Biodiversity of Frog Haemoparasites From Sub-tropical Northern KwaZulu-Natal, South Africa

Biodiversity of frog haemoparasites from sub-tropical northernKwaZulu-Natal South AfricaEdward C Netherlands Courtney A Cook Donnavan JD Kruger Louis H du PreezNico J SmitUnit for Environmental Sciences and Management North-West University Private Bag X6001 Potchefstroom South Africa

A R T I C L E I N F O

Article historyReceived 31 October 2014Revised 13 January 2015Accepted 14 January 2015

KeywordsAmphibianApicomplexanBlood parasite surveyFrog haematozoansHaemoflagellateMicrofilarid

A B S T R A C T

Since South Africa boasts a high biodiversity of frog species a multispecies haemoparasite survey wasconducted by screening the blood from 29 species and 436 individual frogs Frogs were collected at threelocalities in sub-tropical KwaZulu-Natal a hotspot for frog diversity Twenty per cent of the frogs wereinfected with at least one of five groups of parasites recorded Intraerythrocytic parasites comprisingHepatozoon Dactylosoma and viral or bacterial organisms as well as extracellular parasites including try-panosomes and microfilarid nematodes were found A significant difference (P lt 001) in the prevalenceof parasitaemia was found across species those semi-aquatic species demonstrating the highest fol-lowed by semi-terrestrial frog species None of those species described as purely terrestrial and aquaticwere infected Hepatozoon and Trypanosoma species accounted for most of the infections the former dem-onstrating significant differences in intensity of infection across species families and habitat types (P = 0028P = 0006 P = 0007 respectively) Per locality the first the formally protected Ndumo Game Reserve hadthe highest biodiversity of haemoparasite infections with all five groups of parasites recorded The othertwo sites that is the area bordering the reserve and the Kwa Nyamazane Conservancy had a lower di-versity with no parasite infections recorded and only Hepatozoon species recorded respectively Such findingscould be ascribed to the anthropogenic impact on the latter two sites the first by the rural village ac-tivities and the second by the bordering commercial sugar cane agriculture Future studies should includeboth morphological and molecular descriptions of the above parasites as well as the identification ofpotential vectors possibly clarifying the effects human activities may have on frog haemoparasite lifecycles and as such their biodiversitycopy 2015 The Authors Published by Elsevier Ltd on behalf of Australian Society for Parasitology This is an

open access article under the CC BY-NC-ND license (httpcreativecommonsorglicensesby-nc-nd40)

1 Introduction

Amphibians are the most threatened vertebrate group suffer-ing large-scale declines in species diversity since at least accordingto historical data the 1970s (Stuart et al 2004) A decade ago theIUCNrsquos Global Amphibian Assessment indicated that a third of theestimated amphibian species had declined or become extinct (Stuartet al 2004 Beebee and Griffiths 2005) Such declines may be at-tributed to a number of factors ranging from habitat destructionpollution and exploitation to climate change and disease (Beebeeand Griffiths 2005) The disease known as chytridiomycosis (am-phibian chytrid) caused by the fungal pathogen Batrachochytriumdendrobatidis has been responsible for major global amphibian

declines (Readel and Goldberg 2010) Along with chytrid amphib-ians are host to a wide variety of parasites (du Preez and Carruthers2009 Netherlands et al 2014a) including intraerythrocytic and ex-tracellular haemoparasites ranging from protozoans comprising bothintracellular apicomplexans (Davies and Johnston 2000) and ex-tracellular flagellates (Acosta et al 2013) to extracellular nematodemicrofilariae (Baker 2008) as well as those intracellular parasitesof uncertain identity such as the viral and bacterial infections (Daviesand Johnston 2000 Davis et al 2009) The most attention howeverhas been given to those parasites of the first three groups men-tioned most likely due to the frequent findings and thus greaterbasis of knowledge of these organisms in anuran hosts Further-more of these three groups those of the Protozoa particularly theapicomplexans would appear to be the most studied of all (seeDavies and Johnston 2000 Netherlands et al 2014a Netherlandset al 2014b)

However since few parasite surveys on frogs have been carriedout in sub-Saharan Africa the degree of this haemoparasite diver-sity remains unknown (Readel and Goldberg 2010 Netherlands

