By critically appraising open access, educational blogs and podcasts in emergency medicine (EM) using an objective scoring instrument, this installment of the ALiEM (Academic Life in Emergency Medicine) Blog and Podcast Watch series curated and scored relevant posts in the specific areas of pediatric EM.The Approved Instructional Resources – Professional (AIR-Pro) series is a continuously building curriculum covering a new subject area every two months. For each area, six EM chief residents identify 3–5 advanced clinical questions. Using FOAMsearch.net to search blogs and podcasts, relevant posts are scored by eight reviewers from the AIR-Pro Board, which is comprised of EM faculty and chief residents at various institutions. The scoring instrument contains five measurement outcomes based on 7-point Likert scales: recency, accuracy, educational utility, evidence based, and references. The AIR-Pro label is awarded to posts with a score of ≥26 (out of 35) points. An “Honorable Mention” label is awarded if Board members collectively felt that the posts were valuable and the scores were > 20.We included a total of 41 blog posts and podcasts. Key educational pearls from the 10 high quality AIR-Pro posts and four Honorable Mentions are summarized.WestJEM ALiEM Blog and Podcast Watch series is based on the AIR and AIR-Pro series, which attempts to identify high quality educational content on open-access blogs and podcasts. Until more objective quality indicators are developed for learners and educators, this series provides an expert-based, crowdsourced approach towards critically appraising educational social media content for EM clinicians.The  With the rapid rise in the number of social media educational content on blogs and podcasts, especially in emergency medicine (EM),WestJEM presents annotated summaries from the AIR and AIR-Pro Series.To address this need, the Approved Instructional Resources (AIR) SeriesThis installment from the AIR-Pro Series summarizes the best scoring social media educational resources on specific topics within pediatric EM.The AIR-Pro series is a continuously building curriculum covering a new subject area every two months. For each area, six EM chief residents from different U.S. residency programs on the ALiEM-Pro Editorial Board identify 3–5 focused, advanced-level clinical queries within the featured subject area. The topics for this installment included the following:Pediatric arrhythmiasProcedural sedation in pediatricsThe neonate in distressToddlers with a limpPediatric syncopeA broad range of resources were identified using the custom EM search engine FOAMsearch.net, which extracts content from >300 blogs/podcasts relevant to EM as of February 2014.Extracted posts were scored by eight reviewers from the AIR-Pro Editorial Board, which is comprised of EM core faculty and chief residents from various U.S. institutions. The eight reviewers included five chief residents from the AIR-Pro Editorial Board as well as three EM faculty educators, which included one board-certified pediatric EM educator and one pediatric EM fellow.The scoring instrument contains five measurement outcomes using 7-point Likert scales: recency, accuracy, educational utility, evidence based, and references . The sco6An AIR-Pro endorsement is given to posts with a score of ≥26 (out of 35) points. Depending on the redundancy of the highest scoring posts, the best of these are then selected to address each preselected topic. An “Honorable Mention” label is also given to posts specifically felt to be worthwhile, accurate, unbiased, and educationally valuable for advanced clinicians by consensus of the AIR-Pro Board members. These posts must have scored ≥20 (out of 35) points.We initially included a total of 41 blog posts and podcasts. Key educational pearls from the 10 high quality AIR-Pro posts and four Honorable Mentions are summarized.http://www.pemacademy.com/ecgs-long-qt-and-brugada/1. Greene A, Patel S. ECGs: Long QT and Brugada. PEM Academy.  This 10-minute video presentation by Gopwani, Patel, Greene, and Chapman from the Pediatric EM Academy blog covers the pathologies related to Long QT Syndrome  and Brugada Syndrome. Case-based content frames the discussion regarding the various pathologies, emergency department (ED) management, and patient disposition.Long QT Syndrome take-home points: Pediatric patients with a QTc >470 msec should be seen emergently by a cardiologist in the ED. Long QT syndrome is typically either caused by a congenital abnormality or it is acquired (e.g. electrolyte and substance induced). Consider long QT syndrome as an etiology when exertion or emotional stress precedes syncope or seizures.Brugada Syndrome take-home points: The electrocardiogram (ECG) in Brugada Type 1 appears as a “pseudo” right bundle branch block, coupled with ST segment elevation in V1, V2, and V3. The ECG in Brugada Types 2 and 3 has a saddleback-shaped pattern with a positive or biphasic T wave seen in V1, V2, and V3. The difference between Brugada type 2 and 3 is the amount of ST elevation with Type 2 having ≥1 mm of elevation and Type 3 having <1 mm of elevation.Pediatric EM Morsels.  http://pedemmorsels.com/brugada-in-children/2. Fox S. Brugada in Children. This blog discusses the diagnosis and management of Brugada Syndrome. It focuses on the special considerations of Brugada Syndrome in the pediatric population.Take-home points: Febrile illnesses can trigger arrhythmias in pediatric patients with underlying Brugada Syndrome. Children with Brugada Syndrome are more likely to present with complications of monomorphic ventricular tachycardia in comparison to older patients who present with ventricular fibrillation. Although the best available treatment option is an automatic implantable cardioverter-defibrillator (AICD), the decision to place one requires a careful risk-benefit discussion because there is a higher rate of AICD complications in children.http://dontforgetthebubbles.com/syncope-ecgs/3. Forbes E. Syncope Sunday 4: Syncope ECGs. Don’t Forget the Bubbles.  This post discusses the cardiac causes of syncope in pediatric patients including hypertrophic obstructive cardiomyopathy (HOCM), arrhythmogenic right ventricular dysplasia (ARVD), Wolff-Parkinson-White syndrome, Brugada syndrome, long QTc, and catecholaminergic polymorphic ventricular tachycardia (CPTV). A classic ECG is provided for each syndrome, except CPTV.Take-home points: The clinician must maintain a high index of suspicion for subtle ECG findings in the setting of syncope in children. Because HOCM is the leading cause of sudden death in young athletes, look for high left ventricular voltage, Q waves in the inferior-lateral leads, and left atrial enlargement in the setting of a concerning story. ARVD is the second most common cause of sudden death in young athletes. Look for the epsilon waves, T wave inversions in V1–V3, and prolonged S-wave upstroke.http://www.pemacademy.com/ecgs-heart-block-and-sick-sinus/4. Tat S. ECGs: Heart Block and Sick Sinus. PEM Academy.  