Machine Learning Methods and Visual Observations to Categorize Behavior of Grazing Cattle Using Accelerometer Signals.

Accelerometers worn by animals produce distinct behavioral signatures, which can be classified accurately using machine learning methods such as random forest decision trees. The objective of this study was to identify accelerometer signal separation among parsimonious behaviors. We achieved this objective by (1) describing functional differences in accelerometer signals among discrete behaviors, (2) identifying the optimal window size for signal pre-processing, and (3) demonstrating the number of observations required to achieve the desired level of model accuracy,. Crossbred steers (Bos taurus indicus; n = 10) were fitted with GPS collars containing a video camera and tri-axial accelerometers (read-rate = 40 Hz). Distinct behaviors from accelerometer signals, particularly for grazing, were apparent because of the head-down posture. Increasing the smoothing window size to 10 s improved classification accuracy (p < 0.05), but reducing the number of observations below 50% resulted in a decrease in accuracy for all behaviors (p < 0.05). In-pasture observation increased accuracy and precision (0.05 and 0.08 percent, respectively) compared with animal-borne collar video observations.

British societies guideline on the management of emergencies in implantable left ventricular assist device recipients in transplant centres.

An implantable left ventricular assist device (LVAD) is indicated as a bridge to transplantation or recovery in the United Kingdom (UK). The mechanism of action of the LVAD results in a unique state of haemodynamic stability with diminished arterial pulsatility. The clinical assessment of an LVAD recipient can be challenging because non-invasive blood pressure, pulse and oxygen saturation measurements may be hard to obtain. As a result of this unusual situation and complex interplay between the device and the native circulation, resuscitation of LVAD recipients requires bespoke guidelines. Through collaboration with key UK stakeholders, we assessed the current evidence base and developed guidelines for the recognition of clinical deterioration, inadequate circulation and time-critical interventions. Such guidelines, intended for use in transplant centres, are designed to be deployed by those providing immediate care of LVAD patients under conditions of precipitous clinical deterioration. In summary, the Joint British Societies and Transplant Centres LVAD Working Group present the UK guideline on management of emergencies in implantable LVAD recipients for use in advanced heart failure centres. These recommendations have been made with a UK resuscitation focus but are widely applicable to professionals regularly managing patients with implantable LVADs.

Histopathological investigation of four populations of abalone (Haliotis iris) exhibiting divergent growth performance.

The black-foot abalone (paua), Haliotis iris, is a unique and valuable species to New Zealand with cultural importance for Maori. Abalone are marine gastropods that can display a high level of phenotypic variation, including slow-growing or 'stunted' variants. This investigation focused on identifying factors that are associated with growth performance, with particular interest in the slow-growing variants. Tissue alterations in H. iris were examined using histopathological techniques, in relation to growth performance, contrasting populations classified by commercial harvesters as 'stunted' (i.e., slow-growing) and 'non-stunted' (i.e., fast-growing) from four sites around the Chatham Islands (New Zealand). Ten adults and 10 sub-adults were collected from each of the four sites and prepared for histological assessment of condition, tissue alterations, presence of food and presence of parasites. The gut epithelium connective tissue, digestive gland, gill lamellae and right kidney tissues all displayed signs of structural differences between the slow-growing and fast-growing populations. Overall, several factors appear to be correlated to growth performance. The individuals from slow-growing populations were observed to have more degraded macroalgal fragments in the midgut, increased numbers of ceroid granules in multiple tissues, as well as increased prevalence of birefringent mineral crystals and haplosporidian-like parasites in the right kidney. The histopathological approaches presented here complement anecdotal field observations of reduced seaweed availability and increased sand incursion at slow-growing sites, while providing an insight into the health of individual abalone and sub-populations. The approaches described here will ultimately help elucidate the drivers behind variable growth performance which, in turn, supports fisheries management decisions and future surveillance programs.

Histopathological changes in the greenshell mussel, Perna canaliculus, in response to chronic thermal stress.