Corresponding author Unit for Environmental Sciences and ManagementNorth-West University Private Bag X6001 Potchefstroom 2520 South Africa Tel+2718 299 2372 fax +2718 299 2370

E-mail address ecnetherlandsgmailcom (EC Netherlands)

httpdxdoiorg101016jijppaw2015010032213-2244copy 2015 The Authors Published by Elsevier Ltd on behalf of Australian Society for Parasitology This is an open access article under the CC BY-NC-ND license(httpcreativecommonsorglicensesby-nc-nd40)

International Journal for Parasitology Parasites and Wildlife 4 (2015) 135ndash141

Contents lists available at ScienceDirect

International Journal for ParasitologyParasites and Wildlife

journal homepage wwwelseviercom locate i jppaw

et al 2014a 2014b) Yet such diversity of knowledge regarding theseparasites is necessary before further studies can be done on eluci-dating the effects that these parasites may have on their natural hostsand the role these parasites may have in amphibian conservation

Southern Africa currently boasts 159 known species of frogs in33 genera and 13 families (du Preez and Carruthers 2009 Channingand Baptista 2013 Channing et al 2013a 2013b Conradie 2014)This study presents the results of a haemoparasite survey of frogsfrom three localities in KwaZulu-Natal South Africa (see Fig 1) Lo-calities include the formally protected Ndumo Game Reserve (NGR)and the reserversquos anthropogenically impacted surrounds as well asKwa Nyamazane Conservancy (KNC) All three sampling areas fallwithin a sub-tropical region known for its biological richness andas such the province KZN boasts the highest diversity of frog speciesin South Africa (see du Preez and Carruthers 2009) The followingstudy thus aimed to determine and record through a multispecieshaemoparasite survey on frogs if this parasite diversity paralleledthat of its rich frog diversity

2 Materials and methods

21 Study area and frog collection

Ndumo Game Reserve (NGR) (26deg52prime000PrimeS 32deg15prime000PrimeE) issituated in the West of the Maputaland bioregion close to the

borders of South Africa Swaziland and Mozambique The Maputalandbioregion located in northern KwaZulu-Natal (KZN) and crossinginto southern Mozambique is one of the most biologically rich areasin southern Africa (Haddad 2003) and has a sub-tropical climateNdumo is a large reserve holding 10117 ha of diverse habitatsincluding floodplains sub-tropical bush savannah and woodlandto riparian forest (Wesołowska and Haddad 2009) The areadirectly surrounding the NGR (27deg00prime138PrimeS 32deg16prime499PrimeE) isnot formally protected and thus is covered in rural tribal villagescausing the vegetation to be heavily impacted by the villagersrsquolivestock and subsistence farming practices Approximately 80 kmto the south lies the Kwa Nyamazane Conservancy (KNC)(27deg23prime349PrimeS 32deg08prime408PrimeE) a small conservation area runningalong the Phongola River and surrounded by large sects of agricul-tural land most of it utilised for sugar cane farming These localitieswere specifically chosen as all three are located on and are thussupplied by a permanent water source the Phongola River (seeFig 1)

Frogs were collected via active sampling at night in all three lo-calities as mentioned above All these sites were visited during thewarmer and rainy months of February and November 2012 Apriland November 2013 and February 2014 During collection possi-ble invertebrate vectors feeding on frogs were searched for howevernone were observed Captured frogs were held in disposable plasticbags and transported back to a field laboratory either at the NGR

Fig 1 Map displaying the three sampling localities in northern KwaZulu-Natal South Africa Map displaying the three sampling localities at which frogs were surveyed forhaemoparasite biodiversity top to bottom Ndumo Game Reserve (NGR) outside NGR and Kwa Nyamazane Conservancy in northern KwaZulu-Natal South Africa All sam-pling sites were directly or indirectly linked to the Phongolo River

136 EC Netherlands et alInternational Journal for Parasitology Parasites and Wildlife 4 (2015) 135ndash141

or KNC where they were identified to species level using du Preezand Carruthers (2009)

22 Preparation and screening of frog blood

A drop of blood was collected from each frog via cardiac orfemoral venipuncture using a sterile heparinised insulin syringe Aportion of this blood was used to prepare a thin blood smear whichonce air-dried in a dust-proof container was fixed immediately usingabsolute methanol and stained thereafter using a modified solu-tion of Giemsa stain (FLUKA Sigma-Aldrich Steinheim Germany)the other portion was dropped into a microcentrifuge tube with anequal volume of 70 ethanol for future molecular analysis All frogswere processed the morning after collection and were releasedwithin 24 h of capture