This is a 12-minute video presentation covering heart block and sick sinus syndrome created by Gopwani, Patel, Greene, and Chapman. Heart blocks and sick sinus conditions are discussed through real-life cases discussing possible patient presentations, acute management, and appropriate disposition for these patients.Take-home points: Reading the ECG using the same methodical approach every time will aid in identifying subtle atrioventricular (AV) blocks. Second-degree AV blocks typically happen in a set pattern such as 4:3 and 3:2. All patients with a third-degree AV block require an immediate cardiology consultation. In contrast, only symptomatic patients with a second-degree AV block require cardiology consultation in the ED.Pediatric EM Morsels.  http://pedemmorsels.com/ketamine-analgesia/5. Fox S. Ketamine for Analgesia. This blog post highlights current practices using ketamine as a primary pain medication in the pediatric population. Discussion points include indications, dosages, and barriers to use.Take-home points: Ketamine should be considered as a core medication in basic healthcare systems. Ketamine is safe and effective, has a rapid onset, low risk for respiratory depression, and requires little monitoring when administered at analgesic doses (0.1–0.3 mg/kg IV or 0.5–1 mg/kg IM).PEM Currents.  http://www.pemcincinnati.com/podcasts/?p=606. Sobo B. Ketamine. This 8-minute podcast covers the basics of ketamine use in the pediatric population. It focuses on the benefits of ketamine including rapid onset, low side-effect profile, and ease of administration. Discussion points also include parental education regarding tachycardia and hypertension, as well as factors that increase a child’s risk of adverse reactions.Take-home points: Ketamine provides both amnestic and analgesic properties safely and efficiently. Younger patients metabolize ketamine at a faster rate and so may require more frequent re-dosing. Skillfully educating families of the possible effects prior to its use will decrease parental anxiety.EmDocs.  http://www.emdocs.net/sick-neonate/7. Radwine Z. The Sick Neonate. This blog post reviews the initial approach to the evaluation, management, and resuscitation of the sick neonate. The post covers the topics of sepsis, congenital heart diseases, and metabolic disturbances. It includes a stepwise approach to the history and physical exam, as well as a mnemonic to help remember the differential diagnosis for a ill-appearing neonate.Take-home points: The differential diagnosis for the ill-appearing neonate can be remembered using the mnemonic THE MISFITS and NEO SECRETS. Neonates in extremis without a fever have a congenital heart disease until proven otherwise. A pink baby in shock needs prostaglandin (PGE1) and milrinone. In contrast, a cyanotic baby in shock needs epinephrine or norepinephrine.The Blunt Dissection  http://thebluntdissection.org/2013/07/a-knackered-neonate/8. Partyka C. A Knackered Neonate… This blog post covers an initial approach to the evaluation and management of an ill-appearing neonate. The case unfolds as you read with scaffolded questions for you to consider before revealing the answers. In this case, a hypoxic newborn presents with worsening respiratory distress despite being on nasal CPAP. The differential diagnosis is discussed, as well as neonatal intubation and troubleshooting the ventilator.Take-home points: Neonates will become hypothermia very quickly, especially during the prolonged exposure times of endotracheal intubation. Ensure adequate external heating during the resuscitation. Troubleshooting the ventilator can be remembered using the DOPE mnemonic, which stands for Displacement of tubing, Obstruction, Pneumothorax, and Equipment failure.ER CAST.  http://blog.ercast.org/pediatric-limp/9. Orman R. Pediatric Limp - Podcast: 28:22. This 28-minute podcast discusses the workup for a child with a limp as well as a decision rule for distinguishing toxic synovitis from a septic arthritis of the hip. A pediatric orthopedist Dr. Adam Barmada is curbsided for a consultation to give additional pearls for dealing with a limping child.3. The risk for septic arthritis is 0.2% (0 risk factors) and 97% .Take-home points: Examine the entire leg of the limping child, especially a joint above and below the subjectively painful area because, for instance, knee pain may be from hip pathology (and vice versa). The Kocher Criteria for risk stratifying septic hip arthritis includes non-weight bearing status, temperature >38.5°C (101.3°F), erythrocyte sedimentation rate> 40 mm/hr, and serum white blood cell count >12 cells/mmPediatric EM Morsels.  http://pedemmorsels.com/toddlers-fracture10. Fox S. Toddler’s Fracture. This blog post covers an initial approach to the evaluation and management of a toddler’s fracture. It also reviews management strategies for challenging cases, such as children with a normal workup but continue to have symptoms.Take-home points: Toddler’s fractures are non-displaced spiral or oblique fractures of the tibia and may not be visible on initial radiographs in 13–43% of cases. Other areas that may also be initially normal on radiographs include the base of first metatarsal bone, cuboid, and calcaneus and so should be carefully palpated in toddlers with undifferentiated leg pain. A limping pediatric patient is more likely to have a fracture than a ligamentous strain even with a negative radiograph.ER CAST.  http://blog.ercast.org/the-toxic-neonate/1. Orman R. The Toxic Neonate. This 37-minute podcast features an interview with Dr. Tim Horeczko, who was then a pediatric EM fellow. It was chosen as an honorable mention as it gives listeners useful information on the toxic neonate as well as a clear set of clinical priorities to work with for medical decision-making. It was determined to be a must-know resource for evaluating a sick neonate. Topics covered included the pediatric assessment triangle, hypoglycemia, intravenous fluid management, heart disease, and inborn errors of metabolism.Take-home points: With a sick neonate, remember the pediatric assessment triangle  and to continue to reassess after interventions. Always check blood sugar and replete appropriately based on the different age groups: 10% dextrose 5–10 cc/kg (0–1 year old), 25% dextrose 2–4 cc/kg (1–8 years old), and 50% dextrose 1–2 cc/kg (>8 years old). In a “crashing infant” in its first month of life, if your interventions are not working, consider PGE, and if administering PGE, they will likely require intubation due to the risk for apnea.Emergency Medicine Cases.  