Anurans are the world’s most diverse amphibians or batrachians
[1]
. They comprise 55 families divided into 451 genera and 6968 species
[2]
. These animals are vital to man in terms of food, agriculture, and the economy
[3]
. In terms of food, certain Anuran species, such as the African tiger frog (Hoplobatrachus occipitalis Günther), are an important source of animal protein in Nigeria, Benin, and Burkina Faso
[4]
[5]
. This frog (Hoplobatrachus occipitalis) is an important source of animal protein in the diet of the local population of Ganzourgou, Gourma, and southeastern Burkina Faso
[6]
[7]
.
[8]
noted that these Anurans were used to treat hypertension, tuberculosis, conjunctivitis, abortion, and Kwashiorkor. However, amphibian populations are declining worldwide, and many factors, such as habitat loss, toxins, and pathogens, are implicated in this decline
[9]
[10]
. In addition, anthropogenic stressors, such as livestock grazing wetlands, cause amphibians to become more susceptible to infection due to a lowered immune system
[11]
. The causes of these declines are multiple, ranging from habitat destruction and degradation, pollution, and the synergistic action of climate change to the removal of natural resources for human consumption and disease
[12]
. According to
[13]
, pathogens such as Platyhelminthes and new emerging diseases are major causes of the decline of the frog population. These pathogens generally cause significant mortalities, mainly in the ranks of wild amphibian populations in Africa. In addition, high levels of Platyhelminthes infestation cause significant mortalities in African anurans
[14]
. These parasites can cause numerous pathologies and even death in their hosts
[15]
. Both
[16]
and
[17]
provide experimental evidence that a Trematode parasite, Ribeiroia sp., is responsible for the severe limb deformities observed in certain amphibian populations in several regions of North America. However, minimal research on amphibian Platyhelminthes has been conducted thus far in Burkina Faso. This study aims to further the understanding of the Platyhelminthes associated with the African tiger frog (Hoplobatrachus occipitalis) in Burkina Faso, focusing on their diversity and distribution.
2. Materials and Methods2.1. Study Area
The frogs examined in our study were captured in bodies of water in the urban and peri-urban areas of Ouagadougou, Ganzourgou, and Bobo-Dioulasso (
Figure 1
). The study was conducted in the two largest cities of Burkina Faso (Ouagadougou and Bobo Dioulasso) as they concentrate practically two-thirds (65.80%) of the urban population with its corollaries of pollution
[18]
. The Ganzourgou province was chosen as the site because of the high diversity of the frog (Hoplobatrachus occipitalis). Also, the local community consumes this frog a lot.
This species was chosen because amphibians are edible and have a high breeding potential. H. occipitalis is the most widely sold species, as it is easy to harvest and has a large thigh with a high economic value. They are also widely distributed amphibian species in Burkina Faso
[19]
.
The frog collection period ran from June 2022 to November 2023. Sampling was carried out by capture near the ponds between 6 pm and 9 pm and between 4 am and 6 am. Frog species were collected by hand using mist nets and the visual and acoustic approach of
[20]
after examining the frogs’ hiding places on rocks, looking around the ponds with headlamps, and locating them by their croaks. Systematic visual searches consisted of scraping through leaf litter, lifting rocks and logs and looking around or within burrows and termite mounds. Additional supports of acoustic signals were applied
[21]
. Specimens were collected by hand and photographed. Each frog captured alive was placed in a cotton bag and transported to the laboratory for identification and parasite testing. In the laboratory, the frogs were identified using the keys of
[22]
-
[24]
.
2.3. Collection and Identification of Platyhelminthes
Frogs were euthanized by immersion in a benzocaine solution (14% - 20%) for dissection
[25]
. The lungs, bladder, digestive tract, and appendages were dissected and placed separately in a Petri dish containing 0.72% saline solution, then examined to harvest Platyhelminthes. The contents of each organ were excavated in saline solution and examined under a dissecting loupe. Platyhelminthes specimens were fixed with 5% formalin saline, removed after 24 hours, and placed in 70% alcohol. Specimens were stained with acetic carmine and permanently mounted between slides and coverslips in a drop of Canada balsam. After drying in an oven, the preparation was observed under a ZEISS optical microscope (ICS 25 standard) equipped with an optical micrometer. Cestodes and Digenea were identified using the identification keys of
[26]
[27]
.
