A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willibrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. Here we developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167±257 mU/ml) but the VWF was completely incapable of binding FVIII, resulting in 53±23 mU/ml of plasma FVIII levels that were similar to those in VWF deficient (VWF-/-) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4-6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable, but short-lived unprotected in plasma due to dysfunctional 2N-VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF-/- mice. The bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that there are other alternative pathway(s) that can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.

Oncholaimus tripapillatus sp. nov., a New Free-Living Marine Nematode of the Genus Oncholaimus Dujardin, 1845 (Nematoda: Enoplida: Oncholaimidae) from the Subtidal Sediment of Dokdo Island, East Sea, Korea, with a New Record of O. qingdaoensis Zhang & Platt, 1983

During a survey of the free-living marine nematodes of Korea, two species belonging to the genus Oncholaimus Dujardin, 1845 were discovered. A new species, Oncholaimus tripapillatus sp. nov. and a newly recorded species, Oncholaimus qingdaoensis Zhang & Platt, 1983, are reported. Oncholaimus tripapillatus sp. nov. was collected from the washing of subtidal coarse sediments around Dokdo Island of the East Sea, Korea. Oncholaimus tripapillatus sp. nov. is characterized by a relatively long (4071–4435 µm in males and 4514–4661 µm in females) and slender body, a slightly constricted head region, relatively long cephalic setae (10–12 µm), males having a precloacal sexual protuberance bearing two small cone-shaped supplementary spines, five pairs of long cloacal setae (three pairs of precloacal and two pairs of postcloacal setae in subventral position), and three remarkable papillae near the end of the tail, with two pairs of subventral setae. The Korean specimens of Oncholaimus qingdaoensis Zhang & Platt, 1983 are almost identical to the Chinese specimens of the original description from the intertidal sand of Qingdao, China. However, the Korean specimens differ from the Chinese specimens in the longer body length in males (3379–3715 µm vs. 2380–2640 µm), the larger spicule length (47–52 µm vs. 34–36 µm), and the presence of ventral tail papillae situated around 14–16 µm from the tail tip. Detailed morphological features and illustrations of two Oncholaimus species from Korea were obtained by differential interference contrast microscopy and scanning electron microscopy. A pictorial key to the species group with distinct tail papillae among the genus Oncholaimus is also provided.

Two new insect-associated tylenchids, Sychnotylenchus hibisci n. sp. and Neoditylenchus xiphydriae n. sp. (Tylenchomorpha: Anguinidae), from Japan

Summary We isolated two tylenchid species belonging to the Sychnotylenchinae, Sychnotylenchus hibisci n. sp. and Neoditylenchus xiphydriae n. sp., from the bark beetle Ernoporus corpulentus and wood wasp Xiphydria ogasawarai, respectively. Sychnotylenchus hibisci n. sp. is characterised by its moderately stout body in both male and female, gubernaculum to spicule length ratio of one-third or less, crustaformeria structure composed of four columns with six large, rounded cells, and a broad female tail with a rounded terminus. The new species is closely related to S. ulmi, with which it shares body shape and gubernaculum to spicule length ratio, but is distinguished by the female tail shape and several morphometric values. Neoditylenchus xiphydriae n. sp. is characterised by its oval to leaf-shaped male bursa, male tail tip reaching the distal end of, and protruding beyond the bursa, a long post-vulval uterine sac (PUS), and a conical female tail with a narrowly rounded terminus. The species is typologically similar to N. pinophilus but is distinguished by its male spicule shape, gubernaculum to spicule length ratio, and longer PUS. Phylogenetically, the Sychnotylenchinae belongs under the Anguinidae (superfamily Sphaerularioidea) and is closely related to several Ditylenchus, Neotylenchus, Neomisticius, and Ficotylus species.

Reformulation of PULSAR for analysis of pulsatile LH secretion and a revised model of estrogen negative feedback in mice

The recent use of the tail-tip bleeding approach in mice has enabled researchers to generate detailed pulse and surge profiles of luteinizing hormone (LH) secretion in mice. However, the analysis of pulsatile LH secretion is piecemeal across the field with each laboratory using their own methodology. We have reformulated the once popular PULSAR algorithm of Merriam and Wachter to operate on contemporary computer systems and provide down-loadable and on-line pulse analysis platforms. As it is now possible to record the activity of the GnRH pulse generator in freely-behaving mice, we have been able to unambiguously define LH pulses in intact and gonadectomized male and female mice. These data sets were used to determine the appropriate PULSAR parameter sets for analysing pulsatile LH secretion in the mouse. This was then used to establish an accurate model of estrogen negative feedback in the mouse. Intact and ovariectomized mice given Silastic capsules containing 1, 2 and 4 µg 17-β-estradiol/20 gm body weight were tail-tip bled at 6-min intervals and the resultant LH profiles analysed with PULSAR. Only the 4 µg 17-β-estradiol capsule treatment was found to return LH pulse amplitude and frequency to that of intact diestrous mice. Ultra-sensitive mass spec analysis showed that the 4 µg 17-µ-estradiol capsule generated circulating estradiol levels equivalent to that of diestrous mice. It is hoped that the reformulation of PULSAR and generation of a realistic model of estrogen negative feedback will provide a platform for the more uniform assessment of pulsatile hormone secretion in mice.