http://emergencymedicinecases.com/episdode-35-pediatric-orthopedics/2. Helman A. Episode 35: Pediatric Orthopedics Pearls and Pitfalls. This 140-minute podcast was chosen as an honorable mention due to the careful explanation of the injuries they discussed. While a lengthy podcast, it discusses a broad spectrum of pediatric orthopedic conditions. Dr. Sanjay Mehta and Dr. Jonathan Pirie, both Canadian pediatric EM physicians, discuss septic arthritis, toddler’s fracture, tillaux fracture, supracondylar fractures, anterior cruciate ligament tears, non-accidental trauma, pediatric orthopedic pain management, and more.3), C-reactive protein, or ultrasound can be used. Ultimately one must use clinical judgment to differentiate the two conditions, because no one measure is perfect. Remember the CRITOE mnemonic  epicondyle, Trochlea, Olecranon, External  epicondyle) to avoid mistaking an ossification center for an avulsion fracture or vice versa. Comparison of the asymptomatic side is also useful to determine if there is an abnormality.Take-home points: Remember that in children their ligaments are stronger than their bones, making fractures more likely than sprains. To determine the difference between septic arthritis and transient synovitis, the Kocher criteria  http://www.emdocs.net/pediatric-syncope/3. Radwine Z. Pediatric Syncope. This blog post was chosen as an honorable mention because of the practical and well-outlined list of pearls on pediatric syncope. The post discusses red flag considerations, cardiovascular causes, and concerning ECG findings.Take-home points: Remember the 6 P’s when obtaining you syncope history in children: prodrome activities and prodrome symptoms, predisposing and precipitating factors, passerby/witness, and post-ictal phase. Cautionary red flag signs and symptoms include a history of congenital heart disease, no prodromal symptoms, syncope while supine or during exertion, family history of sudden cardiac death, a pathologic heart murmur, and chest pain.Life in the Fastlane.  http://lifeinthefastlane.com/ecg-library/hcm/4. Burns E, Nickson C, Mattu A. Hypertrophic Cardiomyopathy. This blog post was chosen as an honorable mention because of its in-depth review of hypertrophic cardiomyopathy (HOCM). It reviews the background, pathophysiology, and various ECG abnormalities seen with HCOM. Additionally, it includes a link to a 17-minute video presentation by Dr. Amal Mattu on the early recognition and treatments of HOCM as well as multiple brief videos from Dr. Sanjay Sharma discussing classic and subtle ECG findings in HOCM.Take-home points: The chief abnormality associated with HOCM is left ventricular hypertrophy (LVH), occurring in the absence of any inciting stimulus such as hypertension or aortic stenosis.Additional ECG findings are deep, narrow (dagger-like) q waves in both lateral  and inferior  leads. These q waves are <40 msec . HOCM can present with arrhythmias including Wolff-Parkinson-White, atrial fibrillation, supraventricular tachycardia, premature atrial contractions, premature ventricular contractions, and ventricular tachycardia.WestJEM ALiEM Blog and Podcast Watch series serves to identify educational quality blogs and podcasts for EM clinicians through an expert panel using an objective scoring instrument. These social media resources are currently curated in the ALiEM AIR and AIR-Pro Series, originally created to address EM residency needs. These resources are herein shared and summarized to help EM clinicians filter the multitude of blog posts and podcasts that are constantly being published at a rapid rate. Limitations include the fact that the search engine FOAMSearch.net may have omitted valuable websites, and the scoring instrument has not been validated. While these lists are by no means a comprehensive analysis of the entire web for these topics, this series provides a crowdsourced, expert-panel approach towards identifying some high quality, educational social media content for the EM clinician.Until more objective quality indicators are developed, the


AbstractRhinellafestae group is described from montane forests of the Cordillera Azul National Park between 1245 and 1280 m a.s.l. in the Cordillera Oriental, San Martín region, northern Peru. The new species is morphologically and genetically compared with members of the Rhinellaacrolopha group (former genus Rhamphophryne) and members of the R.festae group. The new species is characterized by its large size , eight presacral vertebrae, fusion of the sacrum and coccyx, long protuberant snout, snout directed slightly anteroventral in lateral view, cranial crests moderately developed, absence of occipital crest, presence of tympanic membrane, dorsolateral rows of small conical tubercles extending from parotoid gland to groin, hands and feet with long digits, fingers basally webbed and toes moderately webbed. Phylogenetically it is a member of the R.festae group which is most closely related to R.chavin and R.yanachaga from Peru. Morphologically the new species shares similarities with R.tenrec and R.truebae, members of the R.acrolopha group from Colombia.A new semiarboreal species of the  Bufonidae Gray, 1825 comprises a clade of neobatrachian anurans commonly known as “true toads” of approximately 600 species and more than 50 genera  is one of the most diverse bufonid genera currently composed of 91 species and broadly distributed throughout the Neotropical region (Bufonidae has 54 species assigned to seven genera (AmphibiaWeb 2017): Amazophrynella (4), Atelopus (18), Nannophryne (2), Rhaebo (3), Rhinella (25), Truebella (2). Six similarity-defined groups within the genus Rhinella in South America are traditionally recognized based on anatomical and external characters, as follows: Rhinellaacrolopha, R. crucifer, R.granulosa, R.margaritifera, R.marina, R.spinulosa and R.veraguensis groups  and one species extending also to the upper Amazonian Basin of Ecuador . From early on, the monophyly of the genus was not supported by Rhamphophryne was the anteriorly ossified sphenethmoid forming a protuberant snout, which is variable in some species (R.nicefori and R.rostrata).The former genus Rhamphophryne was synonymized with Rhinella Fitzinger, 1826 by Rhinellaacrolopha group consisting of the species of the former genus Rhamphophryne, nevertheless, they did not formally propose delimitation of this species group. In a phylogenetic analysis based on the mitochondrial 16S rRNA gene of available species, Rhinellafestae group, for a clade of toads containing the following seven species: R.chavin ; R.festae; R.macrorhina; R.manu ; R.nesiotes ; R.rostrata; and R.yanachaga . This monophylum is assumed to be integrated by certain species of the R.acrolopha group and some species of the R.veraguensis group . Herein, we follow Rhinellafestae group.Recently, based on morphological and molecular data, the generic name is group . The RhiCAZNP) located in the Cordillera Oriental of the Andes, Loreto, San Martín, Huánuco and Ucayali regions, is one of the most diverse natural protected areas in northern Peru. CAZNP has an extension of more than 13,000 km2 with an altitudinal range from 200 to 