Climate change associated temperature challenges pose a serious threat to the marine environment. Elevations in average sea surface temperatures are occurring and increasing frequency of marine heatwaves resulting in mortalities of organisms are being reported. In recent years, marine farmers have reported summer mass mortality events of the New Zealand Greenshell mussel, Perna canaliculus, during the summer months; however, the etiological agents have yet to be determined. To elucidate the role of thermal stress, adult P. canaliculus were exposed to three chronic temperature treatments: a benign control of 17 °C and stressful elevations of 21 °C and 24 °C. Eight mussels per treatment were collected each month throughout a 14-month challenge period to identify and investigate histopathological differences among P. canaliculus populations exposed to the three temperatures. Histopathology revealed several significant deleterious alterations to tissues associated with temperature and exposure time. Increasing temperature and progression of time resulted in 1) an increase in the number of focal lipofuscin-ceroid aggregations, 2) an increase in focal hemocytosis, 3) an increase in the thickness of the sub-epithelial layer of the intestinal tract and 4) a decreased energy reserve cell (glycogen) coverage in the mantle. Prolonged exposure, irrespective of temperature, impacted gametogenesis, which was effectively arrested. Furthermore, increased levels of the heat shock protein 70 kDa (HSP 70) were seen in gill and gonad from thermally challenged mussels. The occurrence of the parasite Perkinsus olseni at month 5 in the 24 °C treatment, and month 7 at 21 °C was unexpected and may have exacerbated the fore-mentioned tissue conditions. Prolonged exposure to stable thermal conditions therefore appears to impact P. canaliculus, tissues with implications for broodstock captivity. Mussels experiencing elevated, temperatures of 21 and 24 °C demonstrated more rapid pathological signs. This research provides further insight into the complex host-pathogen-environment interactions for P. canaliculus in response to prolonged elevated temperature.

Bivalve digestive epithelial virosis (DEV): A cause of disease or a natural process?

Epithelial hyperplasia and sloughing of the digestive gland in bivalve mollusks are a global phenomenon and occur in species of commercial interest and cultural significance to indigenous peoples. Where hemocytosis, hyperplasia, and necrosis of digestive tubule cells have been observed associated with electron-dense uncoated virus-like particles (VLPs) 25-45 nm in diameter, the condition has been named digestive epithelial virosis (DEV). This condition has been associated with mortalities of some bivalve species in New Zealand. Similar digestive gland alterations, but without detection of associated VLPs, have been reported in other bivalve species worldwide and are termed "DEV-like" since no virus link has been demonstrated. It remains unclear if DEV is an infectious condition and whether associated VLPs are the cause, a contributor, or simply associated with the observed condition. It is also unclear whether DEV or DEV-like conditions pose a biosecurity or economic threat, or alternatively, whether they reflect a natural cyclic event that does not require disease management. In this mini-review, we summarize the history of digestive epithelial alteration with VLPs (i.e., DEV) or without observation of VLPs (i.e., DEV-like), and we examine the evidence for and against viral-like particles as the cause of DEV in bivalves. We also explore other viral afflictions of bivalves and non-infectious agents, such as harmful algae and xenotoxins, that could elicit similar tissue alterations. Future recommendations for approaches to identify key risk factors that lead to the development of digestive epithelial alterations such as DEV include histological characterization of the digestive gland of marine mollusks; the use of metagenome analysis to design primers that could be used for detection of VLPs and to study host microbiota; disease challenges demonstrating that DEV causes pathology and the relationship between DEV intensity and morbidity/mortality.

SNAPSHOT USA 2020: A second coordinated national camera trap survey of the United States during the COVID-19 pandemic.