Smears were screened using a 100 times immersion oil objective ona Nikon Eclipse E800 compound microscope (Nikon AmsterdamNetherlands) and images were captured with an attached Nikondigital camera The average parasitaemia was calculated per 100erythrocytes with ~104 erythrocytes examined per blood smear fol-lowing Cook et al (2009) The estimated average parasitaemia forextracellular parasites were calculated as number of parasitesper-slide (ps) with an approximate field of 20000 blood cells examinedThis study received the relevant ethical approval (North-West Uni-versity ethics approval no NWU-00005ndash14-S3) as well as approvalto do research from the appropriate conservation authorities(Ezemvelo KZN Wildlife permits OP 6742012 OP 51392012 OP5262014 and OP 8392014)

23 Statistical analysis

The Monte Carlo variant of the Fisherrsquos exact test set to 10000replicates with a confidence interval of 99 was employed to in-vestigate significance in variation of prevalence between speciesfamilies habitat types and sampling periods The habitat types wereestablished based on those described by du Preez and Carruthers(2009) Frog species were classified as terrestrial (those species thriv-ing and breeding away from a permanent water source for most oftheir lives) semi-terrestrial (species thriving away from a perma-nent water source but needing such a source to breed) semi-aquatic (species requiring a position near a permanent water sourcefor most of their lives in order to survive and breed) and aquatic(species permanently living and breeding in a water source rarelyleaving that source) The KruskalndashWallis test was used since it issuitable for non-parametric data and does not assume normal datadistribution and equal sample size It was applied to determine sig-nificance levels (P lt 005) of variation between infection intensityacross species families habitat types and sampling periods It wasfurther employed to determine significance of variation of the overallintensity of Hepatozoon and Trypanosoma across frog species fami-lies habitats and sampling periods A non-parametric Levenesrsquos testwas used to verify the equality of variances in the samples (homo-geneity of variance P gt 005) (Nordstokke and Zumbo 2007 2010)All statistical analyses were performed using IBM SPSS Statistics ver22 (SPSS 2013)

3 Results

Blood smears were collected from 436 frogs of 29 species 6genera and 11 families (Table 1) Of these 1529 (52) of the frogspecies were infected with haemoparasites making up 87436 (20)of the total number of frogs (Table 2) Five groups of haemoparasiteswere recorded including intraerythrocytic haemogregarine andhaemogregarine-like species of the genus Hepatozoon andDactylosoma respectively and intraerythrocytic organisms of a viralor bacterial nature the species of which could not be identified fur-

thermore extracellular flagellate parasite species of the genusTrypanosoma and microfilarid nematode species were observed

Hepatozoon species accounted for most of the infections at 59436 (14) followed by Trypanosoma species at 46436 (11) viralor bacterial infections microfilarid infections and Dactylosomaspecies accounted for 6436 (1) 2436 (05) and 13436 (3) ofthe overall prevalence respectively (Table 2) As for the intensity ofthe groups Hepatozoon showed an overall (all infected frogs pooled)intensity of 5 the Dactylosoma an overall intensity of 1 the viralor bacterial infections an overall intensity of 87 the Trypano-soma an overall intensity of 11 per blood slide and the microfilaridnematode infections an overall intensity of 15 per blood slide(Table 2) The overall prevalence of haemoparasites (all parasitegroups pooled) varied significantly by frog species (χ2 = 163475P lt 001) Ptychadena anchietae demonstrated the highest preva-lence at 4778 (60) and Chiromantis xerampelina the lowest at 144(2) (Table 2) Upon division of the frog species into groups includ-ing aquatic (two species) semi-aquatic (17 species) terrestrial (onespecies) and semi-terrestrial (nine species) (see Table 1) it was ob-served that only the semi-aquatic and semi-terrestrial groupscontained infected species (Table 2) These two groups varied sig-nificantly in prevalence of infection (χ2 = 87000 P lt 001) with 79of the infected individuals from the semi-aquatic group and only21 from the semi-terrestrial group Of the semi-aquatic group thegenus Ptychadena had the highest diversity of haemoparasites in-fected with all types as recorded in Table 2 Furthermore P anchietaeof all the infected frog species revealed the highest prevalence ofparasites making up 4787 (54) of the total with 4778 (60) ofthe P anchietae themselves found to be infected

