Acta Herpetologica 14(2): 109-115, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7748 Occurrence of Batrachochytrium dendrobatidis in the Tensift region, with comments on its spreading in Morocco Redouane Ait El Cadi1, El-Mustapha Laghzaoui1, Angelica Crottini2, Tahar Slimani1, Jaime Bosch3,4, El Hassan EL Mouden1,* 1 Laboratory of Biodiversity and Ecosystem Dynamic, Faculty of Sciences, Semlalia, Cadi Ayyad University, Marrakech, Morocco. *Cor- responding author. Email: elmouden@uca.ac.ma 2 CIBIO Centro de Investigação em Biodiversidade e Recursos Genéticos, Vairão, Portugal 3 Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain 4 UMIB Research Unit of Biodiversity (CSIC, UO, PA), Universidad de Oviedo, Campus de Mieres, Spain Submitted on: 2019, 27th February; revised on: 2019, 4th September; accepted on: 2019, 17th September Editor: Adriana Bellati Abstract. The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a generalist pathogen that affects many amphib- ian species and is responsible of chytridiomycosis onset, considered as the main causes of species extinctions and populations declines worldwide. The chytrid fungal pathogen has been first described in North Africa in 2011. The present work reported the first survey on Bd prevalence and intensity in the Tensift region of Morocco. The survey has been conducted on 11 different localities by collecting skin swabs and tissue samples of 97 individuals. Using a quan- titative Polymerase Chain Reaction (qPCR) protocol, low-intensity of Bd infection has been detected in the area of study. In fact, the chytrid fungal pathogen has been identified in 10 individuals distributed in six of the 11 sites inves- tigated, placing the 95% confidence interval for overall prevalence at 5.5-19.6%. The survey confirmed the occurrence of Bd at both high and low altitude localities, on four species out of seven known to inhabit the region and added two additional species (Pelophylax saharicus and Sclerophrys mauritanica) to the list of Bd susceptible amphibians in Morocco. The present records extended Bd distribution more than 400 km in the South of Morocco, indicating that the chytrid fungal pathogen is more widespread in the country than previously thought. Keywords. Batrachochytrium dendrobatidis, amphibians, Tensift, prevalence, intensity. INTRODUCTION Habitat degradation and overexploitation are the main large-scale factors causing loss of biodiversity worldwide (Hoffmann et al., 2010). While these two fac- tors are to blame for the rapid declines of many verte- brate species, amphibians continue to decline also in undisturbed habitats, due essentially to chytridiomyco- sis, an emerging infectious skin disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) (Berger, 1998; Longcore and Pessier, 1999; Bosch and Martínez-solano, 2006; Lips, 2016). Bd is considered as the first wildlife fungal pathogen to have caused wide- spread species extinctions (Skerratt et al., 2007), and has been implicated in rapid population declines and extinction of several amphibians (Retallick et al., 2004; Schloegel et al., 2006; Skerratt et al., 2007; Kolby and Daszak, 2016; Lips, 2018). It has been detected in about 48% of tested amphibian species (Olson et al., 2013), and is recently known to be rapidly expanding its glob- al range of distribution (Fisher et al., 2009; O’Hanlon et al., 2018). In North Africa, Bd has been detected for the first time in Morocco at three (out of 51) tested locali- ties, and was found in only three species (Discoglos- 110 Ait El Cadi Redouane et alii sus scovazzi, Hyla meridionalis and Pelobates varaldii), with a total prevalence of 6% (El Mouden et al., 2011). The sites where Bd has been detected are in the north- ern part of the country (Tingitana peninsula and its sur- roundings) near South of Spain. El Mouden et al. (2011) explained this presence through a possible arrival from Spain where chytrid fungus is largely distributed, and it’s responsible for severe decline of amphibian popula- tions in mountain areas (Bosch et al., 2001; Bosch et al., 2007; Walker et al., 2010; Lips, 2016). These findings suggest further investigations in other areas were needed to collect more information on the potential extent of the spread of this fungal pathogen in Morocco, and to elucidate its potential source. Morocco is home to many endangered