amphibians are the most threatened vertebrate group , suffering large - scale declines in species diversity since at least , according to historical data , the 1970s ( stuart et  al . , 2004 ) .
a decade ago the iucn 's global amphibian assessment indicated that a third of the estimated amphibian species had declined or become extinct ( stuart et  al . , 2004 ;
such declines may be attributed to a number of factors ranging from habitat destruction , pollution and exploitation , to climate change and disease ( beebee and griffiths , 2005 ) .
the disease known as chytridiomycosis ( amphibian chytrid ) , caused by the fungal pathogen batrachochytrium dendrobatidis , has been responsible for major global amphibian declines ( readel and goldberg , 2010 ) . along with chytrid , amphibians are host to a wide variety of parasites ( du preez and carruthers , 2009 ; netherlands et  al . , 2014a ) , including intraerythrocytic and extracellular haemoparasites ranging from protozoans , comprising both intracellular apicomplexans ( davies and johnston , 2000 ) and extracellular flagellates ( acosta et  al . , 2013 ) , to extracellular nematode microfilariae ( baker , 2008 ) as well as those intracellular parasites of uncertain identity such as the viral and bacterial infections ( davies and johnston , 2000 ; davis et  al . ,
the most attention , however , has been given to those parasites of the first three groups mentioned , most likely due to the frequent findings and thus greater basis of knowledge of these organisms in anuran hosts .
furthermore , of these three groups , those of the protozoa , particularly the apicomplexans , would appear to be the most studied of all ( see davies and johnston , 2000 ; netherlands et  al .
however , since few parasite surveys on frogs have been carried out in sub - saharan africa , the degree of this haemoparasite diversity remains unknown ( readel and goldberg , 2010 ; netherlands et  al .
yet , such diversity of knowledge regarding these parasites is necessary before further studies can be done on elucidating the effects that these parasites may have on their natural hosts , and the role these parasites may have in amphibian conservation .
southern africa currently boasts 159 known species of frogs in 33 genera and 13 families ( du preez and carruthers , 2009 ; channing and baptista , 2013 ; channing et  al .
this study presents the results of a haemoparasite survey of frogs from three localities in kwazulu - natal , south africa ( see fig .  1 ) .
localities include the formally protected ndumo game reserve ( ngr ) and the reserve 's anthropogenically impacted surrounds , as well as kwa nyamazane conservancy ( knc ) .
all three sampling areas fall within a sub - tropical region known for its biological richness and as such the province , kzn , boasts the highest diversity of frog species in south africa ( see du preez and carruthers , 2009 ) .
the following study thus aimed to determine and record , through a multispecies haemoparasite survey on frogs , if this parasite diversity paralleled that of its rich frog diversity .
ndumo game reserve ( ngr ) ( 265200.0s 321500.0e ) is situated in the west of the maputaland bioregion , close to the borders of south africa , swaziland and mozambique .
the maputaland bioregion , located in northern kwazulu - natal ( kzn ) and crossing into southern mozambique , is one of the most biologically rich areas in southern africa ( haddad , 2003 ) and has a sub - tropical climate .
ndumo is a large reserve , holding 10,117 ha of diverse habitats , including floodplains , sub - tropical bush , savannah and woodland , to riparian forest ( wesoowska and haddad , 2009 ) . the area directly surrounding the ngr ( 270013.8s 321649.9e ) is not formally protected and thus is covered in rural tribal villages , causing the vegetation to be heavily impacted by the villagers ' livestock and subsistence farming practices . approximately 80  km to the south
lies the kwa nyamazane conservancy ( knc ) ( 272334.9s 320840.8e ) , a small conservation area running along the phongola river and surrounded by large sects of agricultural land , most of it utilised for sugar cane farming .
these localities were specifically chosen as all three are located on , and are thus supplied by a permanent water source , the phongola river ( see fig .  1 ) .
frogs were collected via active sampling at night in all three localities as mentioned above .
all these sites were visited during the warmer and rainy months of february and november 2012 , april and november 2013 and february 2014 . during collection
captured frogs were held in disposable plastic bags and transported back to a field laboratory either at the ngr or knc , where they were identified to species level using du preez and carruthers ( 2009 ) .
