Abstract
Assessment of parasites and their pathogenicity is essential for studying the ecology of populations and understanding their dynamics. In this study, we investigate the prevalence and intensity of infection of haemogregarines (phylum Apicomplexa) in two sympatric lizard species, Podarcis vaucheri and Scelarcis perspicillata, across three localities in Morocco, and their effect on host immune response. We used the Phytohaemagglutinin (PHA) skin testing technique to relate the level of immune response with parasite infection. Prevalence and intensity levels were estimated with microscopy, and 18S rRNA gene sequences were used to confirm parasite identity. All parasites belong to the haemogregarine lineage found in other North African reptiles. There were differences in prevalence between localities and sexes. Overall, infected lizards were larger than uninfected ones, although we did not detect differences in parasitaemia across species, sex or locality. The swelling response was not related to the presence or number of haemogregarines, or to host body size, body condition, sex or species. We found no evidence of impact for these parasites on the circulating blood cells or the hosts’ immune system, but more data is needed to assess the potential impact of mixed infections, and the possibility of cryptic parasite species.
Introduction
Parasites represent a major component of biodiversity, playing an important role in ecosystems (Pedersen and Fenton, 2007). There is evidence showing that parasites may play an important role in ecosystem dynamics and host community structure (Hudson, Dobson and Lafferty, 2006; Pedersen and Greives, 2008; Poulin, 2017). Knowing the costs for the host to harbour such parasites is thus a central question when studying parasite dynamics and host-parasite interactions.
Apicomplexa Levine 1970 is a variable and widespread group of obligate intracellular parasites (Morrissette and Sibley, 2002; Morrison, 2009; Ruggiero et al., 2015), comprising genera that cause high impact on animal communities and public health, such as species of Plasmodium, Eimeria or Babesia, but also parasites for which pathogenicity is still poorly understood, such as the haemogregarines. Within the haemogregarine group, four genera are known to infect reptiles: Hepatozoon Miller, 1908, Karyolysus Labbé, 1894, Haemogregarina Danilewsky, 1885, and Hemolivia Petit, Landau, Baccam et Lainson, 1990. Hepatozoon has been regarded as one of the most common and widespread of haemogregarines in reptiles (Telford, 2009). Although haemogregarine taxonomy is still under discussion (Karadjian, Chavatte and Landau, 2015; Maia, Carranza and Harris, 2016; Hrazdilová et al., 2021), phylogenetic studies tend to recover a lineage predominantly from lizard hosts including forms identified either as Karyolysus (Haklová et al., 2014) or Hepatozoon (Bartazoon according to Karadjian, Chavatte and Landau, 2015, but see Hrazdilová et al., 2021), which are related to a lineage of Hepatozoon from carnivores (Maia, Carranza and Harris, 2016). Nevertheless, despite being poorly assessed, the use of molecular diagnostic techniques has shed considerable light onto the genetic diversity and phylogenetic relationships of this group of parasites (e.g., Tomé et al., 2014, 2019; Harris et al., 2020a).
Hepatozoon spp. can cause hepatozoonosis in domestic animals, particularly cats and dogs, with severe impact on their health (Baneth et al., 1998; Panciera et al., 2000). However, some haemogregarine species may also be better adapted to the host, producing less severe effects (Baneth et al., 2003; Conradie et al., 2016). On the other hand, there are still many questions regarding haemogregarine pathogenicity. For example, studies on its impact on the fitness of reptile host species show ambiguous results (Telford, 2009). Studies show that water pythons (Liasis fuscus) can be severely impacted by long-term parasitic infection and those harbouring low Hepatozoon intensity are able to achieve older ages (Madsen, Ujvari and Olsson, 2005; Ujvari and Madsen, 2005), larger body size (Madsen and Ujvari, 2006) or exhibit a stronger immune response (Ujvari and Madsen, 2005). In contrast, in the sympatric snake, Tropidonophis mairii, little evidence was found between Hepatozoon intensity and several measures of fitness, indicating an apparently benign association between parasite and host (Brown, Shilton and Shine, 2006). These differences of pathogenicity between sympatric snake species may reflect different levels of coadaptation between parasites and their hosts for the same local conditions.
