Caledonia, the GBR and Ningaloo Reef, and for Sph. acollum from south-east Asia and the GBR. Presumably we can expect more, perhaps most species to prove to be widespread as collecting becomes more comprehensive.Overall, what we know of the distribution of schistorchiine species fits a paradigm of broad Indo-west Pacific distributions of trematodes of tropical marine fishes  from monacanthids of the genus Cantherhines show enigmatic distributions. Paraschistorchis seychellesiensis was described from the Seychelles and P. stenosoma was described and subsequently reported several times form Hawaii; neither species has been reported elsewhere. Here we found both species occurring in the same individual fish on the GBR, more-or-less halfway between the two original description sites. Molecular phylogenetic analysis finds that the two species are sister taxa. This relationship suggests the possibility that the two species diverged in allopatry  and that their distributions subsequently expanded to now overlap. Such hypotheses are clearly speculative and require far more sampling, but the known patterns of distribution suggest that a range of processes may have affected the distributions of schistorchiines.Relative to the broad evidence for widespread species, there are several noteworthy exceptions. First, the two new species of cox1 sequences. This marker was sequenced for what we have interpreted as five species over ranges at least as great as that between Heron and Lizard Islands which are separated by about 1,200 km. Three patterns were recognised. Paraschistorchis seychellesiensis and P. zancli showed negligible cox1 distinctions over range. In contrast, L. aniferum differed at 22 bp between samples from Lizard Island and Ningaloo Reef, from opposite sides of the Australian continent. Finally, S. carneus and P. stenosoma were each represented on the GBR by two cox1 populations, both differing at 48 bp; in the case of P. stenosoma, both populations occur at both Heron and Lizard Islands. The differences of 48 bp are only marginally smaller than the 56–57 bp between the two new species of Blendiella which are easily differentiated by testis number but are otherwise highly similar. Contrasting patterns of regional population structure and its absence have been reported for related species of Aporocotylidae (Cutmore et al., cox1 lineages of the same species infecting the same fish species remains uncommon; it was reported for two species of the lepocreadiid genus Preptetos by Bray et al. (cox1 populations are interpreted as representing the same species on the basis of the species recognition criteria proposed by Bray et al. (cox1 sequences frequently give insight into the recent population history of Indo-Pacific fish trematodes. The extent to which the inconsistency of cox1 similarity is a reliable reflection of population history remains to be explored.Finally, we draw attention to the varying patterns of intraspecific structuring suggested by y et al. . In thaty et al. ; they fa


Candida albicans is a commensal fungus in healthy humans that causes infection in immunocompromised individuals through the secretion of several virulence factors. The successful establishment of infection is owing to elaborate strategies to cope with defensive molecules secreted by the host, including responses toward oxidative stress. Extracellular vesicle (EV) release is considered an alternative to the biomolecule secretory mechanism that favors fungal interactions with the host cells. During candidiasis establishment, the host environment becomes oxidative, and it impacts EV release and cargo. To simulate the host oxidative environment, we added menadione (an oxidative stress inducer) to the culture medium, and we explored C. albicans EV metabolites by metabolomics analysis. This study characterized lipidic molecules transported to an extracellular milieu by C. albicans after menadione exposure. Through Liquid Chromatography coupled with Mass Spectrometry (LC-MS) analyses, we identified biomolecules transported by EVs and supernatant. The identified molecules are related to several biological processes, such as glycerophospholipid and sphingolipid pathways, which may act at different levels by tuning compound production in accordance with cell requirements that favor a myriad of adaptive responses. Taken together, our results provide new insights into the role of EVs in fungal biology and host–pathogen interactions. Candida spp. are the main etiologic agents of systemic fungal infections, with C. albicans being the most prevalent species + at m/z 610.5400. Most of the main fragments of compound (B) matched the