2400 m a.s.l. between the Huallaga and Ucayali rivers  and poison frogs  within the CAZNP have increased the knowledge of the faunal diversity of this region. Most recently, CAZNP. As part of the aforementioned survey, Kaitlin Tasker and Juan C. Cusi conducted fieldworks in the surroundings of the Park Rangers Center N° 53 “Shapaja” at CAZNP, close to Tocache city in 2013, resulting in a discovery of a morphologically distinguishable Rhinella species, which was tentatively reported under the name Rhinellacf.festae by The Cordillera Azul National Park , Lima, Peru.ZNP Fig. . All speZNP Fig. . Tissue Rhinellafestae group or the R.acrolopha group (see Appendix) and original species descriptions. The coordinates of the type and reference localities of species of the R.acrolopha group were obtained using Global Gazetteer version 2.3 . The geographic coordinates were based on the datum WGS84 and maps were designed using ArcGIS version 10.0.The specimens were compared with species previously assigned either to the Morphological characters. Morphometric measurements in millimeters (mm) were taken with a digital caliper Mitutoyo (nearest to 0.1 mm). Measurement abbreviations used throughout the text are:SVL snout–vent length;HW head width (at level of angle of jaw);HL head length (from angle of jaw to tip of snout);ED horizontal eye diameter;IOD interorbital distance;EW upper eyelid width;EL eyelid length (upper eyelid length);IND internarial distance;E–N eye-nostril distance ;NSD nostril-snout distance;SL snout length (between anterior corner of eye and tip of snout);FL forearm length ;HNDL hand length ;FEML femur length;TL tibia length;FOOTL foot length .Fingers and toes are numbered preaxially to postaxially from I–IV and I–V, respectively. We determined comparative lengths of Toes III and V by adpressing both toes against Toe IV; lengths of fingers I and II were determined by adpressing the fingers against each other. Condition of the tympanum was assessed by visual examination under stereoscope. Specimens were sexed by examination of gonads and secondary sex characters. Ovarian eggs number and coloration were observed by dissection of a gravid female. The measurements of each egg were obtained of the maximum diameter, calculating the mean and standard deviation. Format of the description and diagnosis follows the standards of http://www.carestream.es/computed-radiography/vita-cr-systems.html; 44 kV, 32 mAs) and were taken of the dorsal surface of each specimen collected. Images were edited with Adobe Photoshops CS6 for MacBookPro. Coloration in life of specimens was described based on digital photographs and field notes.Webbing formula follows Taxon sampling. Three specimens of the putative new species from the Rhinellafestae group from the Cordillera Azul National Park were compared with the taxa from the dataset from R.festae group available in GenBank for our genetic marker (16S rRNA), i.e. six taxa including one undescribed species “Rhinella sp. C” . We used only one sequence from two available of “R.macrorhina” and “R.rostrata” deposited in GenBank  due to their lower quality (based on comparison with other homologous sequences), and because they are most probably conspecific based on nucleotide sequences in overlapping DNA segments and information on origin of samples. Both samples were collected 0.5 km W of Medellin, Colombia by the same collector (P. Alberch), and are currently stored in the Museum of Vertebrate Zoology under the name “Rhamphophrynemacrorhina” . We therefore used only one sequence (AF375533) of “R.rostrata” and we name it R.macrorhina following David Cannatella’s identification . The same sample (GenBank sequences) was earlier used by R.rostrata” and “R.macrorhina” both represent R.macrorhina and the divergence is probably caused by errors in nucleotide sequences due to their lower qualities. Genetic information for R.rostrata, which might now be extinct , with a particular interest in the affinities of specimens morphologically resembling recently described R.yunga from a new locality, Rio Huatziroki . More detailed information on the new material, and all analyzed samples from the R.festae group, is given in Table  extinct , is thusc marker 6S rRNA, Laboratory protocol and bioinformatics. A fragment of the mitochondrial 16S rRNA gene (~ 550 bp) was targeted. DNA extraction, PCR amplification and sequencing followed the methods described in Phylogenetic analysis and systematics. The maximum likelihood (ML) analysis and Bayesian phylogenetic inference produced trees with the same topologies and strong support for the main clades. Our phylogenetic tree supported the R.festae group (Bayesian posterior probabilities 1.00/ML bootstrap 97) and the distinctiveness of the morphologically identified new species of Rhinella from the Cordillera Azul National Park. The new species was most closely related to a sister group composed of R.yanachaga and R.chavin  from Rio Huatziroki, a buffer zone of the Protected Forest Pui Pui, Junín, Peru, are genetically identical to the type material  and Alto Mayo, San Martín  in the Peruvian Amazonia. Rhinellacf.margaritifera from Bolpebra (Bolivia) is nested together with two samples from Peru and one Bolivian sample of R.cf.paraguayensis. Within the R.veraguensis group, four new samples of R.cf.leptoscelis  from Rio Bravo, Protected Forest Pui Pui, Junín, Peru proved to be closely related to two published sequences from Cordillera Yanachaga, Pasco, Peru .vin Fig. , both frTaxon classificationAnimaliaAnuraBufonidaehttp://zoobank.org/8FD9CFFD-7311-42A5-85E2-2AB00CF4F7E7Rhinellacf.festae : Lily Rodriguez’s Beaked ToadSapo picudo de Lily RodríguezMUSM 32204 , Alto Biavo District, Bellavista Province, San Martín Region, Peru, collected on 27 September 2013 by K. Tasker and J. C. Cusi.MUSM 32206 (field number KT 76), two juveniles MUSM 32211 (field number KT 84), 32213 (field number KT 72) collected with the holotype; two adult females MUSM 32201 (field number KT 89), MUSM 32205 (field number KT 87), same locality as holotype, collected on 28 September 2013 by K. Tasker and J. C. Cusi.Five individuals Figs –8: an adRhinellafestae group confirmed by 16S DNA barcoding. The new species can be diagnosed by the following combination of characters: (1) large size, SVL 47.1–58.3 mm in females (n = 4), males are unknown; PageBreakPageBreak(2) eight presacral vertebrae; (3) sacral vertebrae fused with coccyx; (4) snout long, acuminate, pointed to rounded terminally in dorsal view; snout protuberant, directed slightly anteroventral in profile as a “shark snout”; (5) cranial crests moderately developed; (6) canthal, supraorbital, postorbital and supratympanic crests continuous, distinctly elevated in female, slightly elevated in juveniles; pretympanic crest present, occipital crest absent; (7) tympanic membrane present, tympanic annulus weakly defined; (8) mandibular angle not protruding; (9) parotoid