The lacertid lizards Podarcis vaucheri (Boulenger, 1905) and Scelarcis perspicillata (Duméril and Bibron, 1839) belong to two distinct lineages of Palearctic lacertids (Mendes et al., 2016; García-Porta et al., 2019) and represent good models to investigate the potential impact of haemogregarines on the vertebrate host. These two species show differences in morphology (Martínez del Mármol et al., 2019), diet (Carretero et al., 2006; Perera et al., 2006), and ecophysiology (S’khifa et al., 2020), with notable differences in their microhabitats, since S. perspicillata is primarily rock-dwelling and P. vaucheri is ground-dwelling (Martínez del Mármol et al., 2019). They occur in sympatry in a wide distribution range throughout Morocco (Martínez del Mármol et al., 2019) with haemogregarine infections already documented for both species (Maia, Harris and Perera, 2011; Harris, Maia and Perera, 2012; Maia, Perera and Harris, 2012). While there is some available information on the impact of haemogregarines on the host genus Podarcis (Oppliger et al., 2004; Martín, Amo and López, 2008; Huyghe et al., 2010; Damas-Moreira et al., 2014), nothing is known about their effects on Scelarcis lizards.
Given the impact that haemogregarines have on their hosts (Jacobson, 2007; Telford, 2009), understanding hosts defence mechanisms is of major importance. However, the immune response is complex, involving multiple organs, cells and molecules, and its accurate measurement is challenging (Drake et al., 2019). The Phytohaemagglutinin (PHA) skin-swelling test is commonly used for indirectly measuring T-cells immunocompetence in vertebrates due to its simplicity and easy implementation (Martín et al., 2006). This method has been used in other lizards (e.g., López and Martín, 2005; Calsbeek, Bonneaud and Smith, 2008) and in the genus Podarcis (Oppliger et al., 2004; Sacchi et al., 2007; Huyghe et al., 2010). Besides these approaches, counting the circulating blood cells and comparing differences in their ratio among infected individuals has also been used as indirect evidence for the presence of parasites (Shutler, Smith and Robinson, 2009).
In this study we investigate the haemogregarine diversity, prevalence and intensity of infection in two sympatric lizard host species across three different localities, to assess potential differences in parasite infection levels. Additionally, we determine whether haemogregarines are related to variation in the immune response system, so inferences can be drawn regarding the impact of these parasites. In order to answer these questions immune, blood cell counts and the Phytohaemagglutinin (PHA) skin-testing technique were used as a proxy for the lizards’ immunocompetence, and were compared with haemogregarine prevalence and intensity of infection estimated using microscopy, while sequences of partial 18S rRNA genes were used to confirm the identity of the parasites involved.
Materials and methods
Study sites and species
This study was carried out during May 2012 at three sites in Morocco: Debdou (33.8725°N, 3.0388°W), Oukaimeden (31.2010°N, 7.8554°W) and Mischleiffen (33.4054°N, 5.1033°W). At each locality we studied two lacertid lizard species living in sympatry, Podarcis vaucheri and Scelarcis perspicillata. We collected a total of 160 adult lizards using a noose (see table 1 for details on sampling size per locality and species). After experiments, lizards were released at the capture site.
Descriptive statistics of haemogregarine prevalence and intensity of infection across sites, species and genders.
Citation: Amphibia-Reptilia 43, 1 (2022) ; 10.1163/15685381-bja10078
PHA skin-testing technique
The immune response was measured for each individual using the PHA test. This technique consists of a PHA intradermal application in vivo to one of the hind-foot pads, which triggers a highly complex pro-inflammatory response, inducing a swelling of the foot pad when the lizard exhibits a good inflammatory response (Vinkler, Bainová and Albrecht, 2010). Hence, individuals with a greater swelling pad are considered to have a better immune condition (Vinkler, Bainová and Albrecht, 2010).