fragmentation patterns found in the GNPS database  A and (B) (A) (B) B were id on GNPS  38]. Co. CoA–F  was induced, and the precursor ion [M + H]+ at m/z 480.3080 was annotated with high similarity to the spectral library (m/z 339.2910 is a major fragment that corresponds to dehydrated oleoyl (18:1) glycerol, which was predicted by the HMDB database and previously described + 302.3050. (D) shows the ions at m/z 254.2850, 95.0860, 81.0710, and 60.0450 as the main fragments [2NCHCH2OH]+ is known for compound (D) as the ion at m/z 60.0450 and the base peak according to the HMDB spectral and GNPS database (+ at m/z 318.3000 was annotated in the GNPS database (E), and the MS/MS experiment yielded major fragments at m/z 282.2780 and 60.0450, which corresponded to di-dehydration and to the fragment [H2NCHCH2OH]+, as expected for compound (E) and experimentally found in the HMDB database ) 1-oleoyl-glycero-3-phosphoethanolamine and 1-oleoyl-glycero-3-phosphocholine (Lyso-PC [18:1]) may also be related to glycerophospholipid pathways . These cpsulatum . In addida auris ,70. The da auris ,72. Furtda auris ,73,74. Ada auris . To ensuda auris ,75. The C. albicans EVs may be useful to generate more information about the mechanisms underlying EV communication that may favor the fungal infection process.The EVs can assist in environmental sensing, showing alterations in their sorting of molecules in accordance with cellular requirements , which w


Bacillus cereus is ubiquitous in the environment and a well-known causative agent of foodborne disease. Surprisingly, more and more emerging strains of atypical B. cereus have been identified and related to severe disease in humans and mammals such as chimpanzees, apes, and bovine. Recently, the atypical B. cereus isolates, which mainly derive from North America and Africa, have drawn great attention due to the potential risk of zoonosis. The cluster of B. cereus carries several anthrax-like virulent genes that are implicated in lethal disease. However, in non-mammals, the distribution of atypical B. cereus is still unknown. In this study, we conducted a retrospective screening of the 32 isolates of Bacillus spp. from diseased Chinese soft-shelled turtles from 2016 to 2020. To recognize the causative agent, we used various methods, such as sequencing analysis using PCR-amplification of the 16S rRNA gene, multiplex PCR for discriminating, and colony morphology by following previous studies. Furthermore, the digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated, respectively, below the 70 and 96% cutoff to define species boundaries. According to the summarized results, the pathogen is taxonomically classified as Bacillus tropicus str. JMT . Subsequently, analyses such as targeting the unique genes using PCR and visual observation of the bacteria under various staining techniques were implemented in our study. Our findings show that all  isolates in this retrospective screening share similar phenotypical properties and carry the protective antigen (PA), edema factor (EF), hyaluronic acid (HA), and exopolysaccharide (Bps) genes on their plasmids. In this study, the results indicate that the geographic distribution and host range of B. tropicus were previously underestimated. Tryonyx sinensis is well-adapted to aquatic environments and is favored for commercial production because of its high nutritional and medicinal values. In Taiwan, the main pond-breeding area of turtles is located in Kaohsiung and Pingtung counties. In Taiwan, due to the favorable climate, the period for Chinese soft-shell turtle egg-laying lasts about 9 months. The collection and exportation of eggs to China has produced the most important economic income of this industry. Generally, disease occurs when turtles are cultured at high density as a result of overstocked ponds or when turtles are exposed to poor water quality. In recent years, bacterial disease has increased the difficulty of aquaculture management and has led to limited yields, often resulting in significant economic loss [The Chinese soft-shell turtle, mic loss ,2,3. B. cereus infection were reported for the first time in the pond-cultured farms in Guangdong Province, China [B. cereus, B. thuringiensis also was isolated from diseased Chinese soft-shell turtles [B. cereus group has become a newly emerging bacterial pathogen of Chinese soft-shell turtle in Taiwan. Up to the present date, recurrent epizootic outbreaks have been reported in several pond-cultured farms in Pingtung and Kaohsiung Counties [In 2011, several cases of Chinese soft-shell turtle disease originating from e, China . Soon afe, China . In addi turtles . B. cereCounties . The emeBacillus cereus group, also known as B. cereus sensu lato, are Gram-positive, spore-forming, facultatively anaerobic, rod-shaped bacteria, consisting of at least eight closely related species that are widespread in natural environments [Bacillus cereus group were proposed, including Bacillus tropicus, Bacillus paranthracis, Bacillus pacificus, Bacillus albus, Bacillus mobilis, Bacillus luti, Bacillus proteolyticus, Bacillus nitratireducens, Bacillus paramycoides [Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides. The group has played a critical role in many aspects of human life, such as terrorism [Bacteria within the ronments . Moreovemycoides . The moserrorism , human herrorism , agriculerrorism , aquaculerrorism ,6. B. cereus is an opportunistic pathogen and a well-known causative agent of foodborne bacterial intoxicants that are known to cause an emetic or diarrheal type of food-associated disease, which is generally self-limiting. Most isolates appear to be harmless [harmless .B. cereus G9241 was initially isolated from a Louisiana welder in 1994, and subsequently identified as the causative agent of severe anthrax-like pneumonia [B. anthracis can induce the systemic effects of edema and severe bacteremia, with bacilli often noticed within blood vessels of the various vital organs [Therefore, it was a dramatic turn when neumonia . In prevl organs . The marl organs . HoweverB. cereus have been shown to be associated with the anthrax-like disease in humans and mammals such as chimpanzee, apes, and bovine [B. cereus isolates are capable of causing food-borne illnesses and can cause severe, even fatal, infections in non-immunocompromised humans and various animals [B. cereus G9241 was thought to differ from typical B. cereus isolates by the presence of unique plasmids. The strains harbor two large virulence plasmids, pBCXO1 and pBC210 (or pBCXO2). Since 2004, more and more newly emerging strains of atypical d bovine ,20,21,22 animals . As suchB. cereus G9241, shares a high similarity with plasmid pXO1 of B. anthracis and consists of the toxin genes pagA, lef, and cya for anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. Two components, anthrax toxin and an extracellular capsule, are required for full virulence. The plasmid called pBCXO1, which is the anthrax toxin source of hasACB operon located on pBCXO1, and the exopolysaccharide (Bps) component is encoded by a nine-gene operon, bpsX-H, located on plasmid pBC210. This suspected zoonotic disease has attracted significant attention due to the atypical B. cereus causing an anthrax-like disease in mammals. Additionally, it is worth mentioning that Bacillus tropicus isolated from animals and humans also induced anthrax-like disease in 1941 [For extracellular capsules, the hyaluronic acid (HA) component of the capsule is encoded by the  in 1941 .Bacillus could be our suspect, causing the disease and high mortality in the Chinese soft-shell turtle industry. In the study, we estimated the analytical methods of the previous studies [On the basis of its highly analogous traits, many members of the genus  studies  and prov studies  and digi studies . B. cereus, which has been implicated in lethal disease, is unknown. In this study, we showed the phenotypical and molecular analysis of Bacillus species JMT isolates from Chinese soft-shell turtle with anthrax-like disease. Furthermore, we proposed the taxonomic change for several atypical B. cereus isolates.However, as for non-mammals, the distribution of atypical Bacillus species JMT, from diseased Chinese soft-shell turtles. From 2016 to 2020, the epidemic led by Bacillus spp. JMT among turtles cultured in Kaohsiung (K) and Pingtung (PT), two geographic districts in southern Taiwan, caused severe mortality in up to 100% of infected animal. A total of 32 cases infected by Bacillus spp. JMT were provided by breeders via purposive sampling in the subadult/adult populations of pond-cultured Chinese soft-shell turtle from different ponds. The sources of the Bacillus spp. JMT isolates used in the study are listed in In this study, we collected bacterial isolates, temporarily designated as Isolates JMT107-17 to JMT109-32, were collected for histopathological analysis . Vital oB. thuringiensis ATCC 10792 and B. cereus ATCC 14579, purchased from Bioresource Collection and Research Center , were utilized as the negative positive controls, respectively, to compare with the collected wild strains.Bacteria were isolated from the liver and spleen of Chinese soft-shell turtles by aseptic operation and cultured on trypticase soy agar supplemented with 5% goat blood (BAP) at room temperature (28 °C). The gray–white colonies with β-hemolytic properties can be seen by the naked eye within 24 h. Furthermore, A 0.5 McFarland turbidity standard of the sporulated colony from all isolates was transferred to the microscope slides, and the slides were air-dried before staining. The specimens were stained (0.133% Coomassie Blue stain in 50% acetic acid ), rinsedThe spleen from the diseased Chinese soft-shell turtles was imprinted on a microscope slide and dried by air. The tissue stamp smear was completed using Liu’s Solutions including Liu’s A and B solutions . Liu’s staining was performed according to the manufacturer’s protocol. Then, the smears were washed with tap water and air-dried prior to microscopic examination. The cells from a single colony of each bacterial isolates were scattered into 100 μL of sterilized saline solution and 5 μL of the mixture was pipetted on to a microscope slide and subsequently covered with a coverslip. One drop of India ink  was applied on the edge of the cover slide. The presence of encapsulated cells on the wet mount slide was examined microscopically at 100× oil-immersion magnification. lef3/lef4, JMlef436f/JMlef436r, JMbpsB208f/JMbpsB208r, JMpagA318f/JMpagA318r, JMhasA700f/JMhasA700r, JMcya882f/JMcya882r; genomic DNA was used for the rest of the primer sets  according to the manufacturer’s instructions. Finally, purified DNA was used as a template for subsequent PCR reaction and stored at −20 °C until use. Plasmid DNAwaas used for these primer sets, mer sets .Bacillus spp. [groEL gene were used to amplify 400 bp of expectant product from Bacillus group species. The rest of the sets targeting the gryB yielded 253 bp amplicon, 299 bp amplicon, 475 bp amplicon, and 604 bp amplicon for B. anthracis, B. thuringiensis, B. cereus, and B. mycoides, respectively. Additionally, we further employed a transcriptional regulator (XRE) as the new biomarker of B. thuringiensis [16S rRNA and five sets of primers were used to differentiate between specific species among lus spp. . PCR assngiensis .hasA and bpsB sequences, respectively (GenBank accession number AAEK01000036 and AAEK01000004); whereas the primers for the PA, EF, and LF were designed from the pagA, cya, and lef sequences, respectively (GenBank accession number CP009592). To confirm the results, another published primer set was undertaken to detect the lef gene [In this study, we designed several new primer sets for detecting unique virulence genes. Specific oligonucleotide primers were designed for hyaluronan synthase and exopolysaccharide biosynthesis protein based on the lef gene . The oli2), and 1 U of Top DNA polymerase. The reaction was added to a total volume of 20 µL with nuclease-free water. The reaction parameters were an initial denaturation at 94 °C for 5 min, 35 cycles of amplification with denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, an extension at 72 °C for 60 s, and final extension at 72 °C for 7 min. The PCR conditions could be slightly varied to adapt to each primer set. For each reaction, 50 ng of DNA template and 10 pmole of each primer was added to AccuPower PCR Premix , consisting of 250 µM of each of the nucleotides dATP, dCTP, dGTP, and dTTP, PCR buffer . Finally, the PCR products were analyzed by electrophoresis in 2% agarose gels stained with DNA-View  and immediately obrserved with UVP GelSolo  for analysis.Escherichia coli DH—5α . The recombinant plasmid DNA from the different clones was extracted separately and sequenced using an ABI3730 automatic sequencer . Both forward and reverse strands were sequenced for each individual. For the purpose of ensuring sequence quality, both strands were sequenced as a crosscheck. Determined sequences were compared with the reference strains in GenBank.PCR products amplified by each primer set were cloned into the yT&A vector  according to the manufacturer’s instructions and transformed into hasA fragment (GenBank accession number OP588641), partial bpsB fragment (GenBank accession number OP588640), partial pagA fragment (GenBank accession number OP583942), and partial cya fragment (GenBank accession number OP584757) served as the positive control. In addition, the lef positive control was kindly provided by the Institute of Medical Science, Tzu-Chi University, Taiwan [The partial , Taiwan .Genomic DNA of the representative isolate (JM105-2) was extracted using Pure extraction QIAGEN Genomic-tip 20/G Kit . The gDNA extractions were evaluated for quantity and quality using Qubit , and Q6000B UV-Vis spectrophotometer , following the manufacturer’s instructions. Nanopore DNA Libraries were prepared using 2000 ng of gDNA per sample