amphibian populations that are already threatened by habitat alteration and destruction, pollu- tion, and climatic changes (Fahd et al., 2015; Ben Hass- ine and Nouira, 2012; Escoriza and Ben Hassine, 2017). The aim of this study is to evaluate the chytrid fungal presence and prevalence using qPCR test for amphib- ians naturally occurred in Tensift region, an area located around 400 km South from the localities where Bd was detected for the first time in 2011. This region is char- acterized by a high diversity of habitats from wetlands (High Atlas Mountains) to arid environments (Jbilets), with seven amphibians species known to inhabit the area, two of which are Moroccan endemics (Bons and Geniez, 1996; Beukema et al., 2013). MATERIALS AND METHODS Surveys were conducted between October 2013 and Octo- ber 2017 in the Tensift region (central Morocco). The study area covers about 20,000 km2, surrounded by the southern crest of the High Atlas Mountains (with an altitude up to 4000 m a.s.l.) and the northern Jbilet hills (separated by the Haouz plain), while the coastline of Essaouira extends in the East (Fig. 1). The climate of the region is characterized by strong annual variability, with mean temperatures ranging between a maxi- mum of 37.7 °C and a minimum of 4.9 °C. The rainfall is gen- erally irregular, with occasional prolonged droughts. The mean annual rainfall varies from 800 mm in the mountain to 190 mm in the plain, with significant snowfall between December and March at high elevation (up to 2000 m a.s.l.) (Alaoui Haroni et al., 2009). The semi-arid environment dominates throughout the region, while the sub-humid zones appear at high altitude (up to 1500 m a.s.l.). The Tensift region is characterized by a complex landscape and topography, including escarpments, floodplains, and a great variety of aquatic ecosystems (rivers, permanent ponds and a large network of Mediterranean tempo- rary ponds). This diversity of water ecosystems provides a wide availability of breeding sites for seven native amphibian species: Hyla meridionalis Boettger, 1874, Bufotes boulengeri (Lataste, 1879), Sclerophrys mauritanica (Schlegel, 1841), Bufo spinosus Daudin, 1803 (Near threatened in Morocco), Barbarophryne brongersmai (Hoogmoed, 1972) (endemic to Morocco and Alge- ria), Pelophylax saharicus (Boulenger, 1913) (endemic to North Africa) and Discoglossus scovazzii Camerano, 1878 (endemic to Morocco). Amphibians were searched opportunistically in 11 sites (Table 1; Fig. 1), during the rainy season (when detectabil- ity of amphibians is higher), during nocturnal and occasional diurnal surveys. We extensively searched in all water bod- ies available in the area. After capture, animals were swabbed (using Medical Wire Sterile Dryswab™ - MW100) and released immediately after sampling to the place of capture. To prevent the transmission of diseases by animals, each individual was handled with disposable gloves. In total, 86 individuals were swabbed. Swabs were then air-dried and stored at cool tem- perature (4 °C) until processing. Tissues samples have been collected from 11 specimens that were found dead in two sites Fig. 1. Map of the Northern Morocco showing the result of Batra- chochytrium dendrobatidis (Bd) sampling sites realized during the present study (circles); dark circles indicate populations with Bd- positive samples and open circles indicate populations with Bd- negative samples. Within Bd positive sites, pie charts indicate the proportion of infected individuals in black and uninfected individu- als in white. Squares indicate infected populations as reported by El Mouden et al. (2011). 111Survey of Batrachochytrium dendrobatidis in Morocco (Dam-Oukaimden and Dam d’Ouled Abbas). These 11 tissue samples were preserved in ethanol (Brem et al., 2007; Hyatt et al., 2007). In total, 97 amphibian specimens were sampled belonging to four families and six species. Of these, 56 indi- viduals were sampled in the high Atlas Mountains, 16 in the arid area of Jbilets, 23 in the El Gantour region and two in the Haouz plain (Table 1; Fig. 1). DNA was extracted from both swabs and tissue samples using PrepMan Ultra reagent and extractions were diluted 1:10 before real-time PCR amplification, performed in dupli- cate with a CFX96 thermocycler (Bio-Rad), following Boyle et al. (2004). Each 96-well assay plate