glands moderately large, roughly triangular to rounded in outline, slightly swollen laterally, incorporated into lateral row of tubercles; (10) dorsolateral rows of small, conical tubercles extending from parotoid gland to groin; (11) hands and feet with long digits, fingers basally webbed and toes moderately webbed; (12) skin on dorsum smooth with scattered conical tubercles in females; (13) subarticular tubercles diffuse, round to ovoid; (14) supernumerary tubercles present, round but poorly developed; (15) cloacal sheath absent; (16) in life, dorsum light brown to greenish brown with irregular brown, dark brown or black markings; with or without grey white middorsal stripe; venter cream yellow to brownish grey with minute light cream spots; iris silvery greenish with irregular black mottling.A large species of the Rhinellafestae and R.acrolopha groups. Three species of the R.acrolopha group from Colombia and Panama  are large-sized toads similarly to R.lilyrodriguezae sp. n. . The new species is most similar morphologically to R.truebae and R.tenrec, but is distinguished by lacking the occipital crest (which is low in R.truebae and R.tenrec). Rhinellalilyrodriguezae sp. n. can also be distinguished from R.tenrec by having a sacrum fused with the coccyx, cranial crests moderately developed and tympanic membrane present ; from R.truebae and R.lindae by having a dorsolateral row of small conical tubercles extending from parotoid gland to groin, hands basally webbed, and feet moderately webbed . Rhinellalindae possesses a snout slightly directed upwards .The new species shares similarities with members of the Rhinellaacrolopha group are R.acrolopha, R.nicefori, R.paraguas, and R.ruizi. These species are differentiated from R.lilyrodriguezae sp. n. (characters in parentheses) by the absence of the tympanic membrane (present), hands and feet extensively webbed in R.paraguas and R.ruizi, and reduced webbing in R.acrolopha and R.nicefori , sacrum not fused with the coccyx except in R.paraguas (fused), seven presacral vertebrae except in R.paraguas (eight) and the presence of occipital crest except in R.paraguas (absent). Rhinellanicefori and R.ruizi have hands and feet with short digits (long digits). Rhinellaparaguas and R.ruizi have cranial crests very low (moderately developed). Rhinellanicefori has a dorsolateral row of enlarged tubercles extending from the posterior margin of the parotoid gland to a point about three-fourths the distance between the axilla PageBreakand groin . Rhinellaacrolopha possesses a snout directed markedly anteroventrally  and a dorsolateral row of depressed tubercles extending from the posterior margin of the parotoid gland posteriorly to a point about two-thirds distance between axilla and groin .Other species of the R.acrolopha and R.festae groups from Colombia and Ecuador  are distinguished from R.lilyrodriguezae sp. n. by lacking the tympanic membrane (present), by having hands and feet extensively webbed , seven presacral vertebrae (except for R.macrorhina which has eight), snout directed markedly anteroventrally in R.festae and R.macrorhina and straight in R.rostrata . Rhinellafestae and R.macrorhina have occipital crest well developed (absent). Rhinellamacrorhina is distinct in having a sacrum not fused with the coccyx (fused), and a dorsolateral row of small tubercles extending from posterior margin of parotoid gland posteriorly to a point about one-half distance between axilla and groin (tubercles extending from parotoid gland to groin). Rhinellafestae possesses dorsolateral row of slightly enlarged, conical tubercles extending from posterior margin of skull to a point about three-fourths distance between axilla and groin (tubercles extending from parotoid gland to groin) and hands with short digits (long digits).The remaining species of the R.festae group  are distinguished from R.lilyrodriguezae sp. n. by having smaller females  and by having webbing of hands and feet fleshy (membranous in R.lilyrodriguezae). Rhinellachavin possesses a snout rounded in lateral view , large [about twice ED], ovoid parotoid glands (moderately large [about same size as ED] and triangular), dorsolateral row of large, nearly round elevated tubercles beginning above insertion of forelimb extending to inguinal region , elevated, elongate glands on forearm, tibia and outer dorsal margin of the foot and hand (glands absent) and hands and feet with relatively short digits (long digits). Rhinellanesiotes has a snout rounded in lateral view , lacks cranial crests (moderately developed), low ovoid parotoid glands (moderately large and triangular), lacks dorsolateral row of tubercles (present) and hands and feet with relatively short digits (long digits). Rhinellamanu has a snout pointed in lateral view , large [about twice ED], oblong parotoid glands to the point of being nearly spherical (moderately large [about same size as ED] and triangular) and inconspicuous cranial crests (moderately developed). Rhinellayanachaga is most similar to R.lilyrodriguezae sp. n. from cloud forests of central Peru, both have cranial crests, tympanic membrane distinct, moderately large [about same size as ED] parotoid glands, dorsolateral rows of small, conical tubercles extending from parotoid gland to groin, fingers and toes relatively long and long, slender extremities. Nevertheless, R.yanachaga differs from PageBreakR.lilyrodriguezae sp. n. by having a snout slightly protruding in lateral view , well developed webbing on hands and feet , dorsal skin smooth without keratin-tipped tubercles in females  and dorsal coloration in life is dark brown with small, irregular, green spots and markings .Peruvian species of the SVL 58.3 mm; head triangular in dorsal view; head wider than long (HW 1.12 times HL), head width 30% of SVL; head length 27% of SVL; head narrower than body; snout acuminate, rounded terminally in dorsal view; not bulbous at tip; distance from the nostril to the tip of the snout (3.2 mm) is noticeably less than the distance from the nostril to the eye (5.8 mm), constituting 20% of head length; snout long, protuberant, directed slightly anteroventral as “shark snout” in profile , ED noticeably shorter than E-D; canthal ridges angular evident; cephalic crests moderately developed; pre-, supra-, postorbital crests distinct, continuous; occipital crest absent; supratympanic crest evident, expanded laterally; pretympanic crests well defined; tympanic annulus weakly defined, superficial tympanic membrane present, not in contact with parotoid glands or postorbital crests; tympanum diameter smaller than eye diameter; parotoid glands moderately large (about same size as ED), roughly triangular in outline; upper eyelid covered with many, low, keratin-tipped tubercles; dorsal and lateral surfaces of head bearing many warts; forearms long, slender; forearm length 26% of SVL; dorsal surface of forelimbs spiculate, bearing densely scattered