and preprocessed by Short Read Eliminator Kit  to enhance long-read sequencing read lengths by removing short DNA.Subsequently, libraries construction followed the native barcoding genomic DNA protocol (EXP-NBD114 and SQK-LSK109)  for combined FFPE repair and end-prep, ligation of barcodes, and the ligation of sequencing adapters. Sequencing was performed with MinKNOW software (version 21.05.8) on the GridION Mk1 using Nanopore sequencing platform by Oxford Nanopore Technologies (ONT) . SubsequFinally, genome maps of the chromosome and plasmid of JMT105-2 isolate were drawn by Circos software  and CLCbOrthologous average nucleotide identity (OrthoANI) between a pair of genomes was calculated by using CJ Bioscience’s Orthologous Average Nucleotide Identity Tool (OAT) . http://ggdc.dsmz.de, accessed on 1 December 2022) [4d [Genome-to-genome distance (GGD) analysis was performed using GGD calculator (GGDC) with the GBDP3.0_BLASTPLUS program provided by German Collection of Microorganisms and Cell Cultures  (er 2022) . Distanc022) [d4 . These d022) [d4 ,26. If thttps://www.ncbi.nlm.nih.gov/genome, accessed on 3 December 2022). These accession number included Bacillus tropicus JMT105-2 (GenBank accession number CP115303), Bacillus cereus G9241 (GenBank accession number NZ_CP009590), Bacillus cereus strain 03BB87 (GenBank accession number NZ_CP009941), Bacillus tropicus strain EMB20(GenBank accession number NZ_CP078081), Bacillus cereus ATCC 14579(GenBank accession number NC_004722), Bacillus thuringiensis serovar berliner ATCC 10792 (GenBank accession number NZ_CM000753), Bacillus anthracis str. Ames (GenBank accession number NC_003997), Bacillus mycoides DSM 2048 (GenBank accession number NZ_CM000742).Whole genome sequences of the strains for OrthoANI and dDDH analyses were obtained from the GenBank database  of the JMT105-2 isolate was deposited in GenBank (GenBank accession numbers OP592263) and BLAST was conducted on NCBI database. The result we received shared the highest nucleotide sequence identity of 99~100% within three Bacillus species: B. anthracis, B. cereus, and B. thuringiensis. Therefore, the published multiplex PCR assay was undertaken to discriminate between the Bacillus relatives in the present study by recognizing specific DNA segments. We processed all bacterial isolates in this study, including B. cereus ATCC 14579, B. thuringiensis ATCC 10792 and all Bacillus spp. JMT isolates. One universal and one specific amplicon of each sample in electrophoresis were visible in the PCR reaction of the former two isolates. The rest of the Bacillus spp. JMT isolates were unanimous in presenting amplicons and agreed with B. thuringiensis ATCC 10792 pattern, which amplified the partial groEL fragment (400 bp) and the partial gyrB fragment (299 bp) (The DNA of (299 bp) .B. thuringiensis in a previous study [B. cereus ATCC 14579, B. thuringiensis ATCC 10792, showed the expected results. Thus, all Bacillus spp. JMT isolates were negative and identical in the use of PCR targeting XRE  was described as a reliable and capable tool for identifying us study . The novting XRE . Bacillus spp. JMT isolates from clinical sources were notably identical  and well-demarcated around the bacilli (Bacillus spp. JMT), as shown in Bacillus spp. JMT, only bacilli were observed in B. cereus ATCC 14579 strains. The capsule-producing abilities of 32 dentical . We perfbpsB and hasA were found in all the Bacillus spp. JMT . A total of five primer sets were used for screening in this study, specifically targeting each virulence gene of interest. In the capsule-associated genes, pagA and cya were present  in all Bacillus spp. JMT isolates. Unexpectedly, the results of amplifying the partial lef gene were negative in all the Bacillus spp. JMT isolates using both newly designed and published primer sets. Whether capable of producing the unique capsule or an anthrax-like toxin, all Bacillus spp. JMT isolates retained identical properties. All results of the PCR assay are shown in hasA fragment (GenBank accession number OP588641), partial bpsB fragment (GenBank accession number OP588640), partial pagA fragment (GenBank accession number OP583942), and partial cya fragment (GenBank accession number OP584757).As for anthrax-like toxin-associated genes, 50N value was 5,257,674 bp. The high-quality short-read and long-read sequences were De novo assembled into a complete chromosome and a plasmid using SPAdes Genome Assembler (v. 3.14.1) with default settings. The genome size of JMT105-2 is 5,487,300 bp, containing a chromosome  and a plasmid   were obtained with an average coverage of 236.8. The average read length was 1692.5 bp and