Managing wildlife populations in the face of global change requires regular data on the abundance and distribution of wild animals, but acquiring these over appropriate spatial scales in a sustainable way has proven challenging. Here we present the data from Snapshot USA 2020, a second annual national mammal survey of the USA. This project involved 152 scientists setting camera traps in a standardized protocol at 1485 locations across 103 arrays in 43 states for a total of 52,710 trap-nights of survey effort. Most (58) of these arrays were also sampled during the same months (September and October) in 2019, providing a direct comparison of animal populations in 2 years that includes data from both during and before the COVID-19 pandemic. All data were managed by the eMammal system, with all species identifications checked by at least two reviewers. In total, we recorded 117,415 detections of 78 species of wild mammals, 9236 detections of at least 43 species of birds, 15,851 detections of six domestic animals and 23,825 detections of humans or their vehicles. Spatial differences across arrays explained more variation in the relative abundance than temporal variation across years for all 38 species modeled, although there are examples of significant site-level differences among years for many species. Temporal results show how species allocate their time and can be used to study species interactions, including between humans and wildlife. These data provide a snapshot of the mammal community of the USA for 2020 and will be useful for exploring the drivers of spatial and temporal changes in relative abundance and distribution, and the impacts of species interactions on daily activity patterns. There are no copyright restrictions, and please cite this paper when using these data, or a subset of these data, for publication.

Positive role of continuous positive airway pressure for intensive care unit patients with severe hypoxaemic respiratory failure due to COVID-19 pneumonia: A single centre experience.

OBJECTIVES: Continuous positive airway pressure (CPAP) may be a useful treatment strategy for patients with severe COVID-19 pneumonia but its effectiveness in preventing mechanical ventilation is unknown. We aimed to evaluate the outcomes of COVID-19 patients treated with CPAP and determine predictors of CPAP response. DESIGN: This was a retrospective observational cohort study. SETTING: The study took place in the intensive care unit (ICU) at Royal Papworth Hospital (RPH) in Cambridge, UK. PATIENTS: We included all consecutive patients with confirmed COVID-19 pneumonia who were transferred from neighbouring hospitals between 14th March and 6th May, 2020 for consideration of ventilatory support. INTERVENTION: We instituted the use of CPAP for all patients who arrived in RPH not intubated and were not making satisfactory progress on supplemental oxygen alone. MEASUREMENTS AND MAIN RESULTS: Of 33 self-ventilating patients included in this study, 22 (66.7%) were male and the mean age was 54 ± 13.23 patients received CPAP. They were more hypoxaemic than those treated with oxygen alone (PaO2/FiO2 ratio; 84.3 ± 19.0 vs 170.0 ± 46.0 mmHg, p = 0.001). There was a significant improvement in PaO2/FiO2 ratio 1-2 hours after CPAP initiation (167.4 ± 49.0 from 84.3 ± 19.0 mmHg, p = 0.001). 14 (61%) patients responded to CPAP and 9 required intubation. There was no difference between these two groups in terms of the severity of baseline hypoxaemia (PaO2/FiO2 ratio; 84.5 ± 16.0 vs 83.9 ± 23.0 mmHg, p = 0.94) but CPAP responders had significantly lower C-reactive protein (CRP) (176 ± 83 vs 274 ± 63 mg/L, p = 0.007), interleukin-6 (IL-6) (30 ± 47 vs 139 ± 148 pg/mL, p = 0.037), and D-dimer (321 ± 267 vs 941 + 1990 ng/mL, p = 0.003). CT pulmonary angiogram was performed in 6 out of 9 intubated patients and demonstrated pulmonary emboli in 5 of them. All patients were discharged from ICU and there were no fatalities. CONCLUSIONS: In this cohort, CPAP was an effective treatment modality to improve hypoxaemia and prevent invasive ventilation in a substantial proportion of patients with severe respiratory failure. Accepting the small sample size, we also found raised biomarkers of inflammation (CRP and IL-6) and coagulopathy (D-Dimer) to be more useful predictors of CPAP responsiveness than the severity of hypoxaemia, and could help to guide intubation decisions in this clinical setting.

Observational study of thrombosis and bleeding in COVID-19 VV ECMO patients.