Hepatozoon species accounted for most of the infections fol-lowed by Trypanosoma species significance of intensity calculatedvia the use of the KruskalndashWallis test Hepatozoon intensity acrossfrog species (χ2 = 17683 P = 0028) across families (χ2 = 11717P = 0006) and across the different habitat types (χ2 = 7227 P = 0007)showed a significant difference Hyperolius marmoratus in the semi-aquatic group and Amietophrynus maculatus in the semi-terrestrialgroup accounted for the highest intensities (Table 2) Hepatozoonintensity across the different sampling periods however showedno significant variance (χ2 = 4177 P = 0552) Trypanosoma inten-sity across frog species (χ2 = 11919 P = 0028) showed a significantdifference however across families (χ2 = 3802 P = 0664) habitattypes (χ2 = 0330 P = 0585) and sampling periods (χ2 = 6675P = 0147) no significant difference was observed In this caseHyperolius tuberilinguis in the semi-aquatic group and Chiromantisxerampelina in the semi-terrestrial group accounted for the highestintensities (Table 2)

Per locality it was observed that the NGR with 26 species ex-amined showed a prevalence of 77360 (21) as compared withoutside the NGR with eight species examined and a prevalence of054 (0) and the KNC with seven species examined and a prev-alence of 1022 (45) Furthermore the NGR had a higher diversityof haemoparasites including 50360 (14) infected with Hepatozoonspecies 11360 (3) with Dactylosoma species 5360 (1) with viralor bacterial organisms 46360 (13) with Trypanosoma species and2360 (06) with microfilaria as compared with the KNC frogs thatwere only infected with Hepatozoon

4 Discussion

On the whole 20 (87436) of the frogs in this study were in-fected with at least one haemoparasite group some infected up tofive This was similar to previous comparable studies such as thatof Readel and Goldberg (2010) in western Uganda documenting a17 (30180) prevalence even though this was found to be approx-imately half that of other studies in Africa (see Mohammed andMansour 1959 Ball 1967 Readel and Goldberg 2010) In this study

137EC Netherlands et alInternational Journal for Parasitology Parasites and Wildlife 4 (2015) 135ndash141

Hepatozoon species accounted for most of the infections at 14which was equally true for the survey done in Uganda by Readeland Goldberg (2010) On the contrary Ball (1967) during a surveycompleted in Tanzania and Kenya found a considerably higher prev-alence of 29 Readel and Goldberg (2010) suggested this may beattributable to availability of insect vectors Trypanosome specieswere the second most common parasite infecting frogs in this studyat 11 just slightly higher than the 6 reported by Readel andGoldberg (2010) Both the results of the present and the Readel andGoldberg (2010) studiesrsquo conducted in Africa are in contrast to whathas been recorded in other similar studies but on different conti-nents (see Barta and Desser 1984 Barta et al 1989) in which theTrypanosoma demonstrate a higher prevalence to that of Hepatozoonor any other haemoparasite groups (see Werner 1993) Microfilaridinfections from the South African frogs studied here were also seento 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 andviral or bacterial infections The only other study in Africa to reporton parasite intensities was that by Readel and Goldberg (2010) inwhich Hepatozoon species contained an average intensity of 23Trypanosoma species had an average intensity of 72 parasites andMicrofilariae had an average intensity of 112 parasites In compar-ison the total parasite intensity for the current study was 53 forHepatozoon species 106 for Trypanosoma and 145 for Microfilariae