the ,626 bp) . No evidBacillus spp. JMT-induced bacteremia among 16 clinical cases .By necropsy examination, the diseased Chinese soft-shell turtle exhibited some morphological changes, such as a generalized edema and darkened and enlarged spleen . FurtherBacillus spp. strains, the value of seven paired-genome combinations was higher than both thresholds in terms of orthologous average nucleotide identity and digital DNA–DNA hybridization similarities. The calculation recognized as the same species consisted of the following combinations: 1, Bacillus tropicus JMT105-2(CP115303) and Bacillus cereus G9241(NZ_CP009590.1); 2, Bacillus tropicus JMT105-2(CP115303) and Bacillus cereus strain 03BB87(NZ_CP009941); 3, Bacillus cereus G9241(NZ_CP009590.1) and Bacillus cereus strain 03BB87(NZ_CP009941); 4, Bacillus tropicus JMT105-2(CP115303) and Bacillus tropicus strain EMB20(NZ_CP078081.1); 5, Bacillus cereus G9241(NZ_CP009590.1) and Bacillus tropicus strain EMB20(NZ_CP078081.1); 6, Bacillus cereus strain 03BB87(NZ_CP009941) and Bacillus tropicus JMT105-2(CP115303); 7, Bacillus cereus ATCC 14579(NC_004722) and Bacillus thuringiensis serovar berliner ATCC 10792(NZ_CM000753)  and the multiplex PCR assay results did not match, either. Following molecular analysis, the 16S rRNA fragment was indistinct and not beneficial in distinguishing between species due to its highly conservative DNA sequence. Furthermore, we used a similar strategy as the previous study [B. cereus and B. thuringiensis is that the latter possesses a unique ability to synthesize parasporal crystalline inclusions. Surprisingly, the multiplex PCR primer sets for discriminating in this study apparently failed to identify Bacillus species and cannot address Bacillus spp. JMT isolates.The major difference between B. thuringiensis was the pathogen and highly pathogenic to juvenile Chinese soft-shell turtles. Nevertheless, Bacillus spp. JMT did not possess the essential ability to produce B. thuringiensis crystals in our study.Interestingly, the multiplex PCR results of the study by Chen et al.  were theBacillus species. The digital DNA–DNA hybridization and orthologous average nucleotide identity consequently became a standard technique for the circumscription of bacterial species; we can obtain relatively reliable information to determine genomic similarities. An advantage of these genome-based methods is that they often form sharp, clear clusters of strains compared to those only circumscribed by phenotypic traits [B. cereus (strain G9241 and 03BB87) and Bacillus spp. JMT105-2 isolate were suggested to be the same species as B. tropicus strain EMB20, not B. cereus ATCC 14579 or Bacillus thuringiensis serovar berliner ATCC 10792. It is worth mentioning that although B. cereus and B. thuringiensis share genomic similarities, they were still considered to be different species [Bacillus spp. JMT and B. tropicus strain EMB20, these isolates from diseased Chinese shell-turtle were assigned as Bacillus tropicus JMT. The conflict between the multiplex PCR results and crystal production ability of the assay requires further high-resolution analysis. Genome-sequence-based analyses provided new insight into the similarity between c traits . With in species . On the Bacillus are too close to be distinguished. In previous studies [B. cereus and B. thuringiensis were considered the etiological agents of Chinese soft-shell turtles. Due to the lack of genome sequence-based analyses, it was difficult to address the taxonomic recognition of these close species-level lineages.As previously mentioned, we solved a confusing problem based on the bacterial isolates collected in the study. The members of the genus  studies ,7,38, B.B. cereus-associated researchers unofficially posit the following theory: the origin of the etiological agent is a biological agricultural pesticide. The typical pond-cultured farms are usually located by country roads and surrounded by agricultural farms in the vicinity. It seems rational to assume that B. thuringiensis might be an agent; therefore, it became the most likely suspect. B. thuringiensis is deadly to certain herbivorous insects through the production of crystal toxins and is the most frequently used biological pesticide globally.In Taiwan, many Chinese soft-shell turtle Bacillus spp. JMT isolates provided identical results in several tests. Regardless, the etiological agent that triggered the mass death during outbreaks is more likely to be B. tropicus than either B. cereus or B. thuringiensis in Taiwan. According to the summarized results, Bacillus spp. JMT is referred to as Bacillus tropicus JMT in the following section of