a drop of blood was collected from each frog via cardiac or femoral venipuncture using a sterile heparinised insulin syringe .
a portion of this blood was used to prepare a thin blood smear , which once air - dried in a dust - proof container was fixed immediately using absolute methanol and stained thereafter using a modified solution of giemsa stain ( fluka , sigma - aldrich , steinheim , germany ) ; the other portion was dropped into a microcentrifuge tube with an equal volume of 70% ethanol for future molecular analysis .
all frogs were processed the morning after collection and were released within 24  h of capture .
smears were screened using a 100    immersion oil objective on a nikon eclipse e800 compound microscope ( nikon , amsterdam , netherlands ) , and images were captured with an attached nikon digital camera .
the average parasitaemia was calculated per 100 erythrocytes , with ~10 erythrocytes examined per blood smear following cook et  al .
the estimated average parasitaemia for extracellular parasites were calculated as number of parasites / per - slide ( ps ) with an approximate field of 20,000 blood cells examined .
this study received the relevant ethical approval ( north - west university ethics approval no . :
nwu-0000514-s3 ) , as well as approval to do research from the appropriate conservation authorities ( ezemvelo kzn wildlife , permits : op 674/2012 , op 5139/2012 , op 526/2014 , and op 839/2014 . ) .
the monte carlo variant of the fisher 's exact test , set to 10,000 replicates with a confidence interval of 99% , was employed to investigate significance in variation of prevalence between species , families , habitat types and sampling periods .
the habitat types were established based on those described by du preez and carruthers ( 2009 ) .
frog species were classified as terrestrial ( those species thriving and breeding away from a permanent water source for most of their lives ) , semi - terrestrial ( species thriving away from a permanent water source , but needing such a source to breed ) , semi - aquatic ( species requiring a position near a permanent water source for most of their lives in order to survive and breed ) and aquatic ( species permanently living and breeding in a water source , rarely leaving that source ) . the kruskal 
wallis test was used since it is suitable for non - parametric data and does not assume normal data distribution and equal sample size .
it was applied to determine significance levels ( p  <  0.05 ) of variation between infection intensity across species , families , habitat types , and sampling periods .
it was further employed to determine significance of variation of the overall intensity of hepatozoon and trypanosoma across frog species , families , habitats and sampling periods .
a non - parametric levenes 's test was used to verify the equality of variances in the samples ( homogeneity of variance , p  >  0.05 ) ( nordstokke and zumbo , 2007 , 2010 ) .
ndumo game reserve ( ngr ) ( 265200.0s 321500.0e ) is situated in the west of the maputaland bioregion , close to the borders of south africa , swaziland and mozambique .
the maputaland bioregion , located in northern kwazulu - natal ( kzn ) and crossing into southern mozambique , is one of the most biologically rich areas in southern africa ( haddad , 2003 ) and has a sub - tropical climate .
ndumo is a large reserve , holding 10,117 ha of diverse habitats , including floodplains , sub - tropical bush , savannah and woodland , to riparian forest ( wesoowska and haddad , 2009 ) . the area directly surrounding the ngr ( 270013.8s 321649.9e ) is not formally protected and thus is covered in rural tribal villages , causing the vegetation to be heavily impacted by the villagers ' livestock and subsistence farming practices . approximately 80  km to the south
lies the kwa nyamazane conservancy ( knc ) ( 272334.9s 320840.8e ) , a small conservation area running along the phongola river and surrounded by large sects of agricultural land , most of it utilised for sugar cane farming .
these localities were specifically chosen as all three are located on , and are thus supplied by a permanent water source , the phongola river ( see fig .  1 ) .
frogs were collected via active sampling at night in all three localities as mentioned above .
all these sites were visited during the warmer and rainy months of february and november 2012 , april and november 2013 and february 2014 . during collection
captured frogs were held in disposable plastic bags and transported back to a field laboratory either at the ngr or knc , where they were identified to species level using du preez and carruthers ( 2009 ) .
a drop of blood was collected from each frog via cardiac or femoral venipuncture using a sterile heparinised insulin syringe .