Similar to Readel and Goldberg (2010) significant differences(P lt 001) in the prevalence of parasites among frog species wererecorded during the current study with P anchietae showing thehighest prevalence and C xerampelina the lowest Both frog speciesprefer habitats close to water (classified in this study as being semi-terrestrial) (du Preez and Carruthers 2009) Ptychadena anchietaeis a grass frog and is often found around the waterrsquos edge whilst Cxerampelina is an arboreal frog species Since the abundance of pos-sible vectors associated with water such as mosquitoes and leecheswould be high in such habitats it may explain the high preva-lence of haemoparasites recorded from P anchietae The reason forsuch a low prevalence in C xerampelina particularly for Hepatozoonspecies which may be mosquito transmitted in such an environ-ment (Desser et al 1995 Davies and Johnston 2000) cannot beexplained This result is particularly peculiar since both frog specieswere infected with trypanosomes (only a single individual of Cxerampelina) which are mosquito and leech transmitted (Barta andDesser 1984) One of the only possibilities could be since Hepatozoonis transmitted via the ingestion of the infected invertebrate or ver-tebrate (Davies and Johnston 2000) that C xerampelina prefers adiet not inclusive of mosquitoes and other frogs Future diet studiesmay help to clarify this finding

Division of the frog species into groups showed that only thesemi-aquatic and semi-terrestrial groups contained infected speciesthese two groups varying significantly in prevalence of infection

Table 1Frog species divided into associated habitat types and listed alphabetically with families as well as numbers collected at Ndumo Game Reserve (NGR) the locality border-ing NGR (BNGR) and the Kwa Nyamazane Conservancy (KNC)

Habitat type Frog species Family Locality No collected

Terrestrial (1) Breviceps adspersus Breviciptidae NGR 4Semi-terrestrial (9) Amietophrynus garmani Bufonidae NGR 23

BNGR 2KNC 5

Amietophrynus gutturalis Bufonidae NGR 1KNC 3

Amietophrynus maculatus Bufonidae NGR 9Chiromantis xerampelina Rhacophoridae NGR 43

BNGR 1Leptopelis mossambicus Arthroleptidae NGR 2Schismaderma carens Bufonidae NGR 7Tomopterna cryptotis Pyxicephalidae NGR 6Tomopterna krugerensis Pyxicephalidae NGR 1Tomopterna natalensis Pyxicephalidae NGR 1

Semi-aquatic (17) Afrixalus aureus Hyperoliidae NGR 14Afrixalus delicatus Hyperoliidae NGR 7Afrixalus fornasinii Hyperoliidae NGR 2Cacosternum boettgeri Pyxicephalidae BNGR 11

KNC 1Hemisus marmoratus Hemisotidae NGR 22Hildebrandtia ornata Ptychadenidae NGR 6Hyperolius argus Hyperoliidae BNGR 24Hyperolius marmoratus Hyperoliidae NGR

BNGR2010

KNC 6Hyperolius pusillus Hyperoliidae NGR 10

BNGR 1KNC 3

Hyperolius tuberilinguis Hyperoliidae NGR 12Kassina maculata Hyperoliidae NGR 8Kassina senegalensis Hyperoliidae KNC 3Phrynobatrachus mababiensis Phrynobatrachidae NGR 13Phrynomantis bifasciatus Microhylidae NGR 1Ptychadena anchietae Ptychadenidae NGR 77

KNC 1Ptychadena mascareniensis Ptychadenidae NGR 5

BNGR 2Ptychadena mossambica Ptychadenidae NGR 19

Aquatic (2) Xenopus laevis Pipidae NGR 1Xenopus muelleri Pipidae NGR 46

BNGR 3Total 29 11 3 436


(P lt 001) The semi-aquatic group had the highest prevalence likelyattributable to the Ptychadena species one of them P anchietae ThePtychadena species also showed the highest diversity ofhaemoparasites infected with all five recorded groups Further-more of all the frog species P anchietae was the only species to beparasitised with a species of Dactylosoma These parasites are closelyassociated with water and thus are suggested to be transmitted bya leech vector (Barta 1991) Reports of Dactylosoma parasitising frogsin Africa are numerous accounts of these organisms from at leastfive countries and approximately eight species of frog (see Barta1991) One such report was from South Africa from the bufonid Aregularis (most likely Amietophrynus gutturalis) by Fantham et al(1942) In all these reports the Dactylosoma species are referred toas a single species Dactylosoma ranarum (see Barta 1991) howeveronly future molecular work will be able to clarify if the species hereis one of the same Furthermore the Ptychadenidae were the onlyfrogs found infected with viral or bacterial organisms Viral or bac-terial infections have been recorded from a cosmopolitan distributionof amphibians (see Desser 1987) Unfortunately very little is knownabout the identity classification and effect of these organisms (seeDesser 1987 Davies and Johnston 2000 Davis et al 2009) Alvesde Matos and Paperna (1993) presented the most recent study ofuncertain erythrocyte virus infections from P anchietae in SouthAfrica These virus or bacterial infections were found to be similarto several different viruses of the Frog Erythrocytic Virus (FEV) groupsuch as Toddia Pirhemocyton and other Rickettsiales In contrast tothe above findings in both the semi-aquatic and semi-terrestrialgroups the frog species from the terrestrial as well as aquatic groupswere 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 likelydue to the frequent contact with vectors it was expected that thoseof the aquatic group would be equally parasitised Yet as in Readeland Goldberg (2010) the species of Xenopus (aquatic) here werefound to be uninfected Such a finding is surprising as it would beexpected that Xenopus should contain a rather high prevalence ofparasites particularly as it is well known that leeches feed on thesefrogs (see Badets and Du Preez 2014 Kruger and Du Preez 2015)Possible explanations for this could be that haemoparasites infect-ing Xenopus are extremely host specific or were simply not presentin the area sampled