the study.In this study, all B. tropicus was previously recognized as atypical B. cereus. In this study, we reclarified the taxonomic status of B. tropicus JMT, as well as these atypical B. cereus strains.B. tropicus JMT retains several anthrax-like virulence genes and displays different characteristics, such as B. cereus G9241 and B. cereus 03BB87, not the non-pathogenic B. tropicus strain [B. cereus strains have been widely accepted to overcome host immunity and cause severe symptoms, even death. Since B. cereus G9241 has been identified and characterized, the stereotypes of pathogenic B. cereus isolates not only originate from food-borne incidents but are also related to severe pulmonary disorders. In addition to being an opportunistic pathogen, it is an etiologic agent of emerging disease, requiring vigilance regarding public health issues. These atypical B. cereus strains carry unique virulence toxins encoded on the megaplasmids [B. cereus plasmids have an analogous function to the pXO1 and pXO2 plasmids of B. anthracis and have proven to be sources of pathogenicity [B. cereus-associated strains which induce anthrax-like disease.s strain ,40,41. Iplasmids ,20,42,43genicity . PreviouB. tropicus JMT isolates could be similar to its relative, B. anthracis, which causes a famous zoonotic disease. Anthrax is an acute bacterial disease mainly occurring in wild and domestic herbivores and it occasionally causes acute infection in humans. Like anthrax, atypical B. cereus strains were reported to mainly be isolated from mammals. In the present study, we conducted a retrospective screening of the isolates from 2016 to 2020. Our findings provide solid evidence of the presence of B. tropicus JMT and its critical pathogenic ability against a reptile: Chinese soft-shell turtle. The capsule could escape from phagocytosis via the host immune system, so this has been considered a virulence factor. For instance, the commonly used laboratory strain B. anthracis Sterne harbors plasmid pXO1 only; consequently, its pathogenicity is reduced. Nevertheless, the encapsulated wild B. anthracis strain can inhibit both complement-dependent and complement-independent opsonic phagocytosis. [B. tropicus JMT isolates. The thick and unstainable coat around the bacilli was visualized by microscopy, using India ink staining in all isolates. The capsule was also visible on the smear with Liu’s staining, which has been used for clinical aquatic animal diagnosis in Taiwan. Its application as a capsule stain with excellent discrimination is similar to the standard staining method used for B. anthracis [The infectious mechanism of cytosis. . Our finnthracis . This chB. cereus were usually severe to lethal in mammals in previous studies. We also observed a similar situation in the reptile, Chinese soft-shell turtle. Since 2016, the emerging pathogen has led to a great epidemic of diseases. Bacteremia was recognizable in all vital organs sampled for pathological diagnosis in this study. Previous studies have suggested that the disease may be related to emetic toxin enterotoxins secreted by B. cereus [The anthrax-like diseases induced by atypical . cereus . NeverthB. tropicus JMT, induced similar severe systemic pathological changes in all sampled specimens in this study. The data showed that the diseased animals in this study would suffer from a severe systemic reaction and the circulatory system would eventually be invaded by B. tropicus JMT in the terminal stage. In the aquaculture field, the individuals engage the pathogen, inducing a severe serial immune response in the diseased animals. The diseased individuals usually showed lethargic swimming behavior on water surfaces with twisted necks and rigid legs. Further, they exhibited reduced alertness to the surrounding environment. With the course of disease development, abnormal behaviors were observed, accompanied by prominent reductions in feed intake reduction and general edema of the tissues  are encoded on the same plasmid in atypical B. cereus and B. anthracis. Sequence mutations or incomplete plasmids in B. tropicus JMT probably result in amplification failure. The phenomenon of carrying a partial plasmid occurred in B. cereus FL2013 [The s FL2013 .lef gene. The preliminary result in this study reveals the absence of the lef gene.Furthermore, whole-genome sequencing is a powerful tool to provide a high-resolution view of the entire genome. The data of the de novo assembled genome showed no evidence of the segment of B. tropicus JMT can synthesize astonishingly thick capsules. Both staining visualization methods demonstrated that B. tropicus JMT isolates possess an inherent