PARASITIC NEMATODE INFECTIONS OF ANURANS FROM A DERIVED SAVANNA BIOTOPE IN EDO STATE, NIGERIA

s part of a broader study to increase the information of parasite infections of anurans from the savanna biotope, an investigation of the nematode parasites of anurans in Agbede, a location in the derived savanna of Edo State, Nigeria was undertaken from August 2007 to July 2008. Anurans were collected at night using the visual acoustic encounter survey (VAES) sampling method, dissected and examined for nematode infections. A total of 329 anurans belonging to sixteen taxa, were examined, of which 235 were infected with an overall prevalence of 71.43%. Seasonal prevalences were 85.63% for wet season and 52.48% for the dry season (p<0.05). Ten nematode species, namely: Amplicaecum africanum, Cosmocerca ornata, Oswaldocruzia hoeplii, Physaloptera sp., Rhabdias africanus, Rhabdias sp., Camallanus dimitrovi, an unidentified oxyurid nematode, Ophidascaris larva, and an unidentified nematode were recovered from the hosts examined. The most common nematode species was Amplicaecum africanum (31.31%), whereas the least common was an unidentified oxyurid nematode (0.30%).Physaloptera sp. is a new record for the anurans of the derived savanna.


INTRODUCTION
The various bioclimatic zones in Nigeria range from mangrove and fresh water swamps of the Niger Delta, to the rainforest, derived savannah, guinea savannah, Montane Forest and the Sahel savannah of the extreme North . Investigations of the parasites of amphibians have extensively been carried out in the rainforest biotope (characterized by high levels of precipitation and atmospheric humidity which thus supports high canopy trees and thick undergrowth. These prevailing environmental and atmospheric conditions in the rainforest thus supports the study of the wide diversity of amphibians and their parasites in the country by various authors (Aisien et al., 2001(Aisien et al., , 2003(Aisien et al., , 2004a(Aisien et al., , b, 2009(Aisien et al., , 2011a, Amuzie et al.2018Oniya and Adeyekun, 2019; Edo-Taiwo and Aisien 2020).
The savannah biotope is one characterized by shorter duration of rainfall (six months) when compared to the rainforest. The vegetation consists of grass and deciduous trees which offers very limited canopy cover. These conditions pose great threat to the survival of amphibians in this terrain and impair the life cycle and transmission of nematode parasites. Unlike anurans from the rainforest which have been studied in great detail for their helminth parasitic fauna, not so much attention has been devoted to anuran species from the savanna biotope. Information available in the literature about studies in the savannas of Nigeria include the studies by Avery (1971);Oladimeji (1990); Aisien et al. (2004); Iyaji et al. (2015); Anele et al. (2020).
The only report on the helminth parasites of anurans from the savanna mosaic zone of Edo State is that undertaken by Aisien et al., (2003) from five different locations, excluding Agbede and over a short duration. The latter limitation necessitated the present study to enable a detailed study, carried out over a longer duration. An earlier report dealt with the digenetic trematodes infecting anurans from this study area (Ozemoka and Aisien, 2020). In this paper we report the nematodes parasitizing anurans investigated at Agbede in the derived savannah of Edo State. The study yielded ten nematode parasites with Physaloptera sp. as a new record for the anurans of the derived savanna.

MATERIALS AND METHODS
The anurans examined in this study were collected from Agbede, a location in the derived savanna biotope in Edo State, from August 2007 to July 2008, using the Visual Acoustic Encounter Survey (VAES) techniques (Crump and Scott, 1994). Sampling was carried out at night on a monthly basis (once a month) for twelve months. Agbede is located in Etsako West Local Government Area of Edo State, Nigeria and lies roughly within latitude 06 o 50´N and 06 o 60´N and longitude 06 o 1 0´E and 06 o 20´E (Fig. 1). The anurans collected were identified according to Rödel, (2000). In the laboratory, the anurans were euthanized in Benzocaine solution, dissected and examined. The gastrointestinal tract (oesophagus, stomach, small intestine, large intestine and rectum) was examined for parasites. Other parts examined included the lungs, body cavity and the urinary bladder. The contents were mixed with 0.72% of NaCl solution and examined under a dissecting microscope. The nematode parasites recovered were fixed in hot 70% alcohol and preserved in the same medium. The worms were cleared in lactophenol and examined as temporary mounts. Photomicrographs of the parasites were taken (using a digital camera attached to a binocular microscope) from which we made line drawings. Prevalence rate of parasites was calculated as a percentage of the number of a particular host species infected with a specific helminth parasite divided by the total number of hosts examined. The mean intensity of infection refers to the number of parasites per host (calculated only for the infected hosts examined). Differences in the level of significance of infection according to season of infection, were determined using the one-way ANOVA at a 5% level of significance. All statistical analysis were done using SPSS (Statistical Package for the Social Sciences) version 20 for windows.

RESULTS
In this study, 329 anurans which spread across four families, eight genera and sixteen species were examined. One of the S. maculata examined harboured as many as 24 worms. Amplicaecum africanum parasitized six species out of the 16 species examined. The prevalence values for the parasite in most hosts were very low except for S. regularis (36.78%). As many as 55 worms were recovered from the stomach of one of the S. regularis examined.