included samples, a nega- tive control and standards of 100, 10, 1, and 0.1 Bd zoospore genome equivalents in duplicate. Samples were considered positives when both replicates were ≥ 0.1 and the amplification curves had the typical sigmoidal shape. When only one replicate from any sample amplified, we ran this sample a third time. If the third amplification did not result in an amplification profile, we considered sample as negative for infection. Samples that showed signs of inhibition (non-sigmoidal amplification) were further diluted to 1:100 and re-analyzed. If signs of inhibition remained, the samples were excluded. RESULTS The numbers of screened and testing positive indi- viduals from each population and sampling event, along with geographic information on the sampling site and year of capture are reported in Table 1. The results show that 10 out of 97 screened individuals were Bd infected (10.3%), corresponding to a 95% confidence interval of overall Bd prevalence of 5.5-19.6%. Across the four spe- cies that tested positive, the infection prevalence was 11.9% (95% CI: 5.5-19.6%). This study confirmed Bd occurrence in both the northern and southern parts of the Tensift watershed at elevations between 468 and 2625 m a.s.l (Fig. 1). In the study area, Bd was detected in six out of 11 investigated sites (Table 1), with prevalence values rang- ing between 0 and 25%, and lower GE (genomic equiv- alent) values that varied between 0.3 and 26.8. These results indicate a significant presence of Bd in the Ten- sift region, with low infection prevalence and intensi- ties across all sites that tested positive for Bd. However, Table 1. Surveyed localities of the Tensift region (Morocco) with date and approximate coordinates and altitude (meters a.s.l.) of each site. Species names are abbreviated as follows: Sm, Sclerophrys mauritanica; Bb, Bufotes boulengeri; Br, Barbarophryne brongersmai; Ds, Discoglos- sus scovazzi; Hm, Hyla meridionalis; Ps, Pelophylax saharicus. Life history stage (LHS): Ad, adult; Juv, juvenile. P/S: number of individuals testing positive / Number of analyzed individuals. Bd load in genomic equivalents of zoospores. (*) Indicate individual found dead in the field and tested positive for Bd. Date Locality Coordinates Altitude Species LHS P/S Bd load Oct 2013 Sidi Bouathman 31.9 -7.92 ~ 517 Ps Ad 0/1 Nov 2013 Ijoukak N’fis 31.059 -8.164 ~ 1042 Ps Juv 0/1 Ad 1/3 26.8 Dec 2013 Sidi Bouathman 31.9 -7.92 ~ 517 Ps Ad 0/3 Dec 2013 Dam d’Ouled Abbas 31.966 -8.447 ~ 468 Sm* Ad 1/7 0.3 Dec 2013 Dam-Oukaimeden 31.208 -7.851 ~ 2623 Ps Ad 0/7 Dec 2013 Tighedouine 1 31.4 -7.532 ~ 1145 Ps Ad 0/6 Avr 2014 Tighedouine 2 31.423 -7.524 ~ 1066 Ps Ad 1/4 0.3 Hm Ad 1/7 0.4 Avr 2014 Ijoukak Tizgui 30.971 -8.125 ~ 1142 Ds Ad 0/2 Bb Ad 0/1 Ps Ad 0/5 Dec 2014 El Gantour 1 32.189 -8.335 ~ 402 Ps Ad 0/5 Dec 2014 El Gantour 2 32.187 -8.329 ~ 398 Ds Ad 0/2 Ps Juv 0/1 Ps Ad 1/8 1.3 Jan 2015 Jaidate 31.87 -7.79 ~ 623 Bb Ad 0/9 Br Ad 0/3 Ps Ad 0/2 Avr 2015 Tighedouine 2 31.423 -7.524 ~ 1066 Ps Ad 1/1 1.7 Jun 2016 Tifergine-Oukaimeden 31.207 -7.84 ~ 2565 Ds Ad 1/13 1.3 Oct 2017 Dam-Oukaimeden 31.208 -7.851 ~ 2623 Ps* Ad 3/6 0.6, 3.0, 0.3 112 Ait El Cadi Redouane et alii in October 2017, 50% prevalence was recorded in Dam- Oukaimden, with three testing positive samples out of the six screened individuals, which represents the maxi- mum prevalence obtained in this study. It seems that Bd prevalence can vary among spe- cies and elevations (Table 2). Consequently, 95% confi- dence intervals for Bd prevalence for individuals grouped according to these two parameters were determined. All confidence intervals were overlapping. Thus, no signifi- cant taxonomic and altitudinal difference in Bd preva- lence has been detected given our sample sizes. Howev- er, the obtained results tend towards a more significant prevalence at higher elevations (14.3% vs 4.9%). Likewise, species infection rate varied between 0 and 14.3%. The higher percentage was observed in three species. Bd was found in seven out of 53 P. saharicus across five sampling sites. In the Dam-Oukaimden site, where a large num- bers of dead P. saharicus were found in October 2017, we detected Bd on three out of six tested individuals (95% CI: 11.8-88.2%), which represents the highest preva- lence recorded by species and by site during this study. For both