INTRODUCTION: COVID-19 has been associated with increased risk of thrombosis, heparin resistance and coagulopathy in critically ill patients admitted to intensive care. We report the incidence of thrombotic and bleeding events in a single center cohort of 30 consecutive patients with COVID-19 supported by veno-venous extracorporeal oxygenation (ECMO) and who had a whole body Computed Tomography Scanner (CT) on admission. METHODOLOGY: All patients were initially admitted to other hospitals and later assessed and retrieved by our ECMO team. ECMO was initiated in the referral center and all patients admitted through our CT scan before settling in our intensive care unit. Clinical management was guided by our institutional ECMO guidelines, established since 2011 and applied to at least 40 patients every year. RESULTS: We diagnosed a thrombotic event in 13 patients on the initial CT scan. Two of these 13 patients subsequently developed further thrombotic complications. Five of those 13 patients had a subsequent clinically significant major bleeding. In addition, two patients presented with isolated intracranial bleeds. Of the 11 patients who did not have baseline thrombotic events, one had a subsequent oropharyngeal hemorrhage. When analyzed by ROC analysis, the area under the curve for % time in intended anticoagulation range did not predict thrombosis or bleeding during the ECMO run (0.36 (95% CI 0.10-0.62); and 0.51 (95% CI 0.25-0.78); respectively). CONCLUSION: We observed a high prevalence of VTE and a significant number of hemorrhages in these severely ill patients with COVID-19 requiring veno-venous ECMO support.

Prevalence of Neospora caninum Exposure in Wild Pigs (Sus scrofa) from Oklahoma with Implications of Testing Method on Detection.

Neospora caninum is a protozoan parasite, reported as a leading cause of cattle abortions and reproductive failure worldwide, costing the cattle industry approximately $1.3 billion annually. With wild pig (Sus scrofa) populations estimated at over six million in the United States, contact between wild pigs and livestock is inevitable, mainly because of the widespread geographic co-occurrence of the two species. As a known reservoir for numerous fungal, bacterial, viral and parasitic diseases, wild pigs are of particular importance for human and veterinary health relative to the prevention of infectious diseases. The seroprevalence of N. caninum in wild pig populations was previously documented in the United States, raising the question as to their exposure point of prevalence. This research screened 116 individual wild pigs for N. caninum using a variety of available assays. Using two different commercially available ELISA test kits, seroprevalence ranged from 12.5% to 67.8%. The Indirect Fluorescent Antibody Test resulted in our highest percent seroprevalence for these samples, at 84.1%. However, none of our samples showed any presence of N. caninum or associated pathologies via histological evaluation of representative tissues. Importantly, the assays used in this study were not congruent with all duplicate samples or between the test types used. The implications of these non-congruent results demonstrates that currently available testing assays produce variable results, underscoring the need for more reliable testing kits and a standardized methodology when assessing disease prevalence in wildlife, particularly for N. caninum in wild pigs, which impacts prevalence and comparability across studies.

Xylella fastidiosa in Olive: A Review of Control Attempts and Current Management.

Since 2013, Xylella fastidiosa Wells et al. has been reported to infect several hosts and to be present in different areas of Europe. The main damage has been inflicted on the olive orchards of southern Apulia (Italy), where a severe disease associated with X. fastidiosa subspecies pauca strain De Donno has led to the death of millions of trees. This dramatic and continuously evolving situation has led to European and national (Italian and Spanish) measures being implemented to reduce the spread of the pathogen and the associated olive quick decline syndrome (OQDS). Research has been also carried out to find solutions to better and directly fight the bacterium and its main insect vector, Philaenus spumarius L. In the course of this frantic effort, several treatments based on chemical or biological substances have been tested, in addition to plant breeding techniques and integrated pest management approaches. This review aims to summarize the attempts made so far and describe the prospects for better management of this serious threat, which poses alarming questions for the future of olive cultivation in the Mediterranean basin and beyond.

Effectiveness and Efficiency of Corral Traps, Drop Nets and Suspended Traps for Capturing Wild Pigs (Sus scrofa).

