Page 1 of 13 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Subject Editor: Kaya Klop Toker Editor-in-Chief: Ilse Storch Accepted 16 June 2024 doi: 10.1002/wlb3.01302 00 1–13 2024: e01302 WILDLIFE BIOLOGY Wildlife Biology www.wildlifebiology.org © 2024 The Author(s). Wildlife Biology published by John Wiley & Sons Ltd on behalf of Nordic Society Oikos Many populations of species belonging to the order Crocodilia are threatened due to illegal trafficking, indiscriminate hunting, and habitat loss and degradation affecting crocodilian health and parasitic load. Although several studies have revealed that croc- odiles, caimans, and alligators are frequently infected by Hepatozoon spp., the results from studies exploring the costs of these apicomplexan parasites on the health of their reptilian hosts are still scarce and with inconclusive results. Here, we molecularly assessed the prevalence and genetic diversity of Hepatozoon spp. to explore their pos- sible influence on body condition in captive individuals of two species of Neotropical crocodilians with conservation threats, the spectacled caiman Caiman crocodilus and the American crocodile Crocodylus acutus. Fourteen percent of spectacled caimans were infected by H. caimani, whereas no American crocodiles showed infection. The preva- lence of Hepatozoon in spectacled alligators varied along age, where subadult individu- als were the most frequently parasitized. Surprisingly, the body condition of infected individuals was significantly higher than body condition of uninfected spectacled cai- mans, which suggests greater negative effects of the infection in individuals of poor quality. Also, the body condition of subadult individuals was significantly higher than body condition of juveniles of both alligator species, likely reflecting differences in the occupancy of habitats with higher resource abundance, or variations in the nutritional Host species and age-specific variation on Hepatozoon prevalence and its effect on body condition in two Neotropical crocodiles Alfonso Marzal ✉1,2, Wendy Flores-Saavedra 1,3,4, Sergio Magallanes 1,5,6, Jaime Muriel 1,7, Jefferson Lezama-Briceño 8, Luis Alberto García-Ayachi 9, Esteban Fong 10, Carlos Mora-Rubio 1, Carlos Mendoza11, Blanca Saldaña11, Alazne Díez-Fernández 1, José Martin 12, Carlos Marcial Perea-Sicchar 13 and Manuel González-Blázquez 1,12 1Universidad de Extremadura, Facultad de Biología, Departamento de Anatomía, Biología Celular y Zoología, Badajoz, Spain 2Grupo de Investigaciones en Fauna Silvestre, Universidad Nacional de San Martín, Tarapoto, Peru 3Unidad de Sanidad Animal, Universidad Nacional Agraria la Molina, Lima, Peru 4Facultad de Ciencias Veterinarias y Biológicas, Universidad Científica del Sur, Lima, Peru 5Estación Biológica de Doñana (EBD), CSIC, Departamento de Biología de la Conservación y Cambio Global, Seville, Spain 6CIBER de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain 7Department of Biology, University of Turku, Turku, Finland 8Escuela de Ciencias Veterinarias, Facultad de Ciencias Agropecuarias, Universidad Alas Peruanas, Pachacamac, Lima, Peru 9Instituto Peruano de Herpetología (IPH), Augusto Salazar Bondy, Surco, Lima, Peru 10EverGreen Institute - San Rafael, Distrito de Indiana, Loreto, Peru 11Laboratorio de Análisis Clínico Moraleslab SAC, Morales, San Martín, Peru 12Departamento de Ecología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal, Madrid, Spain 13Centro de Rescate Amazónico (CREA), Carretera Iquitos-Nauta, Iquitos, Peru Correspondence: A. Marzal (amarzal@unex.es) Research article 13 Page 2 of 13 values of the diet between these age classes. These outcomes provide valuable information on disease ecology for developing conservation strategies and the management conservation of wildlife populations of these species. Keywords: blood parasites, body condition, Caiman crocodilus, Crocodilus acutus, Hepatozoon caimani, wildlife conservation. Introduction Haemogregarines are apicomplexan blood parasites that commonly infect all groups of vertebrates. These intracellular parasites show an obligatory heteroxenous life cycle, where asexual reproduction occurs in the vertebrate host, while sexual multiplication occurs in the hematophagous inver- tebrate vector (Telford 2009). They are the most prevalent parasites in reptiles (Smith 1996). Currently, four genera of Haemogregarines are known to parasite reptiles: Karyolysus, Hemolivia, Haemogregarina, and Hepatozoon (Smith 1996, Telford 2009). Among them, the Hepatozoon species are the most common and more widely distributed Haemogregarines in reptiles (Telford 1984, 2009). However, despite the high occurrence and diversity of these parasites among the four major groups of Reptilia (Amphisbaenia, Chelonia, Crocodilia, Serpentes), a large number of new blood apicom- plexan species is yet to be discovered in this group of verte- brates (Duszynski 2021). Moreover, the pathogenicity of infection by these parasites is unclear, and the results of studies analyzing their poten- tial negative effects on their reptilian hosts have revealed mixed results. On one hand, several studies have shown that Hepatozoon infections can be harmful for their reptilian hosts. For example, Knotkova  et  al. (2005) reported anae- mia, low hemoglobin, basophilia, eosinophilia, heterophilia, and azurophilia in Malaysian giant turtles Orlitia borneensis infected with intraerythrocytic haemogregarines. In addition, Miyamoto and Mello (2007) analyzed whether the infection by Hepatozoon spp. affected the incidence of DNA fragmen- tation and cell death in red blood cells from the rattlesnake Crotalus durissus terrificus, showing that the parasite infection was associated with an accelerated destruction of erythrocytes in the reptile host, which may provoke anaemia in these indi- viduals. Also, Wozniak et al. (1996) experimentally infected three lizard species Sceloporus undulatus, Eumeces obsoletus, and Sceloporus poinsetti with Hepatozoon mocassini from a cotton-mouth moccasin Agkistrodon piscivorus leucostoma, showing severe lethargy, anorexia, leukocytosis, and multifo- cal random hepatocellular necrosis in all three lizard species. On the other hand, other studies have failed to report adverse effects associated with Hepatozoon infection in rep- tiles. For example, Brown  et  al. (2006) did not find any negative effect on host fitness (body condition, growth rate, feeding rate, antipredator behaviour, locomotor performance, reproductive status, reproductive output, and recapture rate) in keelback snakes Tropidonophis mairii heavily infected with haemogregarine blood parasites. Similarly, Damas- Moreira  et  al. (2014) estimated the effect of Hepatozoon infection on the flight-initiation distance from a simulated predator in the Andalusian wall lizard Podarcis vaucheri. They showed that the escape distance was not associated with Hepatozoon parasitemia, hence suggesting a limited impact of the parasite infection on their hosts. Also, Marzal  et  al. (2017) assessed the effects of Hepatozoon infection on the Spanish terrapins Mauremys leprosa and found no differences in body size and body condition among infected and unin- fected individuals. Moreover, the effects of Haemogregarines on body condition can vary enormously between related spe- cies of lizards, even between species within the same genus. For example, Amo  et  al. (2004) showed that haemogrega- rine parasitemia had a negative effect on the body condition during the reproductive season in the Iberian rock lizard Lacerta monticola. However, haemogregarine infection was not related to body condition in the ocellated lizard L. lepida (Amo et al. 2005). The order Crocodilia (suborder Eusuchia) consists of 27 species of alligators, caimans, crocodiles, and gharials with an almost cosmopolitan distribution, which inhabit tropical and subtropical areas (Stevenson 2019). Beyond the potential threat posed by infectious diseases (see above), many popu- lations of these reptiles are threatened due to the alteration and destruction of their habitat, indiscriminate hunting, and illegal trafficking of species (Ponce-Campos et al. 2012). In fact, seven crocodilian species are critically endangered, four are vulnerable, and the remaining twelve are considered as low risk, although updated information on these species is needed (IUCN 2021). In a recent review, it was shown that 63% of crocodilian species are frequently infected by apicom- plexan blood parasites, being most diverse and abundant in the genus Hepatozoon (Duszynski et al. 2020). In fact, several species of Hepatozoon have been found infecting crocodilians worldwide, such as H. serrei in the smooth-fronted caiman Paleosuchus trigonatus (Smith 1996), H. crocodilinorum in the American alligator Alligator mississippiensis (Davis  et  al. 2011), H. pettiti in the Nile crocodile Crocodylus niloticus (Leslie  et  al. 2011) and in the marine crocodile C. porosus (Duszynski  et  al. 2020), H. sheppardi in the Nile crocodile (Santos Dias 1952), and H. caimani in the overo caiman Caiman latirostris (Smith 1996), the alligator C. yacare (Viana et al. 2010a, Soares et al. 2017a), and the spectacled caiman C. crocodilus (Lainson et al. 2003, Soares et al. 2017b). However, despite the increasing number of descriptive stud- ies of the prevalence, morphology, diversity, and phyloge- netic relationships of the parasites of the genus Hepatozoon in crocodiles, caimans, and alligators (Bouer  et  al. 2017, Duszynski et al. 2020), the potential negative effects of these parasites on the biological fitness of this group of reptiles have been poorly analyzed. The few studies carried out have found hardly any detrimental effects on their hosts, suggesting that the parasites of the genus Hepatozoon are of low or null patho- genicity in crocodilians (Lovely et al. 2007, Leslie et al. 2011, 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 3 of 13 Soares et al. 2017a). Considering the threats to the survival of many populations of crocodilians, the study of any potential source of mortality or stress, such as these blood parasites, should be a priority for its conservation. Captive breeding facilities, rehabilitation centers, zoos, and other facilities for captive animal management play a key role in species conservation. For example, they provide oppor- tunities for captive breeding programs, and supplementing natural populations (Ziegler et al. 2022). Also, the study of parasites in amphibians and reptiles held or bred in captiv- ity is essential in release programs because they can influence the translocation success or transmit new parasites to wildlife populations (Germano and Bishop 2009, Beckmann  et  al. 2022). For example, recent investigations in captive Orinoco crocodiles Crocodylus intermedius, the most threatened croco- dilian of South America (Balaguera-Reina et al. 2018a), have enabled the deciphering of novel and baseline data for hae- matological and biochemistry values with special importance for the clinical diagnosis and wildlife disease ecology (Barajas- Valero et al. 2021), as well as the evaluation of the genetic characterization determining the viability of reintroduction programs from ex situ populations of this critically endan- gered species (Saldarriaga-Gomez et al. 2023). In this study, we analyzed the relationship between infec- tion by Hepatozoon spp. and body condition, a proxy of health state, in individuals of various age classes of two spe- cies of Neotropical crocodilians with conservation threats, the spectacled caiman Caiman crocodilus and the American crocodile Crocodylus acutus. We used molecular methods for the detection of Hepatozoon parasites in blood samples from individuals of these two species maintained in several fauna recovery centers of the Peruvian Amazon and one aquaculture center from northeast Peru. We predicted that, if Hepatozoon infection were harmful, this would be reflected in a lower body condition of parasitized individuals. Material and methods Study species The spectacled caiman Caiman crocodilus, also known in Peru as common caiman or white caiman, is the alligator with the widest distribution in the Neotropics, inhabiting differ- ent types of freshwater courses and swamps throughout the American intertropical belt, from Mexico to the center of Brazil (Balaguera-Reina and Velasco 2019). This species has been exploited for local food and for commercial purposes, both for national and international trade (Caldwel 2017). For this reason, different specific protocols for its conserva- tion have recently started in several South American coun- tries (Velasco and Balaguera-Reina 2018). The American crocodile Crocodylus acutus, also named the Tumbes crocodile in Peru, has a wide distribution on both coasts of the Neotropical area of the American con- tinent, from Sinaloa (Mexico) to Peru on the Pacific coast, and from the extreme south of Florida to the plains of the Orinoco (northeast Venezuela) in the Atlantic, including Cuba, Jamaica, and Haiti (Ernst et al. 1999, Thorbjarnarson 2010). It is a large crocodile (up to 6 m in length), being the second largest crocodile in America. Despite its wide distribution, it is an endangered species listed as vulnerable by the International Union for the Conservation of Nature (IUCN) due to the population decline caused by habitat destruction and loss, and poaching (Ponce-Campos  et  al. 2012). Importantly, this species is listed as critically endan- gered in Peru because of loss of habitat and massive hunting (SERFOR 2018). Study sites Thirty-five spectacled caimans were seized from illegal wildlife trade by Autoridad Regional Ambiental (ARA) and Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) of the San Martín and Loreto regions (Peru) and were transported and housed in three rescue centers after seizure. Twenty-eight C. crocodilus (21 juveniles and seven neonates) were collected in the Amazon Rescue Center (CREA) (3°53′0ʺS, 73°21′4ʺW) in the Loreto region, located at km 4.5 of the road Iquitos– Nauta, southwest of the city of Iquitos. Juvenile C. crocodilus were housed in individual 3 × 5 m pools of 1.5 m height and a water depth of 0.5 m with several artificial islands. The smaller specimens (neonates) were housed in similar enclosures in groups of 3–5 individuals. In addition, five C. crocodilus (four subadults and one adult) were collected in Selva Viva, located 11.6 km from the center of Tarapoto (6°26′54ʺS, 76°28′19ʺW). Individuals were housed in a large, enclosed area with a surface area of 40 × 100 m and a large central lagoon 1 m deep that occupied 80% of the area. Also, two C. crocodilus (one juvenile and one subadult) were collected in Chullachaqui, located 15.5 km distance from the center of the city of Tarapoto on the road to Yurimaguas (6°27′49ʺS, 76°19′3ʺW). In this center, caimans were indi- vidually housed in 15 m2 wooden pens with a central pool of 0.5 m depth to allow immersion. All spectacled caimans had been housed in these facilities between 1–4 months before we carried out blood sampling. American crocodiles (n = 68: one neonate, 19 juveniles, 34 subadults, and 14 adults) were sampled at Tuna Carranza aquaculture center (3°30ʹ36ʺS, 80°23ʹ46ʺW), an experimen- tal C. acutus hatchery for repopulation purposes belonging to the National Fisheries Development Fund (FONDEPES), located in the district and province of Tumbes in the Tumbes Region. All American crocodiles had been raised in captiv- ity and kept in large enclosures with water pools, according to the recommendations from Ziegler (2001) for enclosure sizes for captive crocodiles. The specimens were separated by age in 15 m × 12 m enclosures, and each pen had a 1 m deep central pool to allow bathing. Neonates and juveniles were housed in two enclosures with ten individuals in each, whereas subadults and adults of C. acutus were housed sepa- rately with a maximum of 11 individuals in each enclosure. All animals were sampled in June 2017, apart from the C. crocodilus at CREA, which were sampled again in June 2018. 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 4 of 13 Handling procedure, sex determination, age, body size, and body condition The crocodiles were restrained and immobilized manually with the help of protocol knots (Rueda-Almonacid  et  al. 2007, Orjuela 2009). Once a crocodile was immobilized out of the water, a wet towel was placed over its eyes and it was held firmly to restrict its movement and minimize stress (Choperena and Ceballos 2016, Dodd 2016). Crocodiles were individually identified with marks on the edge of the dorsal tail plates following the codification proposed by Bolton (1989) (García-Grajales et al. 2011). Sex was determined by exploring the cloaca using a rhino- scope to separate the edges of the cloaca and verify the genita- lia by digital palpation (Webb et al. 1984, Ziegler and Olbort 2007). Age was estimated according to the body size based on previous studies. We established four categories in the case of C. crocodilus: neonate (< 50 cm), juvenile (51–120 cm), subadult (121–180 cm), and adult (> 181 cm) (Ayarzagüena 1983, Guerra-Cárdenas  et  al. 2020). We used five age cat- egories for C. acutus: neonate (< 60 cm), juvenile I (61–120 cm), juvenile II (121–180 cm), subadult (181–240 cm), and adult (> 241 cm) (Lander 2003, Pérez and Escobero-Galván 2007, Seijas 2011). Individual body condition was estimated by calculation of the Fulton body condition factor or Fulton index (K) (Ricker 1975). An assumption of this index is that the weight of a crocodile is proportional to the cube of its length. This index has been widely used to describe the physical state of numerous vertebrates (Hayes and Shonkwiler 2001) includ- ing several species of crocodiles, such as the swamp croco- dile (Cedeño-Vázquez  et  al. 2011, Mazzotti  et  al. 2012), American alligator (Zweig et al. 2014), and spectacled caiman (Grant et al. 2013, Barão-Nóbrega et al. 2018). We measured the total length (TL) of individuals (from the tip of the snout to the end of the tail) using a measuring tape (accuracy ± 1 mm), and the body mass (W) with a digital scale (preci- sion of ± 10 g). Fulton