Before conducting the test, we marked a dot on the pad of lizard’s right hind-foot to ensure that injection and measurements were performed at the same point. The initial thickness of the foot pad was measured three times at this point using a digital calliper (±0.01 mm) and the average value was recorded. Afterwards the foot pad was injected with 0.02 ml of PHA solution (Sigma-Aldrich, Germany), using a new syringe for each animal. To analyse how the lizard’s immune system reacted to the injected PHA, we measured the same point 24 hours later three more times. During this period, animals were kept in individual cloth bags. The difference in swelling between the measurements before and after injection was taken as a metrical index of the intensity of the immune response. To minimize inaccuracies as much as possible, all measurements and injections were performed by the same researchers (IDM and JPM, respectively). After the experiments, species, sex, snout-vent length (SVL) and weight were recorded. Finally, before releasing collected specimens at the sample sites, we collected and stored a small piece of the tail tip in 96% ethanol for genetic analyses, smeared a drop of blood across a glass slide, and stored another drop of blood in Whatman filter paper. This data was used for posterior genetic analyses and blood examination under a microscope in the lab.
Microscopic parasite assessment
Slides were fixed in absolute methanol for 2 minutes and later stained with diluted Giemsa (1:9 distilled water) for 45 minutes. Blood smears were analysed under an Olympus CX41 microscope (Olympus, Hamburg, Germany). We estimated the prevalence as the percentage of infected lizards within a population, and individual parasite intensity as the percentage of infected red blood cells per 2,500 cells (Margolis et al., 1982; Bush et al., 1997). For cell counting, micrographs were randomly taken across the slide at x400 using the CellˆB 3.4 Olympus® software (Olympus, Munster, Germany) and the following cell types were counted using the ImageJ 1.46® program (Abramoff, Magalhães and Ram, 2004): parasitized erythrocytes, un-parasitized erythrocytes, immature erythrocytes, thrombocytes and leukocytes (which include lymphocytes, monocytes, heterophils, eosinophils, azurophils, and basophils) (fig. 1).
Circulating blood cells in P. vaucheri and S. perspicillata lizards: A) uninfected mature erythrocyte (one-headed arrow) and immature erythrocyte (double-headed arrow); B) and C) haemogregarine parasites infecting erythrocytes; D) heterophil cell (double-headed arrow) and two thrombocytes (one-headed arrows); E) an eosinophil (one-headed arrow) and a lymphocyte (double-headed arrow); F) one azurophil; G) one monocyte and H) one basophil. Scale black bar = 10 μm.
Citation: Amphibia-Reptilia 43, 1 (2022) ; 10.1163/15685381-bja10078
Molecular analysis
Twelve samples infected with parasites were randomly selected to confirm parasite identity using molecular methods. DNA was extracted using High Salt methods (Sambrook, Fritsch and Maniatis, 1989; Maia et al., 2014). A fragment of the 18S rRNA gene was amplified using the primers HepF300 and HepR900 (Ujvari, Madsen and Olsson, 2004) following the conditions from Harris et al. (2011). PCR products were purified and both strands were sequenced by a commercial sequencing facility (Macrogen, The Netherlands).
Sequences were analysed using Geneious 6.0.3 (Biomatters Ltd.) and each electropherogram was carefully checked. The new sequences were aligned using the MUSCLE algorithm (Edgar, 2004) implemented in this software, with haemogregarine 18S rRNA gene sequences retrieved from GenBank from various host species. The final dataset contained 92 sequences of 577 bp in length. As outgroups we included Dactylosoma ranarum (HQ224958) and Haemogregarina balli (HQ224959).
We used phylogenetic analyses based on Maximum Likelihood (ML) and Bayesian Inference (BI) methods to estimate the relationships between the parasite sequences we generated and haemogregarine sequences retrieved from GenBank. The TIM1+G model was selected in jModeltest 0.1.1 (Posada, 2008) according to the AIC criterion and implemented in the ML and BI analyses. ML analysis was performed with random sequence addition (100 replicate heuristic searches) using the software PhyML 3.0 (Guindon et al., 2010). Support for nodes was estimated using the bootstrap technique (Felsenstein, 1985) with 1,000 replicates. BI analysis was implemented using Mr. Bayes v.3.1 (Huelsenbeck and Ronquist, 2001) with parameters estimated as part of the analysis. The analysis was run for 107 generations, saving one tree each 1,000 generations. The log-likelihood values of the sample points were plotted against the generation time and all the trees prior to reaching stationarity were discarded. Remaining trees were combined in a 50% majority consensus tree (Huelsenbeck and Ronquist, 2001).