Strategic control and eradication programs for wild pigs (Sus scrofa) are being developed to help curtail the expanding populations of this invasive, alien species. Drop nets and corral traps have a long history of capturing a multitude of wildlife species, so we evaluated the effectiveness and efficiency of these traps for controlling wild pigs in southern Oklahoma. We also developed and evaluated a suspended metal trap that provided real-time monitoring and deployment to capture animals. Effectiveness of each trap type was estimated as the proportion of pigs removed from the total population, whereas efficiency was calculated based on catch per unit effort (CPUE) (i.e., the number of person hours per pig removal). During 3 years of study (2010-2012), we removed 601 pigs, 296 using drop nets, 60 using corral traps, and 245 using suspended traps. Suspended traps removed 88.1% of the estimated population, whereas drop nets removed 85.7% and corral traps removed 48.5%. CPUE was 0.64 person hours/pig using suspended traps followed by 1.9 person hours/pig for drop nets and 2.3 person hours/pig for corral traps. Drop nets and suspended traps were more effective at removing a large proportion of the population (>85%), mainly through whole sounder removal, but the suspended trap with real-time notifications was the most efficient trap type, requiring fewer person hours to operate.

New gregarine species (Apicomplexa) from tunicates show an evolutionary history of host switching and suggest a problem with the systematics of Lankesteria and Lecudina.

Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts.

Investigating the genetic structure of the parasites Anisakis pegreffii and A. berlandi (Nematoda: Anisakidae) in a sympatric area of the southern Pacific Ocean waters using a multilocus genotyping approach: first evidence of their interspecific hybridization.

The southern Pacific Ocean, off the New Zealand coast, has been reported as one sympatric area of the two parasite species Anisakis pegreffii and A. berlandi. Here, a multilocus genotyping approach, based on a panel of eleven DNA microsatellite (SSR) loci plus the sequences analysis of the nuclear nas10 nDNA and the mitochondrial mtDNA cox2 gene loci, was applied to a total of N = 344 adults and larvae of Anisakis spp. from cetacean and fish species, respectively. Out of the newly scored SSR loci, Anisl 15 and Anisl 2 showed fixed alternative alleles between A. pegreffii and A. berlandi resulting as 100% diagnostic loci. Out of SSRs Anisl 00314 and Anisl 7 previously disclosed, two additional loci, i.e., Anisl 4 and Anisl 22, were found to be sex-linked. The Bayesian genotypes clustering approach (STRUCTURE) allowed identification, with a 100% of probability value, N = 208 specimens to the "pure parental" A. pegreffii, N = 133 to the "pure parental" A. berlandi, while one adult and two larval stages showed mixed ancestry between the two groups having, in all cases, a Q-value = 0.50. NEWHYBRIDS analysis assigned (100% of probability) those specimens to their F1 hybrid category. This represents the first evidence of contemporary hybridization between the two parasite species in a sympatric area. The pairwise FST values estimated at intraspecific and interspecific level, inferred from both SSR loci and mitochondrial mtDNA cox2 sequences, have also demonstrated the existence of two distinct panmictic units in this study area, corresponding respectively to A. pegreffii and A. berlandi. The results obtained support the useful application of a multilocus approach in the identification of sibling species and their hybrid categories in sympatric areas. The possible use of sex-linked SSR loci of the two species of the A. simplex (s. l.), for sex determination of their larval stages, is also suggested.

SNAPSHOT USA 2019: a coordinated national camera trap survey of the United States.

With the accelerating pace of global change, it is imperative that we obtain rapid inventories of the status and distribution of wildlife for ecological inferences and conservation planning. To address this challenge, we launched the SNAPSHOT USA project, a collaborative survey of terrestrial wildlife populations using camera traps across the United States. For our first annual survey, we compiled data across all 50 states during a 14-week period (17 August-24 November of 2019). We sampled wildlife at 1,509 camera trap sites from 110 camera trap arrays covering 12 different ecoregions across four development zones. This effort resulted in 166,036 unique detections of 83 species of mammals and 17 species of birds. All images were processed through the Smithsonian's eMammal camera trap data repository and included an expert review phase to ensure taxonomic accuracy of data, resulting in each picture being reviewed at least twice. The results represent a timely and standardized camera trap survey of the United States. All of the 2019 survey data are made available herein. We are currently repeating surveys in fall 2020, opening up the opportunity to other institutions and cooperators to expand coverage of all the urban-wild gradients and ecophysiographic regions of the country. Future data will be available as the database is updated at eMammal.si.edu/snapshot-usa, as will future data paper submissions. These data will be useful for local and macroecological research including the examination of community assembly, effects of environmental and anthropogenic landscape variables, effects of fragmentation and extinction debt dynamics, as well as species-specific population dynamics and conservation action plans. There are no copyright restrictions; please cite this paper when using the data for publication.