index (K) was calculated following the formula: K = W/TL3 × 104 (Barão-Nóbrega et al. 2018, Briggs-González et al. 2021). Blood sampling Blood samples were collected within a maximum period of 5 min after capture to minimize the stress response caused by handling and blood sampling (Guillette et al. 1997), immedi- ately after physical immobilization and before the measuring and weighting procedures described above. Before sampling, we cleaned the puncture skin area with 96% ethanol-soaked cotton wool. Approximately 1 ml of blood was withdrawn from the coccygeal vein, located in the caudal sinus of the tail base, or from post-occipital spinal venous sinus (Jacobson 1993). We used 1- or 5-ml plastic syringes with heparin- ized disposable needles with a variable gauge, from 21G to 27G, depending on the size of the individual (Zhang 2011). After the extraction, the puncture area was pressed with cot- ton with ethanol until checking that there was no bleeding. Blood collected was stored in a vial with 500 µl of SET buffer (0.015 M NaCl, 0.05 M Tris, 0.001 M EDTA, pH = 8.0) (Sambrook et al. 1989) at room temperature for subsequent molecular analyses. All individuals were returned to their enclosures in perfect condition in less than 10 min and con- trolled by the staff of each center in the following days to ensure that they were in a good state of health. Molecular detection of blood parasite infections Genomic DNA was extracted from blood samples using the GeneJET™ Genomic DNA Purification Kit (Thermo Scientific Inc., ref. K0722) according to the manufacturer instructions. Diluted genomic DNA (25 ng µl−1) was used as template to amplify the 18S RNA gene of Hepatozoon spp. as described previously (Harris  et al. 2011). A nested PCR reaction with primers HEMO1 and HEMO2 (Perkins and Keller 2001) for the firsrt PCR, and then primers HepF300 and HepR900 (Ujvari et al. 2004) for the second one, were used for Hepatozoon amplification. The amplification was evaluated by running 2.5 μl of the final PCR product on a 2% agarose gel. To verify that the PCR process was correct, we added one negative control every eight samples and two positive controls every 42 samples. Parasites detected by a positive amplification were sequenced using the procedures described by Bensch et al. (2000). Amplified fragments were sequenced from 5’end with HepF300 Hepatozoon spp. The obtained sequences of 600 bp were edited, aligned, and compared in a sequence identity matrix using the program MEGA (Stecher et al. 2020). A BLAST search was performed to match the product sequence to that of a known haemo- parasite species. Statistical analysis The normality of the data distribution of all continuous vari- ables used in statistic models was assessed with Shapiro–Wilk tests. We used a chi-square test to test for differences in the prevalence of parasites between crocodile species. Also, in the case of C. crocodilus (the only species in which parasites were found; see Results), a logistic regression analysis was used to explore whether sex, age, Fulton index (K factor), year of study, location of sampling, and the interaction between age and Fulton index (K factor) influenced Hepatozoon infection probability (infected/uninfected). A backward stepwise pro- cedure was used to eliminate all non-significant terms (p > 0.05) from our starting maximal model. We used a stepwise backward procedure in a generalized linear model (GLM) with a normal distribution and an identity link function to investigate the effect of age, year of study, location of sam- pling, and Hepatozoon infection (uninfected or infected) on the body condition of C. crocodilus. We also used a stepwise backward procedure in a GLM with a normal distribu- tion and an identity link function to explore the effect of age and sex on the body condition of C. acutus. There were no significant correlations among the predictor variables in any model (Pearson correlation test: all p-values > 0.05). 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 5 of 13 On each GLM, the stepwise procedures started with a full model including all predictors. At each step, the least signifi- cant predictor (based on the highest p-value) was removed from the model until a final model was reached. Only the final models were presented. The adequacy of the models was assessed by examining their explained variances. All analyses were performed using PASW Statistics 22 statistical package for Windows. Results Differences in Hepatozoon infection between crocodile species We found significant differences in the prevalence of infec- tion of Hepatozoon between the two species (chi square test: χ2 = 10.210; df = 1; p = 0.001). Five out of the 35 C. croco- drilus were infected with Hepatozoon (prevalence of infec- tion = 14.28%), while we did not find any C. acutus infected by Hepatozoon. Differences in Hepatozoon infection in relation to age of individuals, sex, body condition, year of study, and sampling location We analysed Hepatozoon infection in relation to sex, age, body condition (Fulton index), year of study, and location of sampling in C. crocodilus. The prevalence of Hepatozoon varied significantly among locations of sampling (Table 1). Two and three crocodiles were infected in CREA (prevalence of infection = 7.1%; n = 28) and Selva Viva (prevalence of infection = 60%; n = 5), respectively, while no C. crocodilus was infected by Hepatozoon in Chullachaqui. We also found differences in the prevalence of Hepatozoon in relation to age of individuals (Table 1). Specifically, three out of five sub- adults were infected (60%), as were two out of 22 juveniles (9.1%). In contrast, no neonate or adult were infected with Hepatozoon. Moreover, the body condition of crocodiles also explained variation in the probability of infection with Hepatozoon (Table 1). Remarkably, infected individuals had significantly higher body condition (n = 5; mean body condi- tion ± SD = 4.527 ± 0.807) than uninfected ones (n = 30, mean body condition ± SD = 3.323 ± 0.732) (Fig. 1). Finally, neither sex nor the interaction between body condi- tion (Fulton index) and age significantly influenced the prob- ability of infection with Hepatozoon in C. crocodilus (Table 1). Genetic diversity of Hepatozoon lineages We compared the parasite sequences obtained from our samples with homologous sequences of other parasite hap- lotypes obtained from GenBank. The obtained 18S rRNA Hepatozoon sequences showed 100% identity with Hepatozoon caimani isolates previously deposited in GenBank from C. crocodilus from Brazil (MF435048, MF322528, MF322539, KJ413132) and Colombia (MW246123). Body condition versus Hepatozoon infection, age, and years of study We analysed the effects of the infection by Hepatozoon in C. crocodilus. GLM analyses showed that Hepatozoon infec- tion and age were related with body condition of crocodiles (Table 2). Specifically, infected individuals had significantly higher body condition than uninfected crocodiles (Fig. 1). Also, body condition from neonates was significantly lower (n = 7, mean body condition ± SD = 2.64 ± 0.45) than the body condition from juveniles (n = 22, mean body condition ± SD = 3.48 ± 0.65), subadults (n = 5, mean body condition ± SD = 4.76 ± 0.56), and adult crocodiles (n = 1, mean body condition = 3.49) (Bonferroni post hoc test: p < 0.010 in all cases). Similarly, the body condition of subadult crocodiles was higher than body condition from neonate, juvenile, and adult crocodiles (Bonferroni post hoc test: p < 0.010 in all cases) (Fig. 2). Neither the year of study nor location of sampling significantly affected body condition in C. crocodilus (Table 2). Finally, we found that body condition varied with the age of individuals in C. acutus (Table 3). Specifically, body condi- tion of subadult crocodiles was significantly higher than body condition of individuals from other age classes (subadults: n = 34, mean body condition ± SD = 5.38 ± 1.25; juveniles I: n = 1, mean body condition = 3.78; juveniles II: n = 19, mean body condition ± SD = 3.88 ± 0.63; adults: n = 14, mean body condition ± SD = 4.46 ± 0.74) (Bonferroni post hoc test: all p < 0.010) (Fig. 3). Male and female American crocodiles did not differ in body condition (Table 2) (males: n = 53, mean body condition ± SD = 4.72 ± 1.14; females: n = 12, mean body condition ± SD = 4.85 ± 1.41). Discussion Investigations of wildlife maintained in captivity are of spe- cial relevance for endangered or poorly studied species. For Table 1. Results from the stepwise backwards logistic regression to predict the probability of Hepatozoon infection in spectacled caimans Caiman crocodilus. Sex, age, Fulton index (K factor), year of study, location of sampling, and the interaction between age and Fulton index (K factor) were included in the analysis as predictor variables. Only independent variables selected by the consensus model are listed. Sample size was 35 individuals. Significant factors are highlighted in bold. Independent variable B SE Wald DF p-value Exp (B) Age 0.774 0.401 3.720 3 0.047 2.152 Location of sampling 