Statistical analysis
Prior to statistical analyses, the body condition (BC) of each individual was calculated as weight/body size residuals. Swelling response was calculated as the ratio between the amount of swelling (difference between final and initial foot thickness) and the initial foot thickness. All continuous variables were tested for normality (Shapiro-Wilk test) and homoscedasticity (Bartlett test) assumptions. Since in several cases variables did not meet the assumptions, we opted for non-parametric approaches.
In order to determine whether snout-vent length (SVL) or body condition (BC) needed to be considered as a covariate in the analysis of parasite prevalence and parasitaemia, we performed permutational ANOVAs with SVL or BC as the dependent variable and the prevalence of infection as independent variable. We also performed non-parametric Spearman correlations between SVL or BC and the variable intensity using the function cor of the R package (R Development Core Team, 2012). Permutational ANOVA was based on 1000 permutations and calculated using the function adonis implemented in the R package Vegan (Oksanen et al., 2012). We used this procedure as an alternative to sum-of-squares-based parametric analysis of variance (Anderson, 2001).
To analyse which factors had a significant effect on parasite prevalence, we built a Generalized Linear Model (GLM) with prevalence (infected/not-infected) as response, and lizard species, localities, sex, SVL and their interactions as factors, using a binomial logistic function (R package MASS; Venables and Ripley, 2002; R Core Team, 2012). We performed a-posteriori pairwise comparisons to identify which group was differentiated from the others using the function lsmeans from the R package lsmeans (Lenth, 2016).
Regarding parasitaemia, a permutational Analysis of Covariance (ANCOVA) was performed with parasite intensity as response variable, and with species, locality and sex, and their interactions, as factors using the adonis function as described above. We also performed a-posteriori permutational pairwise comparison (pairwisePermutationTest function implemented in the R package rcompanion; Mangiafico, 2021) to identify which group was responsible for the differences obtained.
To estimate the relationship between swelling response to PHA, body condition and parasite infection, three analyses were performed. First, we tested whether there was a relationship between lizard body condition and the swelling response to the PHA test. For this, we performed a Spearman correlation between swelling and body condition. Second, we investigated differences in the immune response between infected and uninfected individuals using a permutational ANCOVA with the settings as described above, having the immune response as the dependent variable, SVL as covariate and host characteristics (prevalence, species, locality and sex) as factors. Finally, to understand how the immune response was related to parasite intensity of infection, a similar analysis was run including only infected lizards, using factor intensity instead of prevalence as dependent factor.
The association between immune response and the levels of the circulating blood cell types was assessed with permutational ANCOVAs with either the immune response or the intensity of infection as dependent variables, SVL as a covariate and the number of leukocytes, immature erythrocytes, thrombocytes, and their interactions as factors. Spearman correlations were conducted between significant associations found in the models.
Results
Parasite identification
Consensus sequences for each individual were deposited in the GenBank database (Accession numbers OK244527 to OK254732, see fig. 2). Twelve samples were sequenced, which yielded 6 haplotypes and 3 sequences with heterozygous positions. Our estimate of phylogenetic relationships (fig. 2) confirms that all the parasites analysed belong to a single clade of haemogregarine parasites, identified as Karyolysus clade according to Karadjan et al. (2015) or clade K according to Maia et al. (2015). This clade includes GenBank sequences identified as either Hepatozoon or Karyolysus. We tentatively assign these parasites to Karyolysus, but note that the taxonomy of this lineage remains uncertain.
Estimate of phylogenetic relationships based on a Bayesian analysis of 18S rDNA sequences of haemogregarines. Statistical support is reported for relevant nodes: above, Bayesian Posterior Probabilities > 90; below, bootstrap values for ML > 70; + indicates support of 100%. Clades presented with triangles were collapsed in height by 75%. The sequences from this study are indicated in bold with sample codes given inside parenthesis.