H. meridionalis (Tighedouine region; April 2014) and S. mauritanica (Culinary dam of Ouled Abbas; December 2013), Bd has been detected in one out of seven individuals, corresponding to a confidence interval for the two species of 0.40-57.9%. During our investiga- tion at Ouled Abbas dam, numerous dead specimens of S. mauritanica were observed in the water. Seven tissue samples were collected and screened. The results showed that one sample tested positive for Bd. DISCUSSION In our study area, four out of six amphibian species have been tested as Bd positive, although in comparison with other regions in Marocco, they have lower infection rate. Lower GE values are probably the result of unfa- vorable conditions, such as changes in the abiotic envi- ronment (Ron, 2005; Thorpe et al., 2018). It has been reported that Bd is temperature sensitive, with optimal growth ranging between 17 and 25 °C (Piotrowski et al., 2004; Pounds et al., 2006), with higher temperature (more than 30 °C) reported as unfavorable for its devel- opment (Watve, 2013). Previous studies have shown that lower temperature regime resulted in extended zoospore longevity and in such conditions zoospores numbers in water bodies could be expected to be greater than in warmer water (Voyles et al., 2012; Thorpe et al., 2018). The higher temperatures (more than 30 °C) recorded in the study area expected to have a negative impact on the development of this fungal pathogen. Additionally, opti- mum rainfall for Bd development has been reported to range between 1500 and 2500 mm/year (Thorpe et al., 2018), which are much higher values of the one observed in the Tensift region. Only, Oukaimden approach these rainfall values, but in general remains below to optimum parameters for the development of Bd. In Morocco, Bd surveys were previously conducted only in the northern Tingitana area. El Mouden et al. (2011) reported Bd occurrence in three sampling sites. Through this study, Bd occurrence has been reported in six additional Moroccan localities. Considering all these data, 14.5% of the screened Moroccan localities tested positive to Bd. The finding that Bd was recorded in two separated areas suggested a possible wider distribution across the country. Similarly, Bd was detected in different sites ranging from 400 to over 2600 m a.s.l, indicating a probable wide distribution also along the altitudinal gra- dient. Sample size is still limited and should be extended to have a more robust overview of the pattern of Bd pres- ence across Morocco. The report on the occurrence of Bd in the Tingitana peninsula (North of Morocco) was potentially explained by the extensive commercial trade of products and ani- mals across the straits of Gibraltar with a possible intro- duction of Bd from Spain (El Mouden et al., 2011). This hypothesis is in agreement with the explanations of O’Hanlon et al. (2018) who found Bd spreading out of Asia and dated its emergence on the early 20th century, coinciding with the international expansion of commer- cial trade in amphibians for exotic pet, medical, and food purposes. The presence of Bd in the Tensift region can be explained by a dissemination process from the North Table 2. Bd prevalence with 95% Clopper-Pearson binomial confi- dence intervals for individuals grouped by species and elevation. Group sample size Bd prevalence (%) 95% CI Family/Species Ranidae Pelophylax saharicus 53 13.2 5.6 - 25.8% Alytidae Discoglossus scovazzi 17 5.9 0.1 - 28.7% Bufonidae Bufotes boulengeri 10 0 0 - 30.8 % Sclerophrys mauritanica 7 14.3 0.4 - 57.9% Barbarophryne brongersmai 3 0 0 - 70.8% Hylidae Hyla meridionalis 7 14.3 0.4 - 57.9% Elevation (for all species) Less than 700 m 41 4.9 0.6 - 16.2% More than 700 m 56 14.3 6.1 - 25.4% 113Survey of Batrachochytrium dendrobatidis in Morocco of the country to the South, but also by a possible inde- pendent episode of introduction through other commer- cial routes, such as airports and harbors. The spreading of Bd through the country can be carried out by potential natural vectors, including waterfowl on their feathers or feet (Johnson and Speare, 2005; Garmyn et al., 2012; Bur- rowes and De La Riva, 2017), water (Johnson and Speare, 2003), and non-susceptible to chytridiomycosis amphib- ians acting as carrier (Kolby et al., 2015). In Morocco, as in most regions around the world, the detected Bd