1.126 0.552 4.157 2 0.041 3.082 Fulton index (K factor) 0.399 0.147 7.333 1 0.007 1.491 Constant 1.632 8.362 0.038 1 0.845 5.116 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 6 of 13 example, they have provided valuable information about the life cycle of the parasite (Valkiūnas 2004, Jacobson 2007), the negative impact of the parasite infection on the health of their hosts (Valkiūnas 2004, Jacobson 2007, Santiago- Alarcon and Marzal 2020), and the susceptibility of the host to become infected by a wide range of parasites (Mewius et al. 2021). Furthermore, wildlife birds and reptiles kept in cap- tivity are an excellent model for parasite research and pro- vide valuable results for wildlife management conservation. In this sense, the development of many anti-parasite drugs and chemotherapies has been possible thanks to the use of individuals from zoos and rehabilitation centers (Kent 2004, Palinauskas et al. 2020). Here, we explored the relationships between body condition and Hepatozoon infection in two species of Neotropical crocodiles. The main results were: 1) about 15% of C. crocodilus were infected with H. caimani, whereas no C. acutus were infected with Hepatozoon; 2) age, body condition, and sampling location explained variations in the probability of Hepatozoon infection in C. crocodilus; 3) C. crocodilus infected with Hepatozoon showed higher body condition than uninfected individuals; and 4) body condition varied significantly among age categories in both crocodile species, where subadult individuals showed higher body condition. Prevalence and genetic diversity of Hepatozoon parasites Our results showed that about 15% of C. crocodilus were infected with Hepatozoon. This prevalence of infection is lower than that found in other studies in free-living popula- tions of this crocodile species in Brazil (80%) (Soares  et  al. 2017a) and Peru (90%) (Erazo and Capunay Becerra 2020), but similar (10%) to that found in captive populations in Peru (Rojas et al. 2011). Variations in the prevalence of blood parasite infection between populations are usually attributed to environmental differences that impair the abundance of vectors that transmit these parasites (Martínez-Abraín  et  al. 2004, Martín et al. 2016, Marzal et al. 2017). Alternatively, these differences may also be due to proper handling and low stress conditions that minimize vector presence and immu- nosuppression of captive animals, respectively (Derraik 2005, Manolis and Webb 2016). Although it is assumed that gluco- corticoids levels, the steroid hormones released as a response to stressful challenges, are normally higher in captive animals than in their wild counterparts (Karaer  et  al. 2023), it has Figure 1. Barplots with error bars showing Fulton index (K factor) as an estimate of body condition for infected (n = 5) and uninfected (n = 30) spectacled caimans Caiman crocodilus. Error bars plots show means ± 95% of confidence interval. Table 2. Results from the generalized linear model (GLM) explaining variation in Fulton index (K factor) as an estimate of body condition for spectacled caimans Caiman crocodilus. A backward stepwise procedure was used in the analysis with age, prevalence of Hepatozoon infection, year of study, and location of sampling pre- dictor variables. Only independent variables selected by the back- ward stepwise procedure are listed. Sample size was 35 individuals. Significant factors are highlighted in bold. Independent variable Estimate SE t p-value Age 0.535 0.170 3.151 0.001 Hepatozoon infection 0.653 0.278 2.354 0.028 Figure 2. Barplots with error bars showing Fulton index (K factor) as an estimate of body condition for neonate (n = 7), juvenile (n = 22), subadult (n = 5) and adult (n = 1) spectacled caimans Caiman crocodilus. Error bars plots show means ± 95% of confi- dence interval. Table 3. Results from the generalized linear model (GLM) explaining variation in Fulton index (K factor) as an estimate of body condition for American crocodiles Crocodylus acutus. A backward stepwise procedure was used in the analysis with age and sex of individuals as predictor variables. Only independent variables selected by the backward stepwise procedure are listed. Sample size was 68 indi- viduals. Significant factors are highlighted in bold. Independent variable Estimate SE t p-value Age 0.396 0.194 2.045 0.048 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 7 of 13 recently been reported that captive and wild American alliga- tors show similar values of measurements to assess stress levels (i.e. corticosterone levels and heterophil/lymphocyte ratios) (Merchant et al. 2023), indicating that crocodiles maintained in good captive environments may experience similar chronic stress to wild animals. In addition, variations in blood parasite infections between populations are usually attributed to envi- ronmental differences in the abundance of vectors or density of vertebrate hosts (Grenfell and Dobson 1995). Unfortunately, given that the prepatent period of H. caimani in C. crocodilus ranges between 52 and 82 days (the time elapsed between the transmission of the parasite to the crocodile and detection of gametocytes in its blood) (Lainson et al. 2003), and because spectacled caimans were housed in these facilities between 1 and 4 months before we took blood samples, we could not assess whether Hepatozoon infection was acquired in their nat- ural environment or in captivity after their seizure. In contrast to the observed prevalence of Hepatozoon in C. crocodilus, no C. acutus were infected with this apicom- plexan parasite. These interspecific differences in prevalence of infection may be due to habitat variations in the presence or abundance of transmitting vectors of Hepatozoon. Culex spp. mosquitoes have been identified as natural vectors of this parasite (Lainson et al. 2003, Viana et al. 2010b). However, culicids are not typically eaten by crocodilians, and hence it has been suggested that the main transmission route of H. cai- mani in South America is through the ingestion of paratenic hosts (insectivorous vertebrates such as anurans) (Viana et al. 2012, Pereira et al. 2014, Matta et al. 2022). Caiman crocodilus live in fresh waters in Amazonia (Balaguera-Reina and Velasco 2019), which are also common habitats for Culex mosquitoes (Rueda 2008). In contrast, C. acutus usually inhabits perma- nent bodies of water such as saltwater sections of rivers, coastal lagoons, and estuaries (Thorbjarnarson 2010). The waters of these habitats have higher salinity than those of rivers and other freshwater areas, which decreases the survival of the lar- vae of Culex mosquitoes, among others (Kengne et al. 2019). Therefore, the lower abundance of Hepatozoon transmitting vectors in coastal and brackish areas would decrease the abun- dance of infection in potential paratenic hosts such as anurans and fish, which would explain the absence of infection with H. caimani in C. acutus. Nonetheless, we cannot discard the possibility that the sampled individuals were not infected sim- ply because the conditions of the breeding center were better than in the natural environment. Contrary to our results, a previous study based only on microscopic observations reported a prevalence of 16% of Hepatozoon spp. in C. acutus individuals from the same center where we did our study (zoocriadero Tuna Carranza) (Enríquez  et  al. 2014). To the best of our knowledge, there is no other microscopic or molecular study showing Hepatozoon infection in this species. However, it has been pointed out that the validity of microscopic identification of the genus Hepatozoon in many published studies is ques- tionable because the morphological characteristics of game- tocytes are similar to those of other apicomplexans, such as Haemogregarina (Soares et al. 2017a). Thus, it may be possi- ble that the parasites observed by Enríquez et al. (2014) could be Haemogregarina parasites rather than Hepatozoon, because there has been confusion in assigning parasites to one or another genus in crocodilians for many years (Lainson et al. 2003, Tellez 2014, Duszynski et al. 2020). In support of this idea, C. acutus has been described as being infected by leeches, the main vector of Haemogregarina spp. (García-Grajales and Buenrostro-Silva 2011). To date, 17 out of 27 species of Crocodilia have been examined in search for apicomplexan parasites (Duszynski 2021). Hepatozoon caimani was the only parasite species found in our study. This parasite species is widely distrib- uted among crocodilians in South America, as it has been found infecting C. latirostris (Smith 1996, Duszynski et al. 2020), C. crocodilus (Lainson  et  al. 2003, Duszynski  et  al. 2020), C. yacare (Lainson  et  al. 2003, Viana  et  al. 2010a, Duszynski  et  al. 2020), and the dwarf caiman Paleosuchus palpebrosus (Clemente et al. 2023). Although few species of this parasite genus have been described in South American crocodilians, it is predicted that more than 30 species of api- complexan parasites remain to be discovered from species of Crocodilia (Duszynski 2021). Therefore, further studies exploring the prevalence and diversity of blood parasites in this group of endangered vertebrates are required. Variations in Hepatozoon infection in relation to age, sex, and localities of sampling Subadult C. crocodilus showed a higher prevalence of infec- tion than juveniles. These results agree with those found by Viana  et  al. (2010a) in C. yacare in Pantanal (Brazil), sug- gesting that caimans are first infected at juvenile age, when their diet changes from eating invertebrates to predating on anurans and fish (Viana  et  al. 2010a), which are paratenic hosts of H. caimani (Viana et al. 2012, Pereira et al. 2014). Figure 3. Barplots with error bars showing Fulton index (K factor) as an estimate of body condition for juvenile I (n = 1), juvenile II (n = 19), subadult (n = 34), and adult (n = 14) American crocodiles Crocodylus acutus. Error bars plots show means ± 95% of confi- dence interval. 