Citation: Amphibia-Reptilia 43, 1 (2022) ; 10.1163/15685381-bja10078
Variation in host and parasite parameters
Body size (SVL) differed between species (F1,147 = 49.750, p = 0.001), localities (F2,147 = 18.857, p = 0.001) and sexes (F1,147 = 6.422, p = 0.012). Scelarcis perspicillata individuals were larger than Podarcis vaucheri individuals, with Oukaimeden containing the longest individuals and Debdou the shortest. Furthermore, males were found to be longer than females. Interactions between these factors were not significant (in all cases, p > 0.05). Infected individuals were significantly larger than uninfected ones (F1,157 = 17.65, p = 0.001) (fig. 3). However, considering only infected individuals, we did not find a correlation between body size and the levels of parasitaemia (rs = 0.121, p = 0.273).
Boxplot of the variation in swelling response to PHA (%) among infected individuals by species, localities and sexes. The black line inside the box represents the mean swelling, box represent 50% of the central values, and the extremes represent the minimum and maximum values (excluding outliers).
Citation: Amphibia-Reptilia 43, 1 (2022) ; 10.1163/15685381-bja10078
Body condition differed among localities (F2,147 = 28.930, p = 0.001), sexes (F1,147 = 57.455, p = 0.001), and in the interaction locality*species (F2,147 = 6.489, p = 0.002), and species*sex (F1,147 = 5.205, p = 0.025) but not between species (F1,147 = 0.268, p = 0.591) or any other interaction (in all cases, p > 0.05). Individuals from Mischliffen were in better condition than those from Debdou and Oukaimeden, with males exhibiting better BC compared to females. Body condition did not differ between infected and uninfected lizards (F1,157 = 0.051, p = 0.825), nor was there a correlation between BC and levels of parasitaemia (rs = 0.172, p = 0.118).
Parasite prevalence differed between localities (
Since SVL was not correlated to intensity of parasitism, and was not included in this model. Parasitaemia level was not significantly related to any of the factors included in the model, namely species, sex, and locality, or to their interactions (in all cases, p > 0.05).
Variation in PHA swelling response and blood cell counts
Considering the full dataset, the swelling response triggered by the PHA injection was not correlated with body condition (rs = 0.116, p = 0.146), or with SVL (rs = −0.133, p = 0.093). We also did not find any relation between swelling response and the presence of parasites (F1,157 = 2.28, p = 0.132) or level of parasitaemia (rs = 0.082, p = 0.456).
Locality was the only factor that had a significant influence on swelling variation (F1,147 = 10.637, p = 0.020) when considering the full dataset. Lizards from Debdou triggered the strongest swelling response, whereas those from Oukaimeden showed the lowest swelling response. None of the other factors included in the model, namely species, sex or their interactions was significant (in all cases, p > 0.05). When analysing only infected individuals, the results were mostly congruent with the full dataset; Locality (F2,72 = 5.06, p = 0.007) and the interaction locality*sex (F2,72 = 5.609, p = 0.015) had significant effects on the swelling response. Lizards from Debdou triggered the strongest swelling response, which was particularly accentuated in females, while in the other localities there were no major differences in the swelling response of males and females (fig. 3).
Analysis concerning the circulating blood cells of infected individuals showed that the interaction between SVL*white cells was the only factor associated with parasite load (F = 9.54, df = 1, p = 0.003). Finally, we did not find any significant association between any analysed blood cell counts and the swelling response (in all cases, p > 0.05).