Intensity of Hepatozoon species across frog species and fami-lies as well as across habitat types were found to be significant(P = 0007) Hyperolius marmoratus (Hyperolidae) of the semi-aquatic group and A maculatus (Bufonidae) from the semi-terrestrialgroup had the highest intensity Hyperolius marmoratus may be foundpermanently on the edges of water where vector abundance andcontact rates are likely to be high thus accounting for this high in-tensity Amietophrynus maculatus appears to favour more staticshallow water bodies which are also favoured by mosquito speciesthe high contact rates with possibly Hepatozoon infected mosqui-toes would thus be high Hepatozoon species have been reportedand described from a few Hyperolius species in Africa though themajority have been reported from bufonid species such as Amaculatus (see Netherlands et al 2014b) Trypanosoma species in-tensities varied significantly only across species (P = 0028) beinghighest in the semi-aquatic H tuberilinguis and the semi-terrestrialC xerampelina These two species are permanently associated withwater and thus always in close association with an abundance ofpossible vectors A plethora of trypanosome species have been de-scribed and reported from numerous African frog speciesunfortunately many reports contain inadequate taxonomic descrip-tions and in numerous cases they are simply referred to as aTrypanosoma sp without any morphological data provided on thespecific parasite (see Bardsley and Harmsen 1973 Telford 2009)Ta

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Furthermore since trypanosomes are known to be pleomorphic thetrue diversity seen in this study cannot be realised and thus futuremolecular work along with morphological description is intendedin order to differentiate between species and life stages of these or-ganisms Intensity across sampling periods for both Hepatozoon andTrypanosoma species was insignificant (P = 0552 and P = 0147 re-spectively) likely due to sampling occurring only during the wetseasons

Parasites have always been seen in a negative light especiallywith regards to human and livestock health However within thenatural environment parasites may be seen as a crucial part of afunctional and healthy ecosystem Parasites make a profound impacton the biodiversity of an ecosystem by influencing aspects such ashost competition migration speciation and stability (Combes 1996)Furthermore parasites reflect their host speciesrsquo environmental in-teractions revealing feeding behaviour geographical ranges andsocial systems (Dobson et al 2008) In a stable and healthy naturalecosystem parasites and their hosts have had the opportunity to co-

evolve the parasite causing few pathogenic effects in a healthy hostanimal If however this well established co-existence is disturbedby for example habitat destruction or the indiscriminate move-ment of host animals between habitats pathogenic effects maybecome apparent resulting in the destabilisation of the host pop-ulation (see Combes 1996) Additionally in light of the above theloss of parasite diversity would very likely have unforeseen but graveconsequences especially with respect to regulation of host popu-lations and their abundance within the communities (Dobson et al2008) As aptly put by Dobson et al (2008) if the main aim of con-servation biologists is to conserve fully functional food webs it isimperative that parasites are thus also included within biodiver-sity conservation Ndumo Game Reserve was found to harbour thehighest diversity of both frog species and haemoparasites as com-pared with the other two sites (see Table 1) which are impactedby rural village settlements and peripheral commercial sugar caneagriculture respectively Anthropogenic impacts as found in Readeland Goldberg (2010) may account for the lack of diversity in these