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 8 of 13 Likewise, given the viability and persistence of H. caimani in their hosts (Davis and Johnston 2000), the higher preva- lence of parasites in subadult individuals than in juveniles can be explained by the succession and accumulation of infec- tions throughout the life of the alligator (Viana et al. 2010a), as has been observed in haemoparasite infections in lizards (Amo et al. 2005), snakes (Santos et al. 2005), and other spe- cies of crocodiles (Leslie et al. 2011). We reported differences in prevalence of infection among the fauna recovery centers where C. crocodilus were housed and sampled. These variations are likely to have been caused by differences in the density of vertebrate hosts, as well as divergences in environmental and/or handling conditions potentially influencing the probability of Hepatozoon infec- tions (Grenfell and Dobson 1998, Derraik 2005, Manolis and Webb 2016). Moreover, we cannot exclude the possi- bility that dietary differences between captive centers might have influenced the observed difference in infection rates among these centers. We found no differences in the prevalence of infection between males and females. Although Leslie  et  al. (2011) reported a higher prevalence of infection in female Nile croc- odiles, most studies in crocodiles and alligators found similar probabilities of infection in both sexes (Viana et al. 2010a, Erazo and Capunay Becerra 2020). Body condition versus Hepatozoon infection The negative effects of Hepatozoon infection show great vari- ability among the hosts they infect. For example, in mam- mals, serious pathologies and even mortality associated with Hepatozoon infections have been described in coyotes Canis latrans (Kocan et al. 2000) and spotted hyenas Crocuta cro- cuta (East  et  al. 2008). In some reptile species it has been observed that Hepatozoon infections can cause anaemia and blood cell abnormalities, resulting in immunosuppression (Telford 1984). Furthermore, hepatocellular necrosis has also been observed in three species of lizards infected with Hepatozoon mocassini (Wozniak et al. 1996). Moreover, nega- tive effects on growth, body condition, reproductive success, and survival associated with infection by these blood para- sites have been described in the aquatic python Liasis fuscus (Madsen et al. 2005, Ujvari and Madsen 2006). Although many surveys have shown a wide variety of api- complexan parasites from alligators, caimans, and crocodiles, the low sample size of most of these studies prevented a com- plete taxonomic identification of these parasites, and failed to reveal pathogenic effects on their hosts (Duszynski et al. 2020). Despite the negative effects of Hepatozoon on their hosts presented above, the few studies carried out on croco- dilians have shown that many species can tolerate the pres- ence of infection and hardly suffer any pathogenic effects. For example, two studies in the Nile crocodile did not show sig- nificant differences in clinical hematological values between uninfected and H. pettiti-infected individuals (Lovely  et  al. 2007, Leslie et al. 2011). A greater length in infected adults than in uninfected individuals of the same age class has also been described in this species of crocodiles (Leslie et al. 2011). Furthermore, no histopathological changes were observed in the liver and lungs of C. yacare individuals infected by H. cai- mani (Soares et al. 2017a). Likewise, Viana et al. (2010a) did not find any negative effects of the infection with H. caimani on the body mass of C. yacare individuals. This absence of negative effects has led to the belief that Hepatozoon parasites are not pathogenic in their crocodil- ian hosts (Leslie  et  al. 2011). In our study we found that infected individuals presented higher body condition than the uninfected ones, which could be in line with previous stud- ies reporting an absence of negative effects of Hepatozoon in alligator and crocodiles. Alternatively, another possible expla- nation for these results would be a selective disappearance of low-quality individuals infected in the population, as has been suggested in birds (Marzal et al. 2016, Jiménez-Peñuela et al. 2019, Bichet et al. 2020), mammals (Lynsdale et al. 2020), and other reptile species (Madsen et al. 2005, Marzal et al. 2017). Following this idea, it has been shown in reptiles that indi- viduals with lower body condition presented a lower immune response (Ujvari and Madsen 2006), which can lead to worse defences against blood parasite infections (Schmid-Hempel 2021). If hosts with lower body condition suffer higher mor- tality when infected, only those individuals with good body condition could have good defences that would allow them to counteract the negative effects of the infection and survive, leading to a positive relationship between infection by para- sites and body condition (Budischak et al. 2018, Sánchez et al. 2018). In addition, Balaguera-Reina  et  al. (2023) recently assessed the relationship between body condition and the hae- matological and biochemical (blood) parameters in American alligators from Greater Everglades in Florida, showing that individuals in poorer body condition were likely dehydrated or had an inadequate diet, two of the main contributing fac- tors to musculoskeletal disorders that may increase mortality in crocodilians (Bolton 1989, Nevarez 2006). However, our data should be interpreted with caution because of the low number of infected C. crocodilus in our study (n = 5). Further studies examining haematological and biochemical parameters are needed to complement our findings and determine the patho- genicity of Hepatozoon infection in C. crocodilus. Body condition and age categories The body condition varied significantly among age catego- ries in both alligator species, where subadult C. crocodilus and C. acutus showed higher body condition than juvenile individuals. Similarly, Ojeda-Adame et  al. (2020) reported that adult and subadult C. acutus had higher values of body condition than juvenile individuals. Also, Mazzotti  et  al. (2012) showed that adult swamp crocodiles (Crocodylus moreletii) had higher body condition than younger croco- diles. These differences are likely because adult and subadult crocodiles generally occupy habitats with better and more abundant resources than juveniles, and/or have better access to food with higher nutritional content (Mazzotti  et  al. 2012). Alternatively, the higher body condition in adult and 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 9 of 13 subadult alligators can be explained by differences in diet between age groups, where the consumption of invertebrates (insects and crustaceans) decreases with age in favour of larger prey such as fish and mammals (Wallace and Leslie 2008, Adame  et  al. 2018, Balaguera-Reina  et  al. 2018b), whose protein contribution represents a greater nutritional value (Hernández et al. 2018). In conclusion, we have explored the relationships between Hepatozoon infection and body condition in specimens of two Neotropical alligator species. We found that about 15% of C. crocodilus were infected with Hepatoozon caimani, whereas no C. acutus showed infection. We also have shown a greater infection in subadult individuals in C. crocodilus, which sug- gests that the infection could be provoked by the ingestion of paratenic hosts due the change in diet composition from the juvenile age. This change in diet with greater nutritional con- tribution may also explain the higher body condition of sub- adult individuals in both species of crocodiles. Hepatozoon infection varied significantly among study locations in C. crocodilus, highlighting the importance of handling and feed- ing conditions in captive crocodiles. Finally, C. crocodilus infected with Hepatozoon had higher body condition than the non-infected ones, and hence the link between body condi- tion and Hepatozoon infection requires further investigations to be revealed. Despite our results showed limited negative impact of Hepatozoon infection on C. crocodilus, several studies have described