2.4. Data Analysis
Abundance (A), prevalence (P), and average intensity (IM) were calculated using the method of
[28]
. The parasite abundance (A) is represented by the ratio of the total number of individuals of a parasite species (N) in a sample of hosts to the total number of hosts (H) in a sample. It is, therefore, the average number of individuals of a parasite species (N) per host examined. It is expressed as follows:
The prevalence rate (in %) is calculated as the ratio between the number of host species infected (Ni) by a Platyhelminth species and the total number of hosts examined (Ht). The following mathematical formula expresses it:
Average infection intensity refers to the number of parasites (P) per host (N) (N is the total number of hosts infected by a Platyhelminth species. The subsequent formula expresses it:
The parasite abundance as a function of sites was presented using bar plots. The Venn diagram was used to present the relationship between Platyhelminth species and attachment sites using the “draw.quintuple function” from the “VennDiagram” package. A Spearman correlation test was used to assess the strength of the correlation between parasite abundance and parameters such as weight and height using the ggscatter function from the “ggpubr” package. Parasite abundance as a function of season and sex was presented in boxplots using the ggviolin function in the “ggplot2” package. All statistical tests were performed with R software version 4.2.0 at a significance level of 5%.
3. Results3.1. Platyhelminthes Hosts
Two hundred thirty-eight (238) specimens of Hoplobatrachus occipitalis belonging to the family Dicroglossidae were collected for examination and testing for Platyhelminthes. The 238 collected specimens included 180 female and 58 male frogs, for a total prevalence of 69.75%. By sex, the prevalence of females and males collected was 65.00% and 84.48% respectively. According to the season, the prevalence of infection was 78.30% in the rainy season and 46.11% in the dry season.
3.2. Platyhelminthes
Of the 238 specimens of Hoplobatrachus occipitalis analyzed, a total of 14 species of Platyhelminthes were encountered (
Table 1
). These species belonged to five (05) genera, divided into four (04) genera of Digenea (Diplodiscus, Ganeo, Halipegus, Haematoloechus) and one (01) genus of Cestodes (Cephalochlamys): Diplodiscus fischthalichus, Diplodiscus lali, Diplodiscus subclavatus, Diplodiscus amphichrus, Ganeo africana, Ganeo glottoides, Halipegus sp., Haematoloechus variegatus, Haematoloechus nanchangensis, Haematoloechus parviplexus, Haematoloechus micrurus, Haematoloechus exoterorchis, Haematoloechus sp., Cephalochlamys namaquensis.
Table 1
summarizes the different species inventoried during the study.
<xref ref-type="bibr" rid="scirp.142248-"></xref>Table 1. List of Platyhelminthes, their hosts, and attachment organs.
Classes
Parasite Species
Hosts
Infected Organs
Trematodes
Lungs
Small Intestine
Large Intestine
Stomach
Haematoloechus sp.
Ho. occipitalis
+
-
-
-
Haematoloechus variegatus
Ho. occipitalis
+
-
-
-
Haematoloechus nanchangensis
Ho. occipitalis
+
-
-
-
Haematoloechus exoterorchis
Ho. occipitalis
+
-
-
-
Haematoloechus micrurus
Ho. occipitalis
+
-
-
-
Haematoloechus parviplexus
Ho. occipitalis
+
-
-
-
Diplodiscus fischthalichus
Ho. occipitalis
-
-
+
-
Diplodiscus lali
Ho. occipitalis
-
-
+
-
Diplodiscus subclavatus
Ho. occipitalis
-
-
+
-
Diplodiscus amphichrus
Ho. occipitalis
-
-
+
-
Ganeo africana
Ho. occipitalis
-
+
-
-
Ganeo glottoides
Ho. occipitalis
-
+
-
-
Halipegus sp.