a portion of this blood was used to prepare a thin blood smear , which once air - dried in a dust - proof container was fixed immediately using absolute methanol and stained thereafter using a modified solution of giemsa stain ( fluka , sigma - aldrich , steinheim , germany ) ; the other portion was dropped into a microcentrifuge tube with an equal volume of 70% ethanol for future molecular analysis .
all frogs were processed the morning after collection and were released within 24  h of capture .
smears were screened using a 100    immersion oil objective on a nikon eclipse e800 compound microscope ( nikon , amsterdam , netherlands ) , and images were captured with an attached nikon digital camera .
the average parasitaemia was calculated per 100 erythrocytes , with ~10 erythrocytes examined per blood smear following cook et  al .
the estimated average parasitaemia for extracellular parasites were calculated as number of parasites / per - slide ( ps ) with an approximate field of 20,000 blood cells examined .
this study received the relevant ethical approval ( north - west university ethics approval no . :
nwu-0000514-s3 ) , as well as approval to do research from the appropriate conservation authorities ( ezemvelo kzn wildlife , permits : op 674/2012 , op 5139/2012 , op 526/2014 , and op 839/2014 . ) .
the monte carlo variant of the fisher 's exact test , set to 10,000 replicates with a confidence interval of 99% , was employed to investigate significance in variation of prevalence between species , families , habitat types and sampling periods .
the habitat types were established based on those described by du preez and carruthers ( 2009 ) .
frog species were classified as terrestrial ( those species thriving and breeding away from a permanent water source for most of their lives ) , semi - terrestrial ( species thriving away from a permanent water source , but needing such a source to breed ) , semi - aquatic ( species requiring a position near a permanent water source for most of their lives in order to survive and breed ) and aquatic ( species permanently living and breeding in a water source , rarely leaving that source ) . the kruskal 
wallis test was used since it is suitable for non - parametric data and does not assume normal data distribution and equal sample size .
it was applied to determine significance levels ( p  <  0.05 ) of variation between infection intensity across species , families , habitat types , and sampling periods .
it was further employed to determine significance of variation of the overall intensity of hepatozoon and trypanosoma across frog species , families , habitats and sampling periods .
a non - parametric levenes 's test was used to verify the equality of variances in the samples ( homogeneity of variance , p  >  0.05 ) ( nordstokke and zumbo , 2007 , 2010 ) .
blood smears were collected from 436 frogs of 29 species , 6 genera and 11 families ( table  1 ) . of these
15/29 ( 52% ) of the frog species were infected with haemoparasites , making up 87/436 ( 20% ) of the total number of frogs ( table  2 ) .
five groups of haemoparasites were recorded including intraerythrocytic haemogregarine and haemogregarine - like species of the genus hepatozoon and dactylosoma respectively , and intraerythrocytic organisms of a viral or bacterial nature , the species of which could not be identified ; furthermore , extracellular flagellate parasite species of the genus trypanosoma and microfilarid nematode species were observed .
hepatozoon species accounted for most of the infections at 59/436 ( 14% ) , followed by trypanosoma species at 46/436 ( 11% ) ; viral or bacterial infections , microfilarid infections and dactylosoma species , accounted for 6/436 ( 1% ) , 2/436 ( 0.5% ) and 13/436 ( 3% ) of the overall prevalence respectively ( table  2 ) . as for the intensity of the groups , hepatozoon showed an overall ( all infected frogs pooled ) intensity of 5% , the dactylosoma an overall intensity of 1% , the viral or bacterial infections an overall intensity of 87% , the trypanosoma an overall intensity of 11 per blood slide , and the microfilarid nematode infections an overall intensity of 15 per blood slide ( table  2 ) .
the overall prevalence of haemoparasites ( all parasite groups pooled ) varied significantly by frog species (   =  163.475 , p  <  0.01 ) .
ptychadena anchietae demonstrated the highest prevalence at 47/78 ( 60% ) and chiromantis xerampelina the lowest at 1/44 ( 2% ) ( table  2 ) .