anaemia, lethargy, decreased hemato- crit, and continuous weight loss that may progress to death in crocodiles harbouring Hepatozoon (Viana  et  al. 2010a, Rojas et al. 2011, Campbell 2015). Therefore, longitudinal and experimental studies providing more information on the dynamics of Hepatozoon infection and its effects on fitness of crocodilians would be of great importance for the conserva- tion–management actions of these endangered species. Acknowledgements – We are grateful to technical and human support provided by the Faculty of Bioscience Applied Techniques of SAIUEx (financed by UEX, Junta de Extremadura, MICINN, FEDER, and FSE). We are also grateful to Javier Velasquez y Juan Sánchez from CREA who collaborated during the fieldwork. Funding – This study was funded by line of action LA4 (R + D + I program in the Biodiversity Area financed with the funds of the FEDER Extremadura 2021–2027 Operational Program of the Recovery, Transformation and Resilience Plan). AD-F acknowledges support from the Margarita Salas University of Seville postdoctoral grants funded by the Spanish Ministry of Universities with European Union funds – NextGenerationEU. Permits – All samples were taken in accordance with national Peruvian law (200-2016-SERFOR/DGGSPFFS) and the animal protection laws of the EU (directive 2010/63/EU of the European Parliament). Methods were approved by the Research Ethics and Animal Welfare Committee on Animal Experimentation of the University of Extremadura (reference 101/2016). Author contributions Alfonso Marzal: Conceptualization (lead); Data cura- tion (equal); Formal analysis (equal); Funding acquisition (lead); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (lead); Writing – review and editing (equal). Wendy Flores-Saavedra: Data cura- tion (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Resources (equal); Supervision (equal); Validation (equal); Writing – review and editing (equal). Sergio Magallanes: Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Software (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). Jaime Muriel: Formal analysis (equal); Investigation (equal); Methodology (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review and editing (equal). Jefferson Lezama- Briceño: Data curation (equal); Investigation (equal); Methodology (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). Luis Alberto García-Ayachi: Conceptualization (equal); Data cura- tion (equal); Investigation (equal); Methodology (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). Esteban Fong: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Supervision (equal); Visualization (equal); Writing – review and editing (equal). Carlos Mora-Rubio: Formal analysis (equal); Investigation (equal); Methodology (equal); Software (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). Carlos Mendoza: Data curation (equal); Investigation (equal); Methodology (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). Blanca Saldaña: Data curation (equal); Investigation (equal); Methodology (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). Alazne Díez- Fernández: Formal analysis (equal); Investigation (equal); Methodology (equal); Software (equal); Validation (equal); Visualization (equal); Writing – review and editing (equal). José Martín: Conceptualization (equal); Formal analy- sis (equal); Investigation (equal); Methodology (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review and editing (equal). Carlos Marcial Perea-Sicchar: Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Validation (equal); Writing – review and editing (equal). Manuel González-Blázquez: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review and editing (equal). Transparent peer review The peer review history for this article is available at https:// www.webofscience.com/api/gateway/wos/peer-review/ wlb3.01302. 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 10 of 13 Data availability statement Data are available from the Dryad Digital Repository: https:// doi.org/10.5061/dryad.pc866t1x5 (Marzal et al. 2024). References Adame, M. F., Jardine, T. D., Fry, B., Valdez, D., Lindner, G., Nadji, J. and Bunn, S. E. 2018. Estuarine crocodiles in a trop- ical coastal floodplain obtain nutrition from terrestrial prey. – PLoS One 13: e0197159. Amo, L., López, P. and Martín, J. 2004. Prevalence and intensity of haemogregarinid blood parasites in a population of the Ibe- rian rock lizard, Lacerta monticola. – Parasitol. Res. 94: 290–293. Amo, L., Fargallo, J. A., Martínez-Padilla, J., Millán, J., López, P. and Martín, J. 2005. Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida. – Parasitol. Res. 96: 413–417. Ayarzagüena, J. 1983. Ecología del caimán de anteojos (Caiman crocodilus L.) en los llanos de Apure (Venezuela). – Doñana Acta Vertebr. 10: 45–117. Balaguera-Reina, S. A. and Velasco, A. 2019. Caiman crocodilus. The IUCN Red List of Threatened Species. – e.T46584A3009688, h t tps : / /do i .o rg /10 .2305/IUCN.UK.2019-1 .RLTS. T46584A3009688.en. Balaguera-Reina, S. A., Espinosa-Blanco, A., Antelo, R., Morales- Betancourt, M. and Seijas, A. 2018a. Crocodylus intermedius. The IUCN Red List of Threatened Species. – e.T5661A3044743, https://dx.doi.org/10.2305/IUCN.UK.2018-1.RLTS. T5661A181089024.en. Balaguera‐Reina, S. A., Venegas‐Anaya, M., Beltrán‐López, V., Cris- tancho, A. and Densmore III, L. D. 2018b. Food habits and ontogenetic dietary partitioning of American crocodiles in a tropical Pacific Island in Central America. – Ecosphere 9: e02393. Balaguera-Reina, S. A., Brandt, L. A., Hernandez, N. D., Mason, B. M., Smith, C. D. and Mazzotti, F. J. 2023. Body condition as a descriptor of American alligator (Alligator mississippiensis) health status in the Greater Everglades, Florida, United States. – PLoS One 18: e0295357. Barajas-Valero, S., Rodríguez-Almonacid, C., Rojas-Sereno, Z., Moreno-Torres, C. and Matta, N. E. 2021. Hematology, bio- chemistry reference intervals, and morphological description of peripheral blood cells for a captive population of Crocodylus intermedius in Colombia. – Front. Vet. Sci. 8: 694354. Barão-Nóbrega, J. A. L., Marioni, B., Botero-Arias, R., Nogueira, A. J. A., Lima, E. S., Magnusson, W. E., Da Silveira, R. and Marcon, J. L. 2018. The metabolic cost of nesting: body condi- tion and blood parameters of Sección II. Capítulo 3 Caiman crocodilus and Melanosuchus niger in Central Amazonia. – J. Comp. Physiol. B 188: 127–140. Beckmann, K. M., Cromie, R. L., Sainsbury, A. W., Hilton, G. M., Ewen, J. G., Soorae, P. S. and Kock, R. A. 2022. Wildlife health outcomes and opportunities in conservation translocations. – Ecol. Solut. Evid. 3: e12164. Bensch, S., Stjernman, M., Hasselquist, D., Östman, O., Hansson, B., Westerdahl, H. and Pinheiro, R. T. 2000. Host specificity in avian blood parasites: a study of Plasmodium and Haemopro- teus mitochondrial DNA amplified from birds. – Proc. R. Soc. B 267: 1583–1589. Bichet, C., Brischoux, F., Ribout, C., Parenteau, C., Meillère, A. and Angelier, F. 2020. Physiological and morphological corre- lates of blood parasite infection in urban and non-urban house sparrow populations. – PLoS One 15: e0237170. Bolton, M. 1989. The management of crocodiles in captivity. – Food and Agriculture Organization of the United Nations, ISBN 9251028753. Bouer, A., André, M. R., Gonçalves, L. R., Luzzi, M. C., Oliveira, J. P. D., Rodrigues, A. C., Varani, A. M., Miranda, V. F. O., Perles, L., Werther, K. and Machado, R. Z. 2017. Hepatozoon caimani in Caiman crocodilus yacare (Crocodylia, Alligatoridae) from north Pantanal, Brazil. – Rev. Bras. Parasitol. Vet. 26: 352–358. Briggs-Gonzalez, V. S., Basille, M., Cherkiss, M. S. and Mazzotti, F. J. 2021. American crocodiles (Crocodylus acutus) as restora- tion bioindicators in the Florida Everglades. – PLoS One 16: e0250510. Brown, G. P., Shilton, C. M. and Shine, R. 2006. Do parasites matter? Assessing the fitness consequences of haemogregarine infection in snakes. – Can. J. Zool. 84: 668–676. Budischak, S. A., O'Neal, D., Jolles, A. E. and Ezenwa, V. O. 2018. Differential host responses to parasitism shape divergent fitness costs of infection. – Funct. Ecol. 32: 324–333. Campbell, T. W. 2015. Hematologia dos répteis. – In: Thrall, M., Weiser, G., Allison, R. and Campbell, T. (eds), Hematologia e bioquímica clínica veterinária. – Editora Roca, pp. 239–250. Cedeño-Vázquez, J. R., González-Ávila, F. and Castro-Pérez, J. 2011. Condición corporal del cocodrilo de pantano (Crocodylus moreletii) en el Río Hondo, Quintana Roo, México. – Quehacer Cient. Chiapas 11: 19–26. Choperena, M. C. and Ceballos, C. P. 2016. Guía de manejo vet- erinario de fauna silvestre para las haciendas: Vegas de la Clara, La Candelaria y La Montaña de la Universidad de Antioquia. – Rev. Colomb. Ciencias Pecuarias 32: 312–314. https://doi. org/10.17533/udea.rccp.v32n4a08. Clemente, G. R. C., Gutierrez-Liberato, G. A., Anjos, C. C., Simões, P. I., Mudrek, J. R., Fecchio, A., Lima, J. H. A., Oliveira, P. M. A., Pinho, J. B., Mathias, B. S., Guimarães, L. O. and Kirchgatter, K. 2023. Occurrence of Hepatozoon in some rep- tiles from Brazilian biomes with molecular and morphological characterization of Hepatozoon caimani. – Diversity 15: 1192. Damas-Moreira, I., Harris, D. J., Rosado, D., Tavares, I., Maia, J., Salvi, D. and Perera, A. 2014. Consequences of haemogregarine infection on the escape distance in the lacertid lizard, Podarcis vaucheri. – Acta Herpetol. 