Ho. occipitalis
-
-
-
+
Cestodes
Cephalochlamys namaquensis
Ho. occipitalis
-
+
-
-
Legend: + = present and - = absent; Ho: Hoplobatrachus.
The specimens vary in size from 5 to 150 cm. The correlation between Platyhelminthes abundance and host frog size is shown in
Figure 2
. Analysis of this figure shows that, as frog size increases, so does Platyhelminthes abundance (r = 0.24 and p = 0.00024). The Correlation and Linear Regression Test applied to the data indicates that Platyhelminthes abundance positively correlates with host frog size (p < 0.05). However, frogs between 50 and 60 cm in size hosted the highest number of Platyhelminthes. As a result, this study shows that size significantly affects the abundance of Platyhelminthes in these frogs.
The frog specimens collected weighed between 20 and 195 g. Frogs weighing between 5 and 100g exhibited a significant prevalence of Platyhelminthes (
Figure 3
). The analysis of this image indicates that frog weight correlates positively with the abundance of Platyhelminthes (r = 0.24 and p = 0.00015). The Correlation and Linear Regression Test applied to the data indicates that Platyhelminthes abundance varied with frog weight (p < 0.05). Accordingly, their weight significantly influences the number of Platyhelminthes infesting these frogs.
Figure 2. Correlation line between Platyhelminthes abundance and host frog size.
Figure 3. Correlation line between Platyhelminthes abundance and host frog weight.
The analysis of Platyhelminthes abundance in Hoplobatrachus occipitalis as a function of season showed that there was no significant 2difference in abundance according to season with the Wilcoxon test (p = 0.000087 < 0.05) (
Figure 4
). The abundance of amphibians collected depended on the season. The prevalence of Platyhelminthes in frogs collected in both rainy and dry seasons ranged from 0 to 100%. Comparing this prevalence between the two seasons, as shown in
Figure 4
, we found no significant difference between the rainy and dry seasons (chi-square test, p = 0.16 > 0.05).
Figure 4. Platyhelminthes abundance by season.
The prevalence is higher in males (84.48%) than in females (65.00%).
Figure 5
shows Platyhelminthes abundance as a function of host frog sex. The Wilcoxon test yields a p-value below the significance threshold (p ≤ 0.05). As a result, parasite abundance varies by host sex, although there is no significant difference (
Figure 5
). However, prevalence was higher in males (84.48%) than in females (65.00%).
Figure 5. Platyhelminthes abundance by sex.
Figure 6
illustrates the prevalence of Platyhelminthes as a function of African tiger frog sampling sites, i.e., the urban and peri-urban areas of Ganzourgou, Ouagadougou, and Bobo-Dioulasso. Platyhelminthes prevalence was highest in the commune of Tondogosso (100%), followed by Bama (90.91%) and Koubri (88.00%). Moreover, the lowest prevalence was observed in the commune of Zorgho (34.78%). This outcome indicates that frogs in Tondogosso exhibit a higher prevalence of infection or contact with Platyhelminthes than those in Zorgho.
Figure 6. Prevalence of Platyhelminthes species by site.
Figure 7
shows no relationship between Platyhelminthes species according to their attachment sites within amphibians. Indeed, each genus of Platyhelminthes had its own preferred attachment site. All Cestodes of genus Cephalochlamys were collected from the small intestine, Trematodes of genus Ganeo and genus Haematoloechus from the lungs, those of genus Diplodiscus from the large intestine, and those of genus Halipegus from the stomach.
Figure 7. Relationship between Platyhelminthes species according to attachment sites. A: Lung trematodes; B: Small intestine trematodes; C: Small intestine cestodes; D: Large intestine trematodes; E: Stomach trematodes.