upon division of the frog species into groups including aquatic ( two species ) , semi - aquatic ( 17 species ) , terrestrial ( one species ) and semi - terrestrial ( nine species ) ( see table  1 ) , it was observed that only the semi - aquatic and semi - terrestrial groups contained infected species ( table  2 ) .
these two groups varied significantly in prevalence of infection (   =  87.000 , p  <  0.01 ) , with 79% of the infected individuals from the semi - aquatic group and only 21% from the semi - terrestrial group . of the semi - aquatic group ,
the genus ptychadena had the highest diversity of haemoparasites , infected with all types as recorded in table  2 .
furthermore , p. anchietae , of all the infected frog species , revealed the highest prevalence of parasites , making up 47/87 ( 54% ) of the total with 47/78 ( 60% ) of the p. anchietae themselves found to be infected .
hepatozoon species accounted for most of the infections followed by trypanosoma species , significance of intensity calculated via the use of the kruskal 
=  0.028 ) , across families (   =  11.717 , p  =  0.006 ) , and across the different habitat types (   =  7.227 , p  =  0.007 ) showed a significant difference .
hyperolius marmoratus , in the semi - aquatic group , and amietophrynus maculatus , in the semi - terrestrial group , accounted for the highest intensities ( table  2 ) .
hepatozoon intensity across the different sampling periods , however , showed no significant variance (   =  4.177 , p  =  0.552 ) .
trypanosoma intensity across frog species (   =  11.919 , p  =  0.028 ) showed a significant difference ; however , across families (   =  3.802 , p  =  0.664 ) , habitat types (   =  0.330 , p  =  0.585 ) and sampling periods (   =  6.675 , p  =  0.147 ) , no significant difference was observed . in this case
hyperolius tuberilinguis , in the semi - aquatic group , and chiromantis xerampelina , in the semi - terrestrial group , accounted for the highest intensities ( table  2 ) . per locality
, it was observed that the ngr , with 26 species examined , showed a prevalence of 77/360 ( 21% ) as compared with outside the ngr , with eight species examined and a prevalence of 0/54 ( 0% ) , and the knc , with seven species examined and a prevalence of 10/22 ( 45% ) .
furthermore , the ngr had a higher diversity of haemoparasites , including 50/360 ( 14% ) infected with hepatozoon species , 11/360 ( 3% ) with dactylosoma species , 5/360 ( 1% ) with viral or bacterial organisms , 46/360 ( 13% ) with trypanosoma species and 2/360 ( 0.6% ) with microfilaria as compared with the knc frogs that were only infected with hepatozoon .
on the whole , 20% ( 87/436 ) of the frogs in this study were infected with at least one haemoparasite group , some infected up to five .
this was similar to previous comparable studies such as that of readel and goldberg ( 2010 ) in western uganda documenting a 17% ( 30/180 ) prevalence , even though this was found to be approximately half that of other studies in africa ( see mohammed and mansour , 1959 ; ball , 1967 ; readel and goldberg , 2010 ) . in this study
, hepatozoon species accounted for most of the infections at 14% , which was equally true for the survey done in uganda by readel and goldberg ( 2010 ) .
on the contrary , ball ( 1967 ) , during a survey completed in tanzania and kenya , found a considerably higher prevalence of 29% .
readel and goldberg ( 2010 ) suggested this may be attributable to availability of insect vectors .
trypanosome species were the second most common parasite infecting frogs in this study at 11% , just slightly higher than the 6% reported by readel and goldberg ( 2010 ) .
both the results of the present and the readel and goldberg ( 2010 ) studies ' conducted in africa are in contrast to what has been recorded in other similar studies but on different continents ( see barta and desser , 1984 ; barta et  al . , 1989 ) , in which the trypanosoma demonstrate a higher prevalence to that of hepatozoon or any other haemoparasite groups ( see werner , 1993 ) .
microfilarid infections from the south african frogs studied here , were also seen to occur in low numbers , similar to readel and goldberg ( 2010 ) .
infections not reported by readel and goldberg ( 2010 ) and ball ( 1967 ) , but reported in this study , were a dactylosoma species and viral or bacterial infections .