9: 119–123. Davis, A. K., Horan III, R. V., Grosse, A. M., Harris, B. B., Metts, B. S., Scott, D. E. and Tuberville, T. D. 2011. Gender differ- ences in haemogregarine infections in American alligators (Alli- gator mississippiensis) at Savannah River, South Carolina, USA. – J. Wildl. Dis. 47: 1047–1049. Derraik, J. G. 2005. Recommendations for mosquito control in zoological parks to reduce disease transmission risk. – Weta 29: 16–20. Dodd, C. K. (ed.) 2016. Reptile ecology and conservation: a hand- book of techniques. – Oxford Univ. Press. Duszynski, D. W. 2021. Biodiversity of the coccidia (Apicomplexa: Conoidasida) in vertebrates: what we know, what we do not know, and what needs to be done. – Folia Parasitologica 68, https://doi.org/10.14411/fp.2021.001. Duszynski, D. W., McAllister, C. T. and Tellez, M. 2020. The coc- cidia (Apicomplexa) of the Archosauria (Crocodylia: Eusuchia) of the world. – J. Parasitol. 106: 90–122. East, M. L., Wibbelt, G., Lieckfeldt, D., Ludwig, A., Goller, K., Wilhelm, K., Schares, G., Thierer, D. and Hofer, H. 2008. A 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 11 of 13 Hepatozoon species genetically distinct from H. canis infecting spotted hyenas in the Serengeti ecosystem, Tanzania. – J. Wildl. Dis. 44: 45–52. Enríquez, C., Elías, R., Montes, D. and Saldarriaga, F. 2014. Pres- encia de hemoparásitos de los géneros Hepatozoon y Haemoag- regina en ejemplares de Cocodrilo Americano (Crocodylus acu- tus) mantenidos en cautiverio en Tumbes, Perú. – Zoológica 1: 43–49. Erazo, N. C. and Capuñay Becerra, C. 2020. Presencia de Hepato- zoon spp. en el caimán blanco (Caiman crocodilus) de vida libre en la zona del Bajo Madre de Dios, Perú. – Rev. Investig. Vet. Peru 31: e16106. Ernst, C. H., Ross, F. D. and Ross, C. A. 1999. Crocodylus acutus (Cuvier) American crocodile. – Cat. Am. Amphibians Reptiles (CAAR) 700: 1–17. García-Grajales, J. and Buenrostro-Silva, A. 2011. Infestación y distribución corporal de sanguijuelas en el cocodrilo americano (Crocodylus acutus Cuvier 1807) (Reptilia: Crocodylidae) del estero La Ventanilla, Oaxaca, México. – Acta Zoológica Mex. 27: 565–575. Germano, J. M. and Bishop, P. J. 2009. Suitability of amphibians and reptiles for translocation. – Conserv. Biol. 23: 7–15. Grant, P. B. C., Woudneh, M. B. and Ross, P. S. 2013. Pesticides in blood from spectacled caiman (Caiman crocodilus) down- stream of banana Sección II. Capítulo 3 plantations in Costa Rica. – Environ. Toxicol. Chem. 32: 2576–2583. Grenfell, B. T. and Dobson, A. P. (eds) 1995. Ecology of infectious diseases in natural populations, Vol. 7. – Cambridge Univ. Press. Guerra-Cárdenas, W., Martínez-García, S., Bermúdez, A. and Navas-S, G. R. 2020. Estado poblacional de Crocodylus acutus Cuvier y Caiman crocodilus fuscus (Cope) en Puerto Badel y Correa, Bolívar, Colombia. – Rev. Acad. Colomb. Cienc. Exac- tas Fis. Nat. 44: 182–193. Guillette, L. J., Crain, D. A., Rooney, A. A. and Woodward, A. R. 1997. Effect of acute stress on plasma concentrations of sex and stress hormones in juvenile alligators living in control and con- taminated lakes. – J. Herpetol. 31: 347–353. Harris, D. J., Maia, J. P. and Perera, A. 2011. Molecular charac- terization of Hepatozoon species in reptiles from the Seychelles. – J. Parasitol. 97: 106–110. Hayes, J. P. and Shonkwiler, J. S. 2001. Morphometric indicators of body condition: worthwhile or wishful thinking. – In: Speak- man, J. R. (ed.), Body composition analysis of animals: a hand- book of non-destructive methods. Cambridge Univ. Press, pp. 8–38. Hernandez, P., Soria, H., Farnes, O., Hernandez, H., Garcia, R., Casas, G., Montoya, C. and Vega, F. 2018. Contributions to the nutrition of the American crocodile Crocodylus acutus (Cuvier, 1807) in captivity. – Lat. Am. J. Aquat. Res. 46: 15–19. IUCN 2021. The IUCN Red List of threatened species. Version 2021–1. – https://www.iucnredlist.org. Jacobson, E. 1993. Blood collection techniques in reptiles: labora- tory investigations. – In: Fowler, M. E. (ed.), Zoo and wild animal medicine. Current therapy 3. WB Saunders, pp. 144–146. Jacobson, E. R. 2007. Infectious diseases and pathology of reptiles: color atlas and text. – CRC Press. Jiménez-Peñuela, J., Ferraguti, M., Martínez-de la Puente, J., Soriguer, R. and Figuerola, J. 2019. Urbanization and blood parasite infections affect the body condition of wild birds. – Sci. Total Environ. 651: 3015–3022. Karaer, M. C., Čebulj-Kadunc, N. and Snoj, T. 2023. Stress in wildlife: comparison of the stress response among domestic, captive, and free-ranging animals. – Front. Vet. Sci. 10: 1167016. Kengne, P., Charmantier, G., Blondeau‐Bidet, E., Costantini, C. and Ayala, D. 2019. Tolerance of disease‐vector mosquitoes to brackish water and their osmoregulatory ability. – Ecosphere 10: e02783. Kent, M. S. 2004. The use of chemotherapy in exotic animals. – Vet. Clin. North Am. Exot. Anim. Pract. 7: 807–820. Knotková, Z., Mazánek, S., Hovorka, M., Sloboda, M. and Knotek, Z. 2005. Haematology and plasma chemistry of Bornean river turtles suffering from shell necrosis and haemogregarine para- sites. – Vet. Med. 50: 421–426. Kocan, A. A., Cummings, C. A., Panciera, R. J., Mathew, J. S., Ewing, S. A. and Barker, R. W. 2000. Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma. – J. Wildl. Dis. 36: 149–153. Lainson, R., Paperna, I. and Naiff, R. D. 2003. Development of Hepatozoon caimani (Carini, 1909) Pess a, De Biasi & De Souza, 1972 in the C. crocodilus, the frog Rana catesbeiana and the mosquito Culex fatigans. – Mem. Inst. Oswaldo Cruz 98: 103–113. Lander, A. 2003. Seguimiento de la Población de Caimán de la Costa (Crocodylus acutus Cuvier, 1807) en la bahía de Turiamo. – Estado Aragua, Informe técnico, MARNR. Leslie, A. J., Lovely, C. J. and Pittman, J. M. 2011. A preliminary disease survey in the wild Nile crocodile (Crocodylus niloticus) population in the Okavango Delta, Botswana. – J. S. Afr. Vet. Assoc. 82: 155–159. Lovely, C. J., Pittman, J. M. and Leslie, A. J. 2007. Normal hae- matology and blood biochemistry of wild Nile crocodiles (Croc- odylus niloticus) in the Okavango Delta, Botswana. – J. S. Afr. Vet. Assoc. 78: 137–144. Lynsdale, C. L., Mon, N. O., Dos Santos, D. J. F., Aung, H. H., Nyein, U. K., Htut, W., Childs, D. and Lummaa, V. 2020. Demographic and reproductive associations with nematode infection in a long-lived mammal. – Sci. Rep. 10: 9214. Madsen, T., Ujvari, B. and Olsson, M. 2005. Old pythons stay fit; effects of haematozoan infections on life history traits of a large tropical predator. – Oecologia 142: 407–412. Manolis, S. C. and Webb, G. J. 2016. Best management practices for crocodilian farming. – IUCN-SSC Crocodile Specialist Group. Martin, J., Garrido, M., Ortega, J., Garcia-Roa, R., Ibanez, A. and Marzal, A. 2016. Absence of haemoparasite infection in the fossorial amphisbaenian Trogonophis wiegmanni. – Parasitology 4: 1–4. Martínez-Abraín, A., Esparza, B. and Oro, D. 2004. Lack of blood parasites in bird species: does absence of blood parasite vectors explain it all? – Ardeola 51: 225–232. Marzal, A., Balbontín, J., Reviriego, M., García‐Longoria, L., Relinque, C., Hermosell, I. G., Magallanes, S., López-Calde- rón, C., de Lope, F. and Møller, A. P. 2016. A longitudinal study of age‐related changes in Haemoproteus infection in a passerine bird. – Oikos 125: 1092–1099. Marzal, A., Ibáñez, A., González-Blázquez, M., López, P. and Mar- tín, J. 2017. Prevalence and genetic diversity of blood parasite mixed infections in Spanish terrapins, Mauremys leprosa. – Par- asitology 144: 1449–1457. Marzal, A., Flores-Saavedra, W., Magallanes, S., Muriel, J., Lezama- Briceño, J., García-Ayachi, L. A., Fong, E., Mora-Rubio, C., 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 12 of 13 Mendoza, C., Saldaña, B., Díez-Fernández, A., Martin, J., Perea-Sicchar, C. M. and González-Blázquez, M. 2024. Data from: Host species and age-specific variation on Hepatozoon prevalence and its effect on body condition in two Neotropical crocodiles. – Dryad Digital Repository, https://doi.org/10.5061/ dryad.pc866t1x5. Matta, N. E., Lotta-Arévalo, I. A., Gamboa-Suárez, B. A. and Ber- nal, S. I. 2022. Diptera-borne hemoparasites of herpetofauna: rediscovering its importance. – In: Gutiérrez-López, R., Logan, J. G. and Martínez-de la Puente, J. (eds), Ecology and control of vector-borne diseases. Wageningen Academic Publishers, pp. 39–79, https://doi.org/10.3920/978-90-8686-931-2. Mazzotti, F. J., Cherkiss, M. S., Brandt, L. A., Fujisaki, I., Hart, K., Jeffery, B., McMurry, S. T., Platt, S. G., Rainwater, T. R. and Vinci, J. 2012. Body condition of Morelet's crocodiles (Croco- dylus moreletii) from northern Belize. – J. Herpetol. 