DISCUSSION
The number of species of anurans examined in this study, was sixteen which was more than the number that have been reported in other savannah locations (Avery, 1971;Aisien et al., 2003Aisien et al., , 2004Iyaji et al., 2015;Anele et al., 2020). Xenopus muelleri was the only anuran reported by Avery, (1971). Eleven species were recorded in the previous study undertaken by Aisien et al. (2003) in derived savannah of Edo State. In the Guinea savanna at new Bussa, Aisien et al. (2004) recorded and examined only four species. The numbers recorded were understandably so because of the short durations of the respective studies. Our record is however, still higher than the nine species reported by Anele et al. (2020) from Zaria in an investigation that spanned over two seasons.
The finding of 10 nematodes species from only one location (Agbede) as reported in this study brings two important points to the fore. There is the need to undertake studies of this nature over longer durations to ensure a proper capture of the parasite diversity present in the anurans of any biotope while also avoiding an over harvest of the anuran hosts. It also gives an insight into the influence of atmospheric humidity and vegetation cover in the transmission of nematode parasites. Although the derived savannah in Edo State has only six months of rainfall, the canopy cover is denser than other savanna biotopes further north. Therefore, anurans from areas with comparable rainfall data with lower canopy cover recorded lower nematode diversity: two in Katsina (Oladimeji et al., 1988(Oladimeji et al., -1990; four at New Bussa (Aisien et al., 2004); three from Ayingba (Iyaji et al., 2015) and four from Zaria (Anele et al., 2020) One finding of interest in this study was the occurrence of Physaloptera sp. in five anuran species (P. pumilo, P. bibroni, P. oxyrhynchus, and P. schubotzi and H. occipitalis). This nematode was not recorded in the
oxyrhynchus and P. longirostris, Thereafter, it has been reported in other anurans from different habitats, including, the brackish water environment (Uwasadhiuka, 2010;Aisien et al., 2015), freshwater creek biotope , Guinea savanna (Iyaji et al., (2015) and cocoa plantations (Edo-Taiwo and . In this study, the parasite was recovered from Pty. schubotzi which thus becomes a new host record for the parasite. With regards to site of infection, Physaloptera sp. was recovered from the stomach of the infected hosts. This is in agreement with the reports of Iyaji et al., (2015), Gonzalez and Hamann (2006) and Edo-Taiwo and Aisien, (2020) who all reported the parasite from the stomach. This is in contrast to the report of Aisien et al., (2009), who reported the parasite from the small intestine. Iyaji et al., (2015) attributed the occurrence of the parasite in the small intestine to less nutrition in the stomach of captive host, which may have caused the movement of the parasite down to the small intestine.
Amplicaecum africanum infected more hosts (five) than was previously reported (four) by Aisien et al., (2003) in other locations of the savanna-mosaic in Edo State and three in the Guinea savanna (Aisien et al. (2004).
The prevalence of (31. 31%) in S. regularis was lower than the value recorded for this host in Auchi (43.5%) but much lower when compared with the report for Igarra, Edo State, where 64.9% prevalence was recorded for the same host (Aisien et al., 2003). A much higher prevalence (91.67%) was reported by Iyaji et al. (2015) at Ayingba in the same host (formerly Amietophrynus regularis). Infections with this parasite have been reported in amphibians and reptiles of countries of East, West and Central Africa (Baker, 1987).
The two bufonid anurans in this study (S. regularis and S. maculata) were both infected with R. africanus with an overall prevalence of 15.81% and a mean intensity of 7.15 parasites/ infected host. This prevalence value was however lower than the values recorded in other locations in savanna-mosaic: 36.20% in Igarra, 28.20% in Agenebode in S. regularis. The prevalence value recorded in this study was however higher than the 1.4% recorded at Ihievbe 4.8% in Ozalla and 3.5% in Ogbonna (Aisien et al., 2003). Ophidascaris larva occurring in Pty. mascareniensis and Pty. pumilo has been reported from various anuran species in many locations (Aisien et al., 2001(Aisien et al., , 2003(Aisien et al., , 2004a(Aisien et al., , b, 2009(Aisien et al., , 2011a(Aisien et al., , 2017aImasuen and Aisien, 2015) and Agama colonarum (Odigwe, 1985). A recent study by Edo-Taiwo et al., (2020) reported high prevalence of this parasite in the body cavity of S. regularis (50%), S. maculata (31.8) and Ptychadena oxyrhynchus (50%) in Benin City. These parasites are believed to use anurans as transport or paratenic hosts . Adults of these nematodes have been recovered from snakes in Benin City, Nigeria by Awharitoma et al.

CONCLUSION
In conclusion, all the parasites recorded in this study have previously been reported by other authors in various biotopes. This is the first report of Physaloptera sp. from the savanna mosaic zone. Ptychadena schubotzi is a new host record for this parasite. Hoplobatrachus occipitalis is similarly a new host record for the Rhabdias sp. There is need for further research to properly describe and identify Rhabdias sp. from anurans in Nigeria. There is also the need for appropriate description and identification of the Camallanus specimens from S. regularis. The cooccurrence of A. africanum and Physaloptera sp. in Pty. schubotzi as well as A. africanum and Oswaldocruzia hoeplii in S. regularis and in Pty.bibroni is a new record.

ACKNOWLEDGEMENT
We acknowledge the cooperation of the Traditional Head of Agbede town, the Agbede Vigilante Group and the