Impact of stroke on outcomes following cardiac surgery: Propensity matched analysis.

BACKGROUND: Stroke remains a devastating complication of cardiac surgery. The aim of this study was to characterize the incidence of stroke and analyze the impact of stroke on patient outcomes and survival. METHODS: A retrospective analysis was performed of patients with a computed tomography-confirmed stroke diagnosis between 1 January 2015 and 31 March 2019 at a single center. 2:1 propensity matching was performed to identify a control population. RESULTS: Over the period 165 patients suffered a stroke (1.99%), with an incidence ranging from 0.85% for coronary artery bypass grafting to 8.14% for aortic surgery. The mean age was 70.3 years and 58.8% were male. 18% had experienced a previous stroke or transient ischemic attack. Compared to the comparison group, patients experiencing postoperative stroke had a significantly prolonged period of intensive care unit admission (8.0 vs 1.1 days P < .001) and hospital length of stay (12.94 vs 8.0 days P < .001). Patient survival was also inferior. In-hospital mortality was almost three times as high (17.0% vs 5.9%; P < .001). Longer-term survival was also inferior to Kaplan-Meier estimation (P < .001). The 1-year and 3-year survival were 61.5% and 53.8% respectively compared to 89.4% and 86.1% for the comparison group. CONCLUSION: Perioperative stroke is a devastating complication following cardiac surgery. Perioperative stroke is associated with significantly inferior outcomes in terms of both morbidity and mortality. Notably a 28% reduction in 1-year survival. Efforts should focus on identifying strategies aimed at reducing the incidence, morbidity, and mortality of perioperative stroke following cardiac surgery.

A novel nuclear marker and development of an ARMS-PCR assay targeting the metallopeptidase 10 (nas 10) locus to identify the species of the Anisakis simplex (s. l.) complex (Nematoda, Anisakidae).

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.

TITLE: Un nouveau marqueur nucléaire et développement d'un test ARMS-PCR ciblant le locus de la métallopeptidase 10 (nas 10) pour identifier les espèces du complexe Anisakis simplex (s. l.) (Nematoda, Anisakidae). ABSTRACT: Le genre Anisakis représente l'un des groupes de nématodes ascaridoïdes les plus répandus dans l'écosystème marin. Trois taxons étroitement apparentés sont reconnus dans le complexe Anisakis simplex (s. l.) : A. pegreffii, A. simplex (s. s.) et A. berlandi. Ils sont largement répartis dans les populations de leurs hôtes intermédiaires/paraténiques (poissons et calmars) et définitifs (cétacés). Un nouveau locus de gène nucléaire, la métallopeptidase 10 (nas 10) (451 pb), a été séquencé et validé sur un total de 219 spécimens des trois espèces d'Anisakis, collectés chez des hôtes poissons et cétacés de zones allopatriques incluses dans leur aire de répartition. Les échantillons d'Anisakis ont d'abord été identifiés par des allozymes et une analyse des séquences de l'ADNmt cox2 et de l'ADNn EF1α-1. Le nouveau marqueur nucléaire a montré des positions de nucléotides alternatives fixes dans les trois espèces, c'est-à-dire qu'il a permis un diagnostic à 100%, permettant la détermination de l'espèce d'un grand nombre d'échantillons analysés dans la présente étude. De plus, des amorces à utiliser pour la PCR par système de mutation réfractaire à l'amplification (ARMS) du même locus génique ont été conçues à ces positions nucléotidiques. Ainsi, la détermination directe du génotypage, par double ARMS, a été développée et validée sur 219 spécimens appartenant aux trois espèces. Une concordance complète a été observée entre les dosages ARMS PCR tétra-amorces et les résultats de séquençage direct obtenus pour le locus du gène nas 10. Le nouveau marqueur de diagnostic nucléaire sera utile dans les travaux futurs d'une approche de génotypage multi-locus et également pour étudier les éventuels événements d'hybridation et/ou d'introgression se produisant entre les trois espèces dans des zones sympatriques.