Discussion
To our knowledge, this study is the first to assess the haemogregarine infection parameters and host characteristics (species, sex, size and immune characteristics) in two sympatric lizards across localities with different ecological characteristics. Overall, our results show that hemogregarines are common parasites among species of P. vaucheri and S. perspicillata populations. Parasite prevalence varied between sympatric locations as well as across sexes, but not between lizard species. Local differences may be due to several factors, such as habitat characteristics, or availability and competence of the invertebrate host (Gupta, Bhaskar and Gupta, 2013). Mites are the definitive hosts for this clade (Haklová-Kočíková et al., 2014; Karadjan et al., 2015). Different vector availability or abundance could explain, at least partially, local differences in prevalence. Although species was not considered a main factor by the model, there was an effect in the interaction between locality and species. In Debdou the two lizard species had a similar percentage of individuals infected, but this was not the case for the other two localities: in Oukaimeden P. vaucheri contained higher prevalence than S. perspicillata, while in Mischleiffen we observed the opposite. These differences in the number of infected individuals between species within each site might point to different levels of host resistance (Oppliger, Vernet and Baez, 1999). On the other hand, the lack of differences in parasite intensity between localities, sexes or species, might be indicative of a similar level of virulence in both sympatric hosts. Assessments of parasite load in the gecko Quedenfeldtia trachyblepharus also showed differences between localities, although with a much lower prevalence of haemogregarine infection (7.92%) compared to the lacertid lizards in the present study (Er-Rguibi et al., 2021).
Overall, lizard body size seems to be related to the presence of these blood parasites, with infected lizards being larger than uninfected lizards in both species analyzed. This difference in size can be a consequence of the fact that larger lizards are generally older and thus are exposed for a longer time to haemogregarine vectors, so that older individuals would eventually have a higher probability of being infected than younger ones (Schall, 1986; Amo, López and Martin, 2004; Ujvari and Madsen, 2005). The body condition of the lizards differed among the three studied localities with males generally exhibiting better body condition than females. However, contrary to the body size, body condition did not differ between infected and uninfected lizards, nor was it correlated with the level of parasitaemia. Haemogregarine gametocytes destroy erythrocytes reducing host blood capacity for oxygen transportation (Oppliger et al., 1998). This may have consequences on lizard muscular response to activities such as foraging, and eventually negatively affect body condition (Wozniak et al., 1996). Interestingly, we did not find any association between intensity of parasitism and body condition. Although increasing activity of lizards with better body condition may result in more agonistic encounters, and higher risk of infection by the hematophagous vectors (Amo et al., 2005a; Garrido and Pérez-Mellado, 2013), the association of body condition with haemogregarines is not clear and, in general, results remain incongruent. A few studies found similar results to ours: some observed haemogregarine prevalence, but not the intensity of infection, positively correlated to the adult size, but not to body condition (Amo et al., 2005a), whereas others show both body size and condition positively correlated with blood parasite intensity (Molnár et al., 2013; Maia et al., 2014). In a study with Podarcis muralis, those individuals harbouring higher haemogregarine load had better body condition (Amo, López and Martín, 2005b). However, the opposite was documented for the insular Podarcis lilfordi, where higher prevalence and parasite load was found in individuals with lower body condition (Garrido and Pérez-Mellado, 2013). This ambiguity of results found in a number of studies is worth underlining, as it indicates the highly variable association of haemogregarines with body size and body condition of the host. These differences among studies might also be due to the limited accuracy provided by the body condition index as a proxy for overall body condition (Peig and Green, 2010).
Likewise, assessments of the impact of haemogregarines on the immune system of reptiles produced incongruent results between studies. So far, although a few studies have used the PHA skin-testing technique to investigate haemogregarine effects in lizards (Oppliger et al., 2004; Amo, López and Martín, 2007; Martín et al., 2007; Martín, Amo and López, 2008), only a limited number conducted direct comparisons between lizard immune response and parasite load. To our knowledge, the only studies that compare directly these two variables were performed in Podarcis muralis (Amo, López and Martín, 2006) and Podarcis melisellensis (Huyghe et al., 2010). The first study found a negative correlation between immune response and hemogregarine load. In the latter work, as in our study, the authors did not observe any relationship between the immune response and hemogregarine infection levels, but they did not discuss this particular result, since that test was not the primary objective of their investigation.