the only other study in africa to report on parasite intensities was that by readel and goldberg ( 2010 ) , in which hepatozoon species contained an average intensity of 2.3% , trypanosoma species had an average intensity of 7.2 parasites , and microfilariae had an average intensity of 11.2 parasites . in comparison the total parasite intensity for the current study was 5.3% for hepatozoon species , 10.6 for trypanosoma and 14.5 for microfilariae .
similar to readel and goldberg ( 2010 ) significant differences ( p  <  0.01 ) in the prevalence of parasites among frog species were recorded during the current study , with p. anchietae showing the highest prevalence and c. xerampelina the lowest .
both frog species prefer habitats close to water ( classified in this study as being semi - terrestrial ) ( du preez and carruthers , 2009 ) .
ptychadena anchietae is a grass frog and is often found around the water 's edge whilst c. xerampelina is an arboreal frog species .
since the abundance of possible vectors associated with water , such as mosquitoes and leeches , would be high in such habitats , it may explain the high prevalence of haemoparasites recorded from p. anchietae .
the reason for such a low prevalence in c. xerampelina , particularly for hepatozoon species , which may be mosquito transmitted in such an environment ( desser et  al . , 1995 ; davies and johnston , 2000 ) , can not be explained .
this result is particularly peculiar since both frog species were infected with trypanosomes ( only a single individual of c. xerampelina ) , which are mosquito and leech transmitted ( barta and desser , 1984 ) .
one of the only possibilities could be , since hepatozoon is transmitted via the ingestion of the infected invertebrate or vertebrate ( davies and johnston , 2000 ) , that c. xerampelina prefers a diet not inclusive of mosquitoes and other frogs .
division of the frog species into groups showed that only the semi - aquatic and semi - terrestrial groups contained infected species , these two groups varying significantly in prevalence of infection ( p  <  0.01 ) .
the semi - aquatic group had the highest prevalence , likely attributable to the ptychadena species , one of them p. anchietae .
the ptychadena species also showed the highest diversity of haemoparasites , infected with all five recorded groups . furthermore , of all the frog species , p. anchietae was the only species to be parasitised with a species of dactylosoma .
these parasites are closely associated with water and thus are suggested to be transmitted by a leech vector ( barta , 1991 ) .
reports of dactylosoma parasitising frogs in africa are numerous , accounts of these organisms from at least five countries and approximately eight species of frog ( see barta , 1991 ) .
one such report was from south africa from the bufonid a. regularis ( most likely amietophrynus gutturalis ) by fantham et  al .
are referred to as a single species dactylosoma ranarum ( see barta , 1991 ) ; however , only future molecular work will be able to clarify if the species here is one of the same .
furthermore , the ptychadenidae were the only frogs found infected with viral or bacterial organisms . viral or bacterial infections have been recorded from a cosmopolitan distribution of amphibians ( see desser , 1987 ) . unfortunately ,
very little is known about the identity , classification and effect of these organisms ( see desser , 1987 ; davies and johnston , 2000 ; davis et  al . ,
alves de matos and paperna ( 1993 ) presented the most recent study of uncertain erythrocyte virus infections from p. anchietae in south africa .
these virus or bacterial infections were found to be similar to several different viruses of the frog erythrocytic virus ( fev ) group such as toddia , pirhemocyton and other rickettsiales .
in contrast to the above findings in both the semi - aquatic and semi - terrestrial groups , the frog species from the terrestrial as well as aquatic groups were not observably parasitaemic .
since b. adspersus ( terrestrial ) spends most of its life underground ( du preez and carruthers , 2009 ) , contact with vectors would be rare .
however , since the semi - aquatic group had the highest prevalence of parasites , most likely due to the frequent contact with vectors , it was expected that those of the aquatic group would be equally parasitised . yet , as in readel and goldberg ( 2010 ) , the species of xenopus ( aquatic ) here were found to be uninfected .
such a finding is surprising as it would be expected that xenopus should contain a rather high prevalence of parasites , particularly as it is well known that leeches feed on these frogs ( see badets and du preez , 2014 ; kruger and du preez , 2015 ) .
possible explanations for this could be that haemoparasites infecting xenopus are extremely host specific or were simply not present in the area sampled .