subconical tubercles; hand length 28% of SVL; hands with long fingers; relative lengths of fingers I < II < IV < III; finger tips rounded; fingers basally webbed, extended between fingers II–IV; Finger IV bears well-defined lateral fringes ; tongue narrow, about 2.5 times as long as wide, not notched posteriorly, posterior one half free; choanae small, ovoid, widely separated and partially concealed by palatal shelf of maxilla.Gravid female; body robust; ges Fig. ; palmar ges Fig. ; tarsal SVL: 58.3; HW: 17.8; HL: 15.9; ED: 4.4; IOD: 9.3; EW: 3.9; EL: 6.4; IND: 4.3; E–N: 5.8; NSD: 3.2; SL: 9.1; FL: 15.4; HNDL: 16.3; FEML: 23.7; TL: 23.4; FOOTL: 22.6.Fig. . General dorsal coloration light brown; dorsum and hind limbs with small, irregular, dark brown spots and markings; flanks cream, with a discontinuous, well defined, broad, black ventrolateral band beginning behind tympanum and extending to inguinal region; dark grey transverse bars on shanks; tympanum brown; upper lip cream without bars or spots; dorsolateral row of tubercles reddish brown, sharply contrasting with the black discontinuous ventrolateral band; throat, chest and venter gray with minute light cream spots; ventral surfaces of thighs gray with minute dark grey spots; ventral surfaces of hands and feet dark gray; subarticular and supernumerary tubercles cream on hands.PageBreakPageBreakFig. . Diurnal coloration of dorsum and flanks dark brown; flanks with irregular, dark green dorsolateral blotches extending from behind the parotoid glands to sacral region; dorsolateral row of tubercles light brown; throat dark gray; chest and belly grey with minute light cream spots extending to the thighs; lower side of the belly cream yellow; iris silvery greenish with irregular black mottling.Figs –9. Considerable variation between nocturnal and diurnal coloration was observed. The nocturnal coloration of an adult female  were of similar coloration as MUSM 32205.MUSM 32211, 32213) is light grey with darker irregular markings forming a “dead-leaf pattern” from between eyes to cloacal region. Cranial crests are more prominent in adults than in juveniles. All specimens, except the holotype, have tip of snout acuminate in dorsal view. Radiographs show that all specimens have eight presacral vertebrae and a sacrococcygeal articulation  contained 185 ovarian eggs  with an average diameter of 2.8 ± 0.2 mm , which are pale cream yellow in preservative. The presence of numerous, large, pigmented eggs and association of the individuals with water bodies suggest endotrophic larvae . We classify Rhinellalilyrodriguezae sp. n. as “Data Deficient” according to the IUCN red list criteria  based on external morphology and osteology hypothesized multiple evolutionary histories and complex morphological variation within the genus , its incorporation into the R.festae group is highly likely; as already showed by R.festae + R.chavin] + [R.manu + R.nesiotes]] using combined mitochondrial and nuclear DNA data. On the other hand, the species R.rostrata and R.nesiotes are tentatively removed from the festae group  close to lotic aquatic bodies of medium-size and moderate water flow, and dwelling on the ground during the day. All species of the R.festae and R.acrolopha groups display diverse types of habitat use. Some species are semiarboreal  or arboreal , others are terrestrial . Most members of both groups inhabit rainforests and montane forests at elevations between 1200 m and 3600 m, except for R.tenrec occurring in the Chocó lowlands, and R.festae inhabiting the Amazonian lowlands.Our 16S rRNA analysis supports the monophyly of the up sensu  due to aern Peru  is morphRhinellafestae clade as proposed by R.acrolopha group (i.e. R.macrorhina and Rhinella sp. C), as supported by high morphological similarity and molecular affinity. However, additional molecular data are required for any conclusive observations. Our study constitutes a starting point for understanding the diversification within this radiation. Additional morphological and molecular evidence from a larger number of taxa is needed to reach a better knowledge of the evolution, systematics and biogeography of this interesting South American lineage of bufonid anurans.Finally, the monophyly of the


Edwardsiella ictaluri is a Gram-negative facultative anaerobic rod and the causative agent of enteric septicemia of channel catfish (ESC), which is one of the most prevalent diseases of catfish, causing significant economic losses in the catfish industry. E. ictaluri is resistant to complement system and macrophage killing, which results in rapid systemic septicemia. However, mechanisms of E. ictaluri stress responses under conditions of host environment are not studied well. Therefore, in this work, we report E. ictaluri stress responses during hydrogen peroxide, low pH, and catfish serum stresses as well as during catfish invasion. E. ictaluri stress responses were characterized by identifying expression of 13 universal stress protein (USP) genes (usp01-usp13) and seven USP-interacting protein genes . Data indicated that three usp genes  were highly expressed in all stress conditions. Similarly, E. ictaluri heat shock proteins groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Also, E. ictaluri grpE and relA were highly expressed in catfish spleen and head kidney. These findings contribute to our understanding of stress response mechanisms in E. ictaluri stress response, and stress-related proteins that are essential for E. ictaluri could be potential targets for live attenuated vaccine development against ESC. E. ictaluri is a Gram-negative bacterium causing enteric septicemia of channel catfish (ESC), one of the most important diseases of farm-raised channel catfish in the US [E. ictaluri is a facultative intracellular pathogen capable of surviving inside catfish macrophages [E. ictaluri virulence factors include flagella [E. ictaluri lipopolysaccharide oligo-polysaccharide (LPS O-PS) plays a major role during catfish infection [E. ictaluri tricarboxylic acid (TCA) cycle and ferric hydroxamate uptake systems also contribute to E. ictaluri virulence in catfish [n the US , 2. E. irophages , which crophages . E. ictaflagella , extraceflagella , lipopolflagella –11 outerflagella , hemolysflagella  and chonflagella . Recentlnfection , and E.  catfish –18.Bacteria often have to cope with stressful conditions such as nutrient limitation, abiotic stresses, and predation. Bacterial adaptation to stress includes regulation of mRNA stability and translation, protein quality control systems, and specific and general stress responses. Understanding bacterial stress responses contribute to our knowledge of host microenvironments, mechanisms of virulence, and potential targets for treatment of infectious diseases .E. coli has six usp genes whose products are involved in several functions including oxidative stress, adhesion, and motility [usp genes is regulated