Using the PHA skin-swelling test, our study did not determine the relationship between the immune response and either the presence of infection or parasite intensity in infected individuals, for any of the three localities and for both species. The lack of evidence for a negative impact of hemogregarines in the hosts that we found is in accordance with the work of Brown, Shilton and Shine (2006), who suggested that Hepatozoon infection often may have insignificant consequences on host fitness. Likewise, there are documented cases of reptile species capable of tolerating high levels of hemogregarine infections, experiencing little or no negative impact (Wozniak et al., 1996), as well as studies showing only trivial consequences of hemogregarine infections on reptile health (Caudell, Whittier and Conover, 2002; Sperry et al., 2009). Such a low impact is also supported by the lack of association between parasitism and the increase in the number of leukocyte cells, that provide a measure of the immune function (Norris and Evans, 2000), or with an increase in the number of immature erythrocytes, that are indicators of anemia and possibly of a weaker immune system (Oppliger, Celerier and Clobert, 1996; Telford, 2009). The immune response was also not correlated with any circulating blood cell value and was found to be significantly lower in larger individuals, although a decline in immune function as individuals grow older could be related to gradual alterations of the immune system and increased vulnerability to infections (Miller, 1996).
The finding of mixed infections inducing an increased immune response in terrapins (Marzal et al., 2017) highlights several aspects regarding these parasites that require further investigation. Mixed infections with multiple species of Hepatozoon seem to be common in some host groups, for example felines (Harris et al., 2020a). The Sanger sequencing technique used in almost all parasitological studies in reptiles may not identify mixed infections where one parasite is more prevalent. While next generation sequencing approaches can be more effective in this aspect, they are still rarely employed in non-model systems (Harris et al., 2020a). A further impediment is that “cryptic” Hepatozoon species are common within reptiles, with recent molecular assessments uncovering notable diversity (Tomé et al., 2019, 2021). Our genetic assessment identified 6 different haemogregarine haplotypes from the lizards sampled, and while these all belonged to the same large clade, it is not clear if they correspond to the same species. Further assessments are needed to determine if mixed infections by undescribed cryptic species occur, and if they have a greater impact on the host. As an additional complication, the inability of reptiles to regulate internally their body temperature may have profound consequences on the allocation of resources for different activities including the immune response (Zimmerman, Vogel and Bowden, 2010). This response is energetically very costly, and as such, seasonality and temperature may indirectly play a role in triggering their immune response (Zimmerman, 2016). One reason for the differences in identified immune responses may therefore be related to the seasonal timing of the different studies. Assessments of parasitaemia levels within lizard hosts across seasons are needed to better determine this impact.
In conclusion, our results point to a non-significant effect of parasitism by haemogregarines on the immune system of wild P. vaucheri and S. perspicillata from Morocco, which is in agreement with previous works on reptiles and amphibians (e.g., Brown, Shilton and Shine, 2006; Shutler, Smith and Robinson, 2009). This study does not support the common assumption that reptiles harbouring these parasites may have relatively lower fitness. The results also highlight the need for the application of more precise approaches for measuring immune response and host body condition, as well as more accurate genetic tools to improve our understanding regarding the impact of haemogregarines on their hosts, in particular the role of mixed infections and of cryptic species.
Corresponding author; e-mail: perera@cibio.up.pt
Acknowledgements
We wish to thank Le Haut Commissaire aux Eaux et Forêts et à la Lutte Contre la Désertification of Morocco for providing permits for sampling (10/2014/HCEFLCD/DLCDPN/DPRN/CFF), and to Soumia Fahd (Abdelmalek Essaâdi University, Tetouan, Morocco) and Antigoni Kaliontzopoulou (UB, Spain) for the support. Fieldwork for this study was partially supported by the Percy Sladen Memorial Fund (to DJH). DJH was supported by FEDER through the compete program, the project “Genomics and Evolutionary Biology” co-financed by North Portugal Regional Operational Program (ON.2) under NSRF through the European Regional Development Fund. JPM, DS, and AP were supported by grants and contracts by Fundação para a Ciência e Tecnologia (FCT, Portugal) under the Programa Operacional Potencial Humano – Quadro de Referência Estratégico Nacional funds (POPH-QREN) from the European Social Fund (ESF) and Portuguese Ministério da Educação e Ciência (JPM: PhD grant SFRH/BD/74305/2010; DS: post-doctoral grant SFRH/BPD/66592/2009; AP: contracts IF/01257/2012 and PTDC/BIA-EVL/28090/2017, POCI-01-0145-FEDER-028090).
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Footnotes
Associate Editor: Uwe Fritz