Cross-species utility of microsatellite loci for the genetic characterisation of Anisakis berlandi (Nematoda: Anisakidae).

Eight microsatellite loci, recently developed in the species Anisakis pegreffii, were successfully amplified in Anisakis berlandi, sibling species of the A. simplex (s. l.) complex. They were validated on adult specimens (n = 46) of the parasite species, collected from two individuals of the definitive host, the long-finned pilot whale Globicephala melas from New Zealand waters. Among the eight loci scored, one, Anisl 07132, had null alleles in A. berlandi and was thus excluded from the subsequent genetic analysis. Two loci, Anisl 00314 and Anisl 10535, were monomorphic. In addition, as also previously detected in the other species of the A. simplex (s. l.) complex, the Anisl 7 locus was seen to be sex-linked, showing hemizygosity in male specimens. Differential allele frequency distributions of A. berlandi, with respect to those previously observed in A. pegreffii and A. simplex (s. s.), were found at some microsatellite loci. The Anisl 7 locus provided 100% diagnosis between A. berlandi and A. pegreffii, while others resulted in 99% diagnosis between A. berlandi and the other two species. Simple sequence repeat (SSR) loci also allowed us to estimate the genetic differentiation of A. berlandi from A. pegreffii (F st ≈ 0.45, Dc = 0.82) and A. simplex (s. s.) (F st ≈ 0.57, Dc = 0.73). The results suggest that SSRs provide a set of candidate markers for population genetics analysis of A. berlandi, as well as for the investigation, through a multi-locus genotyping approach, of possible patterns of hybridisation/introgression events between A. berlandi and the other two Anisakis species in sympatric conditions.

TITLE: Utilité des loci microsatellites pour la caractérisation génétique interspécifique d'Anisakis berlandi (Nematoda, Anisakidae). ABSTRACT: Huit loci microsatellites, récemment développés chez l'espèce Anisakis pegreffii, ont été amplifiés avec succès chez Anisakis berlandi, espèce sœur du complexe A. simplex (s. l.). Ils ont été validés sur des spécimens adultes (n = 46) de l'espèce, récoltés chez deux individus de l'hôte définitif, le globicéphale commun Globicephala melas, des eaux néo-zélandaises. Parmi les huit loci notés, l'un, Anisl 07132, avait des allèles nuls chez A. berlandi et a donc été exclu de l'analyse génétique ultérieure. Deux loci, Anisl 00314 et Anisl 10535, étaient monomorphes. De plus, comme cela a également été détecté précédemment dans les autres espèces du complexe A. simplex (s. l.), le locus Anisl 7 était lié au sexe, montrant une hémizygosité chez les spécimens mâles. Chez A. berlandi, des distributions de fréquences d'allèles, différentielles par rapport à celles précédemment observées chez A. pegreffii et A. simplex (s. s.), ont été trouvées pour certains loci microsatellites. Le locus Anisl 7 a fourni un diagnostic à 100 % entre A. berlandi et A. pegreffii, tandis que d'autres ont abouti à un diagnostic à 99 % entre A. berlandi et les deux autres espèces. Les loci des SSR ont également permis d'estimer la différenciation génétique d'A. berlandi par rapport à A. pegreffii (F st ≈ 0,45, Dc = 0,82) et A. simplex (s. s.) (F st ≈ 0,57, Dc = 0,73). Les résultats suggèrent que les répétitions de séquences simples (SSR) fournissent un ensemble de marqueurs candidats pour l'analyse génétique des populations d'A. berlandi, ainsi que pour l'investigation, dans une approche de génotypage multilocus, des modèles possibles d'hybridation/introgression entre A. berlandi et les deux autres espèces d'Anisakis dans des conditions sympatriques.