Pemphigus foliaceus in Arabian oryx (Oryx leucoryx)

Pemphigus foliaceus (PF) is an autoimmune acantholytic skin disease described in humans, dogs, cats, horses, goats, and sheep. From 2003 to 2016, six Arabian oryx ( Oryx leucoryx) at the National Zoological Garden in Pretoria, South Africa, developed progressive, bilaterally symmetrical, hyperkeratotic skin lesions and pustules consistent with PF. Lesions were similar to those observed in domestic animals and primarily affected the pinnae, face and nasal planum, distal legs, and tail tip. Histological evaluation of suspect PF skin lesions in affected animals, evaluation of medical records for treatments received, causative agents in the diet and environment, and special stains for infectious organisms yielded no consistent inciting cause. The Arabian oryx is a species highly adapted to arid environments of the desert and has recently survived from a severe genetic bottleneck; both of these factors may have contributed to the development of PF in these animals.

Description of Ditylenchus acantholimonis n. sp. (Rhabditida: Anguinidae) from Iran, a morphological and molecular phylogenetic study

Summary Ditylenchus acantholimonis n. sp. is described based on morphological, morphometric and molecular characters. It was isolated from the rhizosphere soil of Acantholimon sp. in Golestan province, Iran, and is mainly characterised by having four lines in the lateral field, a pyriform to bottle-shaped offset pharyngeal bulb, post-vulval uterine sac 36.6-56.1% of the vulva to anus distance long, and a subcylindrical to conical tail with widely rounded tip. It is further characterised by short to medium-sized females, 480-617 μm long, with a fine stylet having small rounded knobs, V = 80.8-83.6, c = 11.0-13.8, c′ = 3.3-4.6, and males with 16.0-17.0 μm long spicules. The new species was morphologically compared with six species having four lines in their lateral field, rounded tail tip and comparable morphometric data namely: D. dipsacoideus, D. emus, D. exilis, D. paraparvus, D. sturhani, and D. solani. It was also compared with two species, D. ferepolitor and D. angustus, forming a maximally supported clade in the 18S tree. The phylogenetic analyses using the maximal number of Anguinidae and several Sphaerularioidea genera based upon partial 18S and 28S rDNA D2-D3 sequences revealed that Ditylenchus is polyphyletic. In the 18S tree, the new species formed a clade with D. ferepolitor (KJ636374) and D. angustus (AJ966483); in the 28S tree it formed a poorly supported clade with D. phyllobios (KT192618) and Ditylenchus sp. (MG865719).

Gynogenetic diploids, tetraploids, or octoploids, and a path to polyploidy in Anuran Amphibians

Unreduced gametes have been implicated in the evolution of polyploid species of plants and animals and are normally produced by female anuran amphibians. Such eggs may initiate the evolution of polyploid species that have independently arisen in several anuran families. Polyploid females could also produce unreduced eggs that might lead to species with higher ploidy levels or their eggs may develop gynogenetically to reduce the ploidy level. Diploid Hyla chrysoscelis (2n=24) and tetraploid H. versicolor (4n=48) are sibling cryptic species of North American Grey Treefrogs. Artificial crosses using H. versicolor females and genetically distant diploid males were performed to produce haploid H. versicolor and to assess the production of unreduced eggs in this tetraploid species. Gynogenetic diploid (haploid H. versicolor), allotriploid, gynogenetic tetraploid, allopentaploid, autohexaploid, and gynogenetic octoploid tadpoles were confirmed using chromosome counts from tadpole tail tip squashes. Transformation and survival of the different ploidies varied. Gynogenetic diploids transformed but expressed aspects of the haploid syndrome and died before or shortly after transformation.

Cryptaphelenchus abietis n. sp. (Tylenchomorpha: Aphelenchoididae) isolated from Cryphalus piceae (Ratzeburg) (Coleoptera: Scolytinae) emerged from Abies veitchii Lindl. (Pinaceae) from Nagano, Japan

Summary An undescribed Cryptaphelenchus species was isolated from a bark beetle, Cryphalus piceae that had emerged from a dead log of the Veitch’s fir, Abies veitchii, collected from Sugadaira Montane Research Center, University of Tsukuba, Nagano, Japan. The new species is characterised by its female post-vulval uterine sac (PUS) of less than one vulval body diam. in length and female posterior body end (tail) conical with elongate posterior part and variably shaped tip, seven male genital papillae, which are composed/arranged as a precloacal P1, P2 pair slightly anterior to cloacal opening, and two small pairs of glandpapillae near tail tip and very narrow bursal flap-like cuticular extension surrounding the male tail. In addition to these characters, the male apophysis appears wing-like in ventral view. In possessing an elongate posterior part, Cryptaphelenchus abietis n. sp. shares the female tail shape with C. borlossi, C. leptocaudus and C. sutoricus, and shares a short PUS with C. iranicus, C. varicaudatus, C. baujardi and C. paravaricaudatus. However, the new species can be distinguished from these species by other typological and morphometric characters and its molecular phylogenetic status. Phylogenetically, the new species is closest to Bursaphelenchus minutus, but can be clearly distinguished by morphological and molecular sequence characters.

Dnmt2-null sperm block maternal transmission of a paramutant phenotype

Previous studies have shown that Dnmt2-null sperm block the paternal transmission (through sperm) of certain acquired traits, e.g., high fat diet-induced metabolic disorders or white tails due to a Kit paramutation. Here, we report that DNMT2 is also required for the transmission of a Kit paramutant phenotype (white tail tip) through the female germline (i.e., oocytes). Specifically, ablation of Dnmt2 led to aberrant profiles of tRNA-derived small RNAs (tsRNAs) and other small noncoding RNAs (sncRNAs) in sperm, which correlate with altered mRNA transcriptomes in pronuclear zygotes derived from wild type oocytes carrying the Kit paramutation and a complete blockage of transmission of the paramutant phenotype through oocytes. Together, the present study suggests that both paternal and maternal transmission of epigenetic phenotypes requires intact DNMT2 functions in the male germline.