ability to produce capsules.B. tropicus JMT. The hasACB genes are encoded on the plasmid of B. anthrax and atypical B. cereus. In contrast to B. anthracis, atypical B. cereus contains non-mutated hasACB genes and is functional for capsule production [B. anthracis is considered to help avoid opsonization and phagocytosis of the host immune system [B. cereus possess this ability due to their two unique natural gifts. Both the virulence plasmids make the required contributions for synthesizing a unique capsule structured with hyaluronic acid (hasACB) and polysaccharide (bpsX-H). Thus, they differ from the poly-γ-d-glutamic acid (PGA) capsule produced by B. anthracis.As for the capsule production, the PCR results correspond to the above-mentioned phenotypic characteristics of oduction . The cape system ,51. SimiB. cereus G9241 [B. tropicus JMT isolates carry both the hasA and bpsB genes related to the formation of capsules. According to the PCR results, capsule-associated genes were present in all B. tropicus JMT isolates, implying that they could possibly provide ancillary pathogenic factors. However, whether they have an analogous effect on capsule production compared to previous atypical B. cereus isolates remains unknown.In terms of pathogenicity and the thickness of the produced capsule, hyaluronic acid is thought to be a more influential capsule componant than exopolysaccharide in us G9241 . B. tropB. tropicus JMT isolates. B. cereus group members are too similar to be recognized as B. tropicus JMT in phenotypic analysis. Despite its high pathogenicity and infectivity against Chinese soft-shell turtle, there is no evidence that any other animals or humans could naturally acquire the disease and no medical cases have been documented to date. The living organisms have varying innate susceptibility to the pathogen. Generally, individual species and genetic background are critical determinants of pathogenicity [B. tropicus JMT carries anthrax-like virulence genes, which inevitably give rise to concerns about zoonotic infectious diseases. A thorough understanding of the host range and geographic distribution of B. tropicus JMT is essential for the control and prevention of disease outbreaks. Based on previous studies, the distribution of atypical B. cereus, which usually infects mammals, has been restricted to North America and Africa. This study is the first retrospective screening report of anthrax-like virulent genes in B. tropicus  isolated from a reptile and reveals that the atypical strains are pathogens of not only mammals but also reptiles. There is an imperceptible threat that is expanding to affect human and animal health. Additionally, the geographic distribution and host range of B. tropicus were underestimated. Extensive surveillance will allow us to better understand and evaluate the epidemic cross-species risk. Furthermore, it will help raise alertness and develop strategies to prevent potential threats. Likewise, for the uphill challenges faced by the aquaculture industry, precise and rapid identification of the etiologic agent is also increasingly important to recover economic losses. Consequently, a reliable tool is needed to assess potentially contagious hazards.The powerful primer sets used in the present study are vital for detecting pathogenic genicity . Using tB. tropicus JMT. The powerful PCR assay based on the anthrax-like gene primer sets JMbpsB208f/JMbpsB208r, JMpagA318f/JMpagA318r, JMhasA700f/JMhasA700r, and JMcya882f/JMcya882r displayed a much stronger ability to identify B. tropicus JMT isolates, with the highest detection rate (32/32 = 100%). These results help to confirm that this method is likely to be a viable candidate for screening the causative agents.The amplification of each specific virulence gene by PCR can be a valuable tool to distinguish and identify pathogenic B. tropicus str. JMT in terms of taxonomic classification. They harbor anthrax-like virulence genes, as shown using a PCR assay (32/32 = 100%). Furthermore, genome sequence-based analyses with high resolution can successfully unveil the lurking emerging pathogen in turtle aquaculture.In this study, the bacterial isolates collected from Chinese soft-shell turtles cultured in southern Taiwan during the epizootics of 2016–2020 were B. cereus is not only in North America and Africa, but also in Asia. Consequently, the clinical observations imply that B. tropicus JMT is a frank pathogen against its host, Chinese soft-shell turtle. Lastly, further studies are needed to elucidate B. tropicus JMT’s pathogenic ability against other animals.Our findings demonstrate that the geographic distribution of atypical