intensity of hepatozoon species across frog species and families , as well as across habitat types were found to be significant ( p  =  0.007 ) .
hyperolius marmoratus ( hyperolidae ) of the semi - aquatic group , and a. maculatus ( bufonidae ) from the semi - terrestrial group , had the highest intensity .
hyperolius marmoratus may be found permanently on the edges of water , where vector abundance and contact rates are likely to be high , thus accounting for this high intensity .
amietophrynus maculatus appears to favour more static , shallow water bodies , which are also favoured by mosquito species , the high contact rates with possibly hepatozoon infected mosquitoes would thus be high .
hepatozoon species have been reported and described from a few hyperolius species in africa , though the majority have been reported from bufonid species such as a. maculatus ( see netherlands et  al .
trypanosoma species intensities varied significantly only across species ( p  =  0.028 ) , being highest in the semi - aquatic h. tuberilinguis and the semi - terrestrial c. xerampelina . these two species are permanently associated with water , and thus always in close association with an abundance of possible vectors . a plethora of trypanosome species have been described and reported from numerous african frog species ,
unfortunately many reports contain inadequate taxonomic descriptions and in numerous cases they are simply referred to as a trypanosoma sp . without any morphological data provided on the specific parasite ( see bardsley and harmsen , 1973 ; telford , 2009 ) .
furthermore , since trypanosomes are known to be pleomorphic , the true diversity seen in this study can not be realised , and thus future molecular work along with morphological description is intended in order to differentiate between species and life stages of these organisms .
intensity across sampling periods for both hepatozoon and trypanosoma species was insignificant ( p  =  0.552 and p  =  0.147 respectively ) , likely due to sampling occurring only during the wet seasons .
parasites have always been seen in a negative light , especially with regards to human and livestock health .
however , within the natural environment parasites may be seen as a crucial part of a functional and healthy ecosystem .
parasites make a profound impact on the biodiversity of an ecosystem by influencing aspects such as host competition , migration , speciation and stability ( combes , 1996 ) .
furthermore , parasites reflect their host species ' environmental interactions , revealing feeding behaviour , geographical ranges and social systems ( dobson et  al . , 2008 ) . in a stable and healthy natural ecosystem parasites and their hosts
have had the opportunity to co - evolve , the parasite causing few pathogenic effects in a healthy host animal . if however , this well established co - existence is disturbed by , for example , habitat destruction or the indiscriminate movement of host animals between habitats ; pathogenic effects may become apparent resulting in the destabilisation of the host population ( see combes , 1996 ) .
additionally , in light of the above , the loss of parasite diversity would very likely have unforeseen , but grave consequences , especially with respect to regulation of host populations and their abundance within the communities ( dobson et  al . , 2008 ) .
( 2008 ) , if the main aim of conservation biologists is to conserve fully functional food webs , it is imperative that parasites are thus also included within biodiversity conservation .
ndumo game reserve was found to harbour the highest diversity of both frog species and haemoparasites as compared with the other two sites ( see table  1 ) , which are impacted by rural village settlements and peripheral commercial sugar cane agriculture respectively .
anthropogenic impacts , as found in readel and goldberg ( 2010 ) , may account for the lack of diversity in these two sites , affecting vector distributions and contact rates between frog hosts and vectors .
it is anticipated that through future work , including both morphological and molecular descriptions of the parasites reported in this study , that the biodiversity of this region will be elucidated .
furthermore , it is hoped that with this biodiversity knowledge and the identification of potential vectors , the effects human activities may have on frog haemoparasite life cycles and as such their biodiversity will be clarified .
 highlightsfirst frog ( 11 families , 6 genera , 29 spp . ) 
 haemoparasite survey in south africa.showing a higher biodiversity as compared with similar research in africa.intraerythrocytic and extracellular haemoparasites of five groups found.hepatozoon and trypanosoma spp . 
 were the most prevalent.protected areas boast higher parasite diversity compared with human impacted sites . 
Introduction
Materials and methods
Study area and frog collection
Preparation and screening of frog blood
Statistical analysis
Results
Discussion
Conflict of interest