46: 356–362. Merchant, M., Joanen, T., Berkin, J. and Courville, C. 2023. A comparison of stress levels in farmed and wild American alliga- tors: an indicator of animal health and best farming practices. – J. Appl. Aquacult. 35: 687–702. Mewius, A., Lusa, E. R., Pertille, J. G., Reis, T. D. D., Pletsch, J. A., França, R. T. and Castro, L. L. D. 2021. Endoparasites in group of wild animals raised in captivity. – Pesq. Vet. Bras. 41: https://doi.org/10.1590/1678-5150-PVB-6758. Miyamoto, M. and Mello, M. L. S. 2007. Chromatin supraor- ganization, DNA fragmentation, and cell death in erythrocytes of the rattlesnake, Crotalus durissus terrificus (Serpentes, Viperi- dae), infected with the protozoan, Hepatozoon spp. (Apicompl- exa, Hepatozoidae). – Cell Biol. Int. 31: 494–499. Nevarez, J. 2006. Crocodilian differential diagnosis. Reptile medi- cine and surgery. – In: Mader, D. R. (ed.), Reptile medicine and surgery. Saunders Elsevier, pp. 705–714. https://doi. org/10.1016/B0-72-169327-X/50045-6. Ojeda-Adame, R. A., Hernández-Hurtado, H., Ramírez-Martinez, M. M. and Iñiguez-Davalos, L. I. 2020. A body condition score for crocodilians. – S. Am. J. Herpetol. 16: 10–15. Orjuela Acosta, D. 2009. Introducción a la medicina de fauna sil- vestre en Latinoamérica. –Serrano Editores. Palinauskas, V., Martínez de la Puente, J., Hernández-Soto, S. R. and Marzal, A. 2020. Experimental parasitology and ecoim- munology: concepts and opportunities in avian haemosporid- ian studies. – In: Santiago-Alarcón, D. and Marzal, A. (eds), Avian malaria and related parasites in the tropics: ecology, evo- lution and systematics. Springer, pp. 527–558. Pereira, G. R., Soares, P., Gomes, M. Q., Viana, L. A., Manso, P. P., Machado, M. P., Paiva, F. and Lourenço-de-Oliveira, R. 2014. Are fish paratenic natural hosts of the caiman haemopar- asite Hepatozoon caimani? – Parasitol. Res. 113: 39–45. Pérez, O. and Escobedo-Galván, A. H. 2007. Crecimiento en cau- tiverio de Crocodylus acutus (Cuvier, 1807) en Tumbes, Perú. – Rev. Peru. Biol. 14: 221–223. Perkins, S. L. and Keller, A. K. 2001. Phylogeny of nuclear small subunit rRNA genes of hemogregarines amplified with specific primers. – J. Parasitol. 87: 870–876. Ponce-Campos, P., Thorbjarnarson, J. and Velasco, A. 2012. Croco- dylus acutus. The IUCN Red List of threatened species e. T5659A3043244. – IUCN SSC Crocodile Specialist Group. Ricker, W. E. 1975. Computation and interpretation of biological statistics of fish populations. – Bull. Fish. Res. Bd. Can. 191: 1–382. Rojas, G., Alvis, R., Pino, J. and Shiga, B. 2011. Presencia de Hepa- tozoon caimani (Apicomplexa: Hepatozoidae) en el caimán blanco Caiman crocodilus (Linneus 1758) en un zoológico de Lima, Perú. – Rev. Investig. Vet. Peru 22: 56–60. Rueda, L. M. 2008. Global diversity of mosquitoes (Insecta: Dip- tera: Culicidae) in freshwater. – Hydrobiologia 595: 477–487. Rueda-Almonacid, J. V., Carr, J. L., Mittermeier, R. A., Rodríguez- Mahecha, J. V., Mast, R. B., Vogt, R. C. and Mittermeier, C. G. 2007. Las tortugas y los cocodrilianos de los países andinos del trópico. – S. Guías Trop. Campo 6: 412–423. Saldarriaga-Gómez, A. M., Ardila-Robayo, M. C., Medem, F. and Vargas-Ramírez, M. 2023. Hope is the last thing lost: Colom- bian captive-bred population of the critically endangered Ori- noco crocodile (Crocodylus intermedius) is a genetic reservoir that could help to save the species from extinction. – Nat. Con- serv. 53: 85–103. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular clon- ing: a laboratory manual. Plainview. – Cold Spring Harbor Laboratory Press. Sánchez, C. A., Becker, D. J., Teitelbaum, C. S., Barriga, P., Brown, L. M., Majewska, A. A., Hall, R. J. and Altizer, S. 2018. On the relationship between body condition and parasite infection in wildlife: a review and meta‐analysis. – Ecol. Lett. 21: 1869–1884. Santiago-Alarcon, D. and Marzal, A. 2020. Avian malaria and related parasites in the tropics. – Springer. Santos, M. M. D. V., O’Dwyer, L. H. and da Silva, R. J. 2005. Seasonal variation of Hepatozoon spp. (Apicomplexa, Hepato- zoidae) parasitemia from boa constrictor amarali (Serpentes, Boidae) and Hydrodynastes gigas (Serpentes, Colubridae). – Par- asitol. Res. 97: 94–97. Santos Dias, J. A. T. 1952. Acerca de uma nova espécie de Haemogregarina, parasita dos eritrocitos do Crocodilus niloticus em Moçambique: Haemogregarina sheppardi. – An. Inst. Med. Trop. 9: 181. Schmid-Hempel, P. 2021. Evolutionary parasitology: the integrated study of infections, immunology, ecology, and genetics. – Oxford Univ. Press. Seijas, A. E. 2011. Los crocodylia de Venezuela. Academia de cien- cias físicas, matemáticas y naturales. Ecología y conservación. – Colección Estudios y Divulgación Científica y Tecnológica. SERFOR 2018. Libro Rojo de la fauna silvestre Amenazada del Perú. Primera edición. – SERFOR (Servicio Nacional forestal y de fauna silvestre). Smith, T. G. 1996. The genus Hepatozoon (Apicomplexa: Adeleina). – J. Parasitol. 82: 565–585. Soares, H. S., Marcili, A., Barbieri, A. R., Minervino, A. H., Moreira, T. R., Gennari, S. M. and Labruna, M. B. 2017a. Novel piroplasmid and Hepatozoon organisms infecting the wildlife of two regions of the Brazilian Amazon. – Int. J. Para- sitol. Parasites Wildl. 6: 115–121. Soares, P., Borghesan, T. C., Tavares, L. E. R., Ferreira, V. L., Teix- eira, M. M. G. and Paiva, F. 2017b. Hepatozoon caimani Carini, 1909 (Adeleina: Hepatozoidae) in wild population of Caiman yacare Daudin, 1801 (Crocodylia: Alligatoridae), Pantanal, Bra- zil. – Parasitol. Res. 116: 1907–1916. Stecher, G., Tamura, K. and Kumar, S. 2020. Molecular evolution- ary genetics analysis (MEGA) for macOS. – Mol. Biol. Evol. 37: 1237–1239. Stevenson, C. 2019. Crocodiles of the world. – New Holland Pub- lishers. Telford, S. R. 1984. Haemoparasites of reptiles. – In: Hoff, G. L., Frye, F. L. and Jacobson, E. R. (eds), Diseases of amphibians and reptiles. Plenum Press, pp. 385–517. 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Page 13 of 13 Telford Jr, S. R. 2009. Hemoparasites of the Reptilia: color atlas and text. – CRC Press. Tellez, M. 2014. A checklist of host-parasite interactions of the order Crocodylia. Vol. 136. – Univ. of California Press. Thorbjarnarson, J. B. 2010. American crocodile Crocodylus acutus. – In: Manolis, S. C. and Stevenson, C. (eds), Crocodiles. Status survey and conservation action plan. IUCN-SSC Crocodile Specialist Group, pp. 46–53. Ujvari, B. and Madsen, T. 2006. Age, parasites, and condition affect humoral immune response in tropical pythons. – Behav. Ecol. 17: 20–24. Ujvari, B., Madsen, T. and Olsson, M. 2004. High prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia. – J. Para- sitol. 90: 670–672. Valkiūnas, G. 2004. Avian malaria parasites and other haemos- poridia. – CRC Press. Velasco, A. and Balaguera-Reina, S. A. 2018. Spectacled caiman (Caiman crocodilus). – In: Manolis, S. C. and Stevenson, C. (eds), Crocodiles. Status survey and conservation action plan. IUCN-SSC Crocodile Specialist Group, pp. 10–15. Viana, L. A., Paiva, F., Coutinho, M. E. and Lourenço-de-Oliveira, R. 2010a. Hepatozoon caimani (Apicomplexa: Hepatozoidae) in wild caiman, Caiman yacare, from the Pantanal region, Brazil. – J. Parasitol. 96: 83–88. Viana, L. A., Soares, P., Paiva, F. and Lourenço-De-Oliveira, R. 2010b. Caiman-biting mosquitoes and the natural vectors of Hepatozoon caimani in Brazil. – J. Med. Entomol. 47: 670–676. Viana, L. A., Soares, P., Silva, J. E., Paiva, F. and Coutinho, M. E. 2012. Anurans as paratenic hosts in the transmission of Hepa- tozoon caimani to caimans Caiman yacare and Caiman latiro- stris. – Parasitol. Res. 110: 883–886. Wallace, K. M. and Leslie, A. J. 2008. Diet of the Nile crocodile (Crocodylus niloticus) in the Okavango Delta, Botswana. – J. Herpetol. 42: 361–368. Webb, G. J., Manolis, S. C. and Sack, G. C. 1984. Cloacal sexual of hatchling crocodiles. – Wildl. Res. 11: 201–202. Wozniak, E. J., Kazacos, K. R., Telford Jr, S. R. and McLaughlin, G. L. 1996. Characterization of the clinical and anatomical pathological changes associated with Hepatozoon mocassini infections in unnatural reptilian hosts. – Int. J. Parasitol. 26: 141–146. Zhang, Z. Q. 2011. Animal biodiversity: an outline of higher-level classification and survey of taxonomic richness. – Magnolia Press. Ziegler, T. and Olbort, S. 2007. Genital structures and sex identi- fication in crocodiles. – Crocodile Spec. Group Newsl. 26: 16–17. Ziegler, T., Kamphausen, J., Glaw, F., Crottini, A., Garcia, G., Röd- der, D., Rauhaus, A., Stenger, L. M. and Wahle, A. 2022. Threatened Malagasy amphibians and reptiles in zoos–a call for enhanced implementation of the IUCN’s one plan approach. – Zool. Garten 90: 21–69. Zweig, C. L., Rice, K. G., Percival, F. and Mazzotti, F. J. 2014. Body condition factor analysis for the American alligator (Alli- gator mississippiensis). – Herpetol. Rev. 45: 216–219. 1903220x, 0, D ow nloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1002/wlb3.01302 by Duodecim Medical Publications Ltd, W iley Online Library on [28/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License