by sigma factors within RNA polymerases, and ppGpp is another important regulator of USPs [Universal stress proteins (USPs) are a conserved group of stress proteins that are generally between 140–160 amino acids. They are present in archaea, bacteria, plants, and fungi, but not humans . E. colimotility . Express of USPs . Under s of USPs , 23.Salmonella, Klebsiella, and Mycobacterium need USPs to invade the host [Mycobacterium tuberculosis [Salmonella [Burkholderia pseudomallei [Listeria monocytogenes [Staphylococcus aureus, virulence factors were downregulated in vivo while expression of uspA increased [Acinetobacter baumannii, uspA is essential for pneumonia and sepsis pathogenesis [USPs are important for pathogenic bacteria. For example, the host . USPs arrculosis ; growth lmonella ; virulendomallei ; and intytogenes . In Stapncreased . In Acinogenesis .usp (usp01 to usp13) and seven USP-interacting genes, including six genes that encode heat shock proteins  and a gene that encodes guanosine pentaphosphate synthetase (relA). We expect that expression analysis of these genes will help us understand stress responses in E. ictaluri, and stress-related proteins that are essential for E. ictaluri could be potential targets for live attenuated vaccine development against ESC.USPs can interact with several heat shock proteins (HSPs) and other stress-related proteins to help bacterial virulence and stress response. Therefore, we determined the expression of 13 Animal work was performed in accordance with the Institutional Animal Care and Use Committee at Mississippi State University. The Office of Laboratory Animal Resources at Mississippi State University is required to adhere to applicable federal, state, local and institutional laws and policies governing animal research.E. ictaluri strain 93–146 was grown at 30°C using brain heart infusion (BHI) broth and agar . E. coli were cultured at 37°C using Luria-Bertani (LB) broth and agar (Difco). E. coli SM10λpir were used for transferring pAKgfplux1 into E. ictaluri. Ampicillin (100 μg/ml) and colistin (12.5 μg/ml) antibiotics  were used when needed.Bacteria and plasmids used in this study are shown in E. ictaluri USP genes and proteins were obtained from the E. ictaluri genome [http://string-db.org) provides pre-calculated protein-protein interactions for organisms, including E. ictaluri. By querying protein sequences of the E. ictaluri USPs, the interacting proteins were identified, and the interaction network was visualized. Because of its regulatory role for some USPs, ppGpp synthetase (relA) was also included in our analysis.Sequences of the i genome , and sixi genome . The STRE. ictaluri strain 93–146 colony was inoculated in 5 ml of BHI broth, followed by 16–18 h incubation at 30°C with shaking. The next day, 40 ml of BHI broth was inoculated and grown to an optical density (OD600) of 0.4. Then each culture was divided into four aliquots, 10 ml each, and bacteria were harvested by centrifugation at 6,000 x g for 15 m. Supernatant was removed, and bacteria were resuspended in acidic BHI broth  and standard BHI broth (pH 7.0). Cultures were incubated with shaking (180 rpm) at 30°C for 30 m., and pellets were collected by centrifugation (7000 x g for 15 m). RNAlater was added, and bacteria were stored for a week at -20°C until RNA was isolated.An E. ictaluri 93–146 was streaked on BHI agar containing 12.5 μg/ml of colistin and incubated at 30°C for 48 h. Small cultures were prepared by inoculating 5 ml of BHI broth with 12.5 μg/ml colistin and incubating for 16-18h at 30°C with shaking at 200 rpm. 40 ml of BHI broth were inoculated and grown to an optical density (OD600) of 0.4. Each culture was divided into four aliquots, 10 ml each, and bacteria were harvested by centrifugation at 6,000 x g for 15 m. Supernatant was removed, and bacteria were resuspended in two types of BHI broth: 20 ml standard BHI broth and 20 ml BHI broth supplemented with 1.5 mM (0.05%) H2O2. Cultures were incubated with shaking (180 rpm) at 30°C for 30 m. Bacteria were harvested by centrifugation at 7000 x g for 15 m., and pellets were suspended in RNAlater and stored a week at -20°C until RNA isolation.E. ictaluri were exposed to naïve catfish serum and heat-inactivated catfish serum. Each treatment included four biological replicates. E. ictaluri cultures were washed three times using 1.25 ml of cell wash buffer (10 mM TrisCl and 5 mM magnesium acetate). Either normal serum or heat-inactivated serum (1.25 ml) was added to each E. ictaluri pellet. Tubes were inverted to mix bacteria and serum thoroughly followed by incubation for 30 m at 30°C. Serum-bacteria mixture was used for total RNA isolation.E. ictaluri were injected into intraperitoneal space in 100 μl PBS (approximately 1×104 CFU). Negative control fish were injected with 100 μl PBS.Approximately 18-month-old SPF channel catfish  were stocked into two tanks at a rate of 7 fish/tank. After one week of acclimation, fish were anesthetized in water containing 100 mg/L tricaine methanesulfonate (MS-222), and bioluminescent Bacteria were visualized in live catfish using an IVIS Lumina XRMS In Vivo Imaging System  by following the procedures described previously . BiolumiAt 30 h post-exposure, fish were euthanized with a high dose of MS-222 (300 mg/L), and anterior kidney and spleen tissues were collected immediately in RNAlater Stabilization Solution. Then, RNA was isolated from four fish with an RNeasy Mini Kit (Qiagen).In each experiment, total RNA was isolated from four biological replicates using RNeasy Protect Bacteria Mini Kit (Qiagen). Contaminating bacterial DNA was eliminated by DNase I treatment with RNase-Free DNase Set (Qiagen). The concentration and quality of the isolated total RNA were measured by NanoDrop 1000 (Thermo Scientific). First-strand cDNA was produced from 1 μg total RNA using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific) by following manufacturer’s instructions.Primers sequences and expected PCR fragment sizes are shown in -ΔΔCt. ΔΔCt was calculated by ΔΔCt = ΔCt (stress condition) - ΔCt (non-stress condition), where ΔCt is the normalized signal level in a sample (ΔCt = Ct of target gene–Ct of reference gene).Relative expression values were calculated by the threshold cycle (Ct) changes in sample and control using the ΔΔCt method . All expOne-way Analysis of Variance (ANOVA) was used to compare gene expression among different stress conditions with SAS 9.1.4 . P value cutoff for statistical significance was < 0.05. To increase statistical reliability, four replicates were run for each sample, and the mean Ct considered for each gene.E. ictaluri genome [We identified 13 USPs and seven stress-related proteins encoded in the i genome  Fig 1A)E. ictalui genome  Fig 1B)E. ictaluE. ictaluri in live catfish allowed us to determine the best time point for tissue collection , which are involved in several functions including