4. Discussion
The study concerned the only frog species belonging to the Dicroglossidae family in Burkina Faso (Hoplobatrachus occipitalis). Specimens of this species are not only consumed in Burkina Faso
[6]
but also in other African countries such as Nigeria
[4]
, Côte d’Ivoire
[29]
, and Benin
[6]
. This study revealed an overall prevalence of 69.75% of Platyhelminthes infection in African tiger frogs. This study enabled us to identify one species of Cestode, Cephalochlamys namaquensis, and 13 species of Digenea, six of which were recorded for the first time in Burkina Faso. The recorded prevalence indicates a significant parasite infection among the sampled frogs. In fact, out of 238 frog specimens examined, 166 species were infested with Platyhelminthes. Fourteen species of Platyhelminthes were identified in the examined frogs; however, Cephalochlamys namaquensis has previously been documented in Hoplobatrachus occipitalis in Burkina
[30]
. These Platyhelminthes recovered from these amphibians were similar to those reported from other African countries
[31]
-
[34]
, and the prevalence was of the order of that observed in amphibians from the Nigerian savannah
[33]
[34]
. Ganeo africana has been reported in Hoplobatrachus occipitalis from Porto Novo in southern Benin
[32]
and recorded in this host in the Pendjari Biosphere Reserve.
Among the Haematoloechus species described by
[35]
, two (02) were subsequently reported by
[31]
on D. occipitalis in Ghana. Diplodiscus fischthalicus was also found in D. occipitalis in the Guinean savannah at New Bussa, Nigeria. Cephalochlamys compactus is an intestinal parasite of Hoplobatrachus occipitalis. It has so far been recovered from H. occipitalis collected in the Niger Delta region of Nigeria, namely from Warri and Ase in Delta State, as well as from Egbeda and Degema in River State
[33]
[36]
[37]
. Among the Diplodiscidae encountered in this study, Diplodiscus fischthalicus appears to be a multi-host or heteroxenous parasite.
[34]
collected D. fischthalicus from Hoplobatrachus occipitalis, while
[38]
originally described the parasite from Rana angolensis in Ethiopia. Additionally,
[39]
collected and identified Diplodiscus specimens in 1916 as D. subclavatus from H. occipitalis in Ghana.
[40]
also identified Diplodiscus specimens collected from H. occipitalis and Ptychadena mascareniensis from Cameroon as D. subclavatus.
Our study examined the impact of sex on parasite abundance in amphibians from different environments, demonstrating that sex significantly affects parasitism. Indeed, males exhibit a higher prevalence of infestation than females. This disparity may be partially attributed to hormonal variations and divergences in the evolution of immune defense mechanisms corresponding to specific reproductive roles
[41]
.
It is worth noting that
[42]
showed that parasitism levels in Bangladesh were frequently elevated in male toads compared to females. They believed that sex hormones impede parasite establishment in female amphibians. The effect of biological parameters such as size and mass on the diversity and abundance of Platyhelminthes in amphibians in the course of this work showed the existence of significant correlations between the abundance and diversity of Platyhelminthes species collected and the size, as well as the mass, of the host body. Amphibian size and mass are key determinants of parasite abundance and prevalence. This suggests that larger amphibians have a more varied diet, ingest more significant quantities of food, and provide more surface area for parasite colonization
[43]
. Larger size is also linked to a longer life span and more infection time, which favors parasitism
[44]
. Indeed, previous studies demonstrated that large amphibians harbored more Platyhelminthes species than small amphibians
[45]
-
[47]
.
While capturing frogs, we noted the use of pesticides on these sites, which could explain the difference in infestation between sites. Indeed,
[48]
demonstrated that pesticides are stressful for aquatic organisms, including amphibians. As anurans become compromised, they become more vulnerable to predation and colonization by new parasitic species.
[49]
have shown that pesticides have an immunodeficient effect on frogs, which can make them more susceptible to infestation by Platyhelminthes.
5. Conclusion
The present study revealed the presence of five genera of Platyhelminthes in the African tiger frog (Hoplobatrachus occipitalis): One (1) species of Cestodes (Cephalochlamys namaquensis) and Thirteen (13) species of Digenea. These Platyhelminthes’ overall prevalence or infestation rate was low (69.75%) across the three sites surveyed. The preferred organs for infestation of these Platyhelminthes are the lungs, stomach, small intestine, and large intestine of frogs. These findings may serve as a foundation for extensive research to determine how these Platyhelminthes affect amphibian morbidity and mortality.