belongs to the Global Panzootic Lineage (J. Bosch, unpublished data). This is a highly virulent and highly transmissible chytrid fungus, which is currently infecting more that 700 amphibian species worldwide (Olson and Ronnenberg, 2014; Lips, 2016). We detect- ed a mass mortality of amphibians in two localities (S. mauritanica in Dam d’Ouled Abbas and P. saharicus in Dam-Oukaimden) and some of the sampled individuals tested positive for Bd infection. However, in the absence of a detailed study, we have no evidence of Bd-associated amphibian mortality especially because mortality of indi- viduals generally occurred with higher infection rates than those found in the present study. Other biotic and abiotic factors can be the cause of the witnessed mass mortality (e.g., Croteau et al., 2008; Hayes et al., 2010; Relyea et al., 2012; Whittaker et al., 2013; Budzik et al., 2014; De Wijer et al., 2018). However, it is worth noting that the two new species found infected by Bd (P. saha- ricus and S. mauritanica) have a wide distribution and abundance in the southern Mediterranean region (Bons and Geniez, 1996; Schleich et al., 1996; Mateo et al., 2013), which can make Bd dissemination faster. A systematic survey to determine the impact of Bd occurrence in Morocco is crucial to better characterize its impact on the amphibian’s population dynamic. In the mean time, other Bd positive sites located in Tens- fit region have been recorded and helped extending the known Bd occurrence area in Morocco, both at the high and the low altitudes (486-2625 m a.s.l.). In addition, having detected Bd at multiple sites and in two new amphibian species contributes to the growing knowledge on the global pattern of Bd distribution in Morocco and North Africa. 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Acta Herpetologica Vol. 14, n. 2 - December 2019 Firenze University Press Podarcis siculus latastei (Bedriaga, 1879) of the Western Pontine Islands (Italy) raised to the species rank, and a brief taxonomic overview of Podarcis lizards Gabriele Senczuk1,2,*, Riccardo Castiglia2, Wolfgang Böhme3, Claudia Corti1 Substrate type has a limited impact on the sprint performance of a Mediterranean lizard Pantelis Savvides1,*, Eleni Georgiou1, Panayiotis Pafilis2,3, Spyros Sfenthourakis1 Coping with aliens: how a native gecko manages to persist on Mediterranean islands despite the Black rat? Michel-Jean Delaugerre1,*, Roberto Sacchi2, Marta Biaggini3, Pietro Lo Cascio4, Ridha Ouni5, Claudia Corti 3 PIT-Tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian Trogonophis wiegmanni Pablo Recio, Gonzalo Rodríguez-Ruiz, Jesús Ortega, José Martín* Occurrence of Batrachochytrium dendrobatidis in the Tensift region, with comments on its spreading in Morocco Ait El Cadi Redouane1, Laghzaoui El-Mustapha1, Angelica Crottini2, Slimani Tahar1, Bosch Jaime3,4, EL Mouden El Hassan1,* Hematological parameters of the Bolson tortoise Gopherus flavomarginatus in Mexico Cristina García-De la Peña1,*, Roger Iván Rodríguez-Vivas2, Jorge A. Zegbe-Domínguez3, Luis Manuel Valenzuela-Núñez1, César A. Meza Herrera4, Quetzaly Siller-Rodríguez1, Verónica Ávila-Rodríguez1 Ontogenetic and interspecific variation in skull morphology of two closely related species of toad, Bufo bufo and B. spinosus (Anura: Bufonidae) Giovanni Sanna Visible Implant Alphanumeric (VIA) as a marking method in the lesser snouted treefrog Scinax nasicus Andrea Caballero-Gini1,2,3,*, Diego Bueno Villafañe2,3, Lía Romero2, Marcela Ferreira2,3, Lucas Cañete4, Rafaela Laino2, Karim Musalem2,5 Morphological variation of the newly confirmed population of the Javelin sand boa, Eryx jaculus (Linnaeus, 1758) (Serpentes, Erycidae) in Sicily, Italy Francesco P. Faraone1,*, Salvatore Russotto2, Salvatore A. Barra3, Roberto Chiara3, Gabriele Giacalone4, Mario Lo Valvo3 Variability in the dorsal pattern of the Sardinian grass snake (Natrix natrix cetti) with notes on its ecology Enrico Lunghi1,2,3,4,*, Simone Giachello5, Manuela Mulargia6, Pier Paolo Dore7, Roberto Cogoni8, Claudia Corti1 Estimating abundance of the Stripeless tree-frog Hyla meridionalis by means of replicated call counts Federico Crovetto, Sebastiano Salvidio, Andrea Costa* AT-rich microsatellite loci development for Fejervarya multistriata by Illumina HiSeq sequencing Yan-Mei Wang, Jing-Yi Chen, Guo-Hua Ding*, Zhi-Hua Lin