oxidative stress, adhesion, and motility [In this research, we aimed to determine the expression of 20 stress-related genes under different stress conditions. There are different types of universal stress proteins (USP) in archaea, bacteria, and plants . E. colimotility .E. ictaluri can resist macrophage killing and replicate in phagocytes, which is considered one of the key mechanisms for rapid spread of the pathogen in the host. The low pH and oxidative stress we applied mimic the conditions present in the phagolysosome [usp05, usp13, dnaK, groEL, groES, and grpE may indicate their involvement in E. ictaluri survival in harsh phagolysosomal conditions.Following bacterial phagocytosis by macrophages, fusion of the lysosome with the phagosome causes release of acid-activated enzymes and toxic metabolites and acidification of the phagolysosome to pH 4–5. As a result, bacteria are typically inactivated and broken down by the enzymes and toxins. lysosome . Expressusp genes in serum was upregulated, while expression of HSPs was mostly down-regulated. In particular, usp13 expression was 62.2-fold compared to control. The complement system in serum lyses and opsonizes bacteria for phagocytosis. The complement system of fish can also inactivate bacterial exotoxins [E. ictaluri is resistant to serum [E. ictaluri, sialic acid-induced suppression of ACP activation was thought to be the mechanism for E. ictaluri resistance to normal catfish serum [E. ictaluri’s ability to survive in serum, we were not expecting a high expression of stress response genes in normal serum. Only usp13 showed significant up-regulation, suggesting that USP13 might be associated with serum tolerance in E. ictaluri.Expression of xotoxins . In fishxotoxins . E. ictato serum . In E. ish serum . Besidessh serum . BecauseE. ictaluri indicated a similar infection pattern shown in our previous studies [E. ictaluri. Later, photon intensity increases faster due to rapid colonization of catfish by the pathogen. This rapid increase may indicate overwhelming of catfish immune system by the pathogen, which results in death of catfish in a couple of days. Therefore, 30 h post-infection seems a suitable time point for tissue collection because E. ictaluri is in a rapid increase stage while catfish immune responses are not overwhelmed completely.Imaging of live catfish injected with bioluminescent  studies , 36. Biousp05, usp08, usp09, and usp11 were highly expressed under in vitro stress conditions, usp04-08, usp10, usp13, grpE, and relA were upregulated in the host. Overall, usp05, usp07, usp13, and grpE may be critical for E. ictaluri to cope with several different stressors. Previously, our group identified a transposon insertion mutant of usp05 that showed attenuated virulence in catfish and provided protection against ESC. The usp05 gene is known as uspA, and it is an important regulator of survival and virulence in many pathogens [E. coli uspA mutant caused a survival defect under a variety of growth-arrested conditions, whereas overexpression induces growth in the growth-arrested state. Our data suggest that usp05 could be an important virulence gene in E. ictaluri.Whereas athogens . An E. c+ transporter system in E. coli, and KdpD/E control the expression of the kdpABC operon, which is one of two-component sensor-regulator [Staphylococcus aureus [Salmonella enterica serovar Typhimurium kdpD mutant is attenuated in host and macrophage survival experiment. Also, it is promoting resistance to osmotic, oxidative, and antimicrobial stresses [usp07 showed very high expression, especially when E. ictaluri is in the catfish head kidney and spleen, indicating that it may involve in E. ictaluri survival in the host.Kdp is a Kegulator . USP07 iegulator . We incls aureus . KdpD fus aureus . Salmonestresses . KdpD isstresses . In our E. ictaluri, CpxP showed very high expression in all stress conditions except oxidative stress. CpxP is an important regulator of cell membrane stress in bacteria during host infection, and it might promote the virulence and survival mechanisms related to distinct salt and acidic conditions.USP13 was described as a universal stress protein and CpxP like protein in NCBI. USP13, extracytoplasmic adaptor protein (CpxP), is placed in the inner membrane with histidine kinase CpxA and CpxR, a response regulator , 48. CpxE. ictaluri. Protein-protein interactions play critical roles in protein function, and in a large scale protein-protein interaction study in E. coli showed that a USPA has 83 target proteins, which mostly overlap with DnaK, DnaJ, and GroEL, also interactions with GroEL and DnaK (HSP70) [E. tarda [E. tarda with different degrees of virulence, authors showed that GroEL was highly expressed in pathogenic strain compared to nonpathogenic strain [groEL and groES were high in the oxidative stress, and dnaK was highly upregulated. Therefore, highly expressed heat shock proteins in oxidative stress should further investigated to explain the folding and unfolding process in oxidative stress of E. ictaluri. ClpB plays an important role in protein control to protein disaggregation in collaboration with the DnaK, DnaJ, and GrpE [E. tarda, protein expression of ClpB was higher in virulent strain than non-virulent [clpB was high in oxidative stress like dnaK, groEL, and groES. ClpB is part of a stress-induced system involved in the recovery of the cell from damage in cooperation with DnaJ, DnaK, and GrpE. The grpE was also shown similar high expression in oxidative stress and the internal organs. Further, it has very high expression in low pH. Thus, grpE may be an important heat shock protein to cope with stress in E. ictaluri.Other seven stress proteins were chosen by a possible regulation of USPs or interaction with USPs in  (HSP70) . GroEL a (HSP70) . The fun (HSP70) . DnaK/J  (HSP70) . GroEL wE. tarda . In anotc strain . Our resand GrpE , and it and GrpE , 62. In virulent . In our relA) was increased when E. ictaluri was in kidney and spleen. Similarly, serum affects the expression of relA. Therefore, we think that relA could be essential for E. ictaluri adaptation in the host. Further studies should be conducted in relA to better understand its role in E. ictaluri virulence and the regulation of USPs.The stringent response (SR) coordinates adaptations to nutritional starvation and various stress conditions , 64. SR usp genes (usp 05-07-13) were highly expressed in many stress conditions, and five heat shock proteins were highly expressed in oxidative stress , and grpE was highly expressed in spleen, kidney, and low pH. Finally, relA was highly expressed in kidney and spleen. As a result, we expect these findings will help us understand the role of stress proteins in E. ictaluri virulence. Furthermore, essential USPs and stress regulators in E. ictaluri can be potential targets for live vaccine development against ESC.In this study, we identified that three