Fig 2 Micrographs of various frog haemoparasites encountered in the current study Haemoparasites from the peripheral blood of 15 frog species collected from threelocalities in northern KwaZulu-Natal stained with Giemsa stain (AndashE) gamonts of Hepatozoon species (FndashG) primary and secondary stage gamonts of Dactylosoma species(HndashJ) viral or bacterial inclusions (K) microfilarid nematode species (LndashT) Trypanosoma species Scale bar 10 μm


two sites affecting vector distributions and contact rates betweenfrog hosts and vectors

This study represents the first multispecies haemoparasite surveydone on frogs in South Africa It is anticipated that through futurework including both morphological and molecular descriptions ofthe parasites reported in this study that the biodiversity of this regionwill be elucidated Furthermore it is hoped that with this biodi-versity knowledge and the identification of potential vectors theeffects human activities may have on frog haemoparasite life cyclesand as such their biodiversity will be clarified

Acknowledgements

We are thankful to Ndumo Game Reserve and Kwa NyamazaneConservancy for allowing us to collect samples as well as to bothDr Leon Meyer and Mr Nico Wolmerans for their much appreci-ated help during collection Prof John R Barta from the Universityof Guelph Canada for aiding in the identification of some of thehaemogregarines as well as providing us with numerous sourcesof relevant literature In addition we are grateful for the financialassistance of the National Research Foundation (NRF) of South Africato CAC (NRF Scarce Skills Postdoctoral Scholarship-GrantSFP13090332476) and to ECN (NRF Scarce Skills Masters Scholarship-Grant UID 89924) Opinions expressed and conclusions arrived atare those of the authors and are not necessarily to be attributed tothe NRF The fieldwork and running expenses of this research wasfunded by the Water Research Commission (WRC) of South Africa(Project K5-2185 NJ Smit PI) Ezemvelo KZN Wildlife is thankedfor research permits OP 6742012 OP 51392012 OP 5262014 andOP 8392014

Conflict of interest

The authors declared that there is no conflict of interest

References

Acosta ICL Costa AP Nunes PH Gondim MFN Gatti A Rossi JL et al 2013Morphological and molecular characterization and phylogenetic relationshipsof a new species of trypanosome in Tapirus terrestris (lowland tapir) Trypanosomaterrestris sp nov from Atlantic Rainforest of southeastern Brazil Parasit Vectors6 349

Alves de Matos AP Paperna I 1993 Ultrastructure of erythrocytic virus of the SouthAfrican anuran Ptychadena anchietae Dis Aquat Org 16 105ndash109

Badets M Du Preez L 2014 Phoretic interaction between the kangaroo leechMarsupiobdella africana (Hirudinea Glossiphoniidae) and the cape river crabPotamonautes perlatus (Decapoda Potamonautidae) Int J Parasitol ParasitesWildl 3 6ndash11

Baker DG 2008 Flynnrsquos Parasites of Laboratory Animals John Wiley amp Sons pp149ndash150

Ball GH 1967 Blood sporozoans from East African Amphibia J Eukaryot Microbiol14 521ndash527

Bardsley JE Harmsen R 1973 The trypanosomes of anura Adv Parasitol 11 1ndash73Barta JR 1991 The dactylosomatidae Adv Parasitol 30 1ndash37Barta JR Desser SS 1984 Blood parasites of amphibians from Algonquin park

Ontario J Wildl Dis 20 180ndash189Barta JR Boulard Y Desser SS 1989 Blood parasites of Rana esculenta from

Corsica comparison of its parasites with those of eastern North American ranidsin the context of host phylogeny Trans Am Microsc Soc 108 6ndash20

Beebee TJC Griffiths RA 2005 The amphibian decline crisis A watershed forconservation biology Biol Conserv 125 271ndash285

Channing A Baptista N 2013 Amietia angolensis and A fuscigula (AnuraPyxicephalidae) in southern Africa a cold case reheated Zootaxa 3640 501ndash520

Channing A Hillers A Loumltters S Roumldel MO Schick S Conradie W et al 2013aTaxonomy of the super-cryptic Hyperolius nasutus group of long reed frogs ofAfrica (Anura Hyperoliidae) with descriptions of six new species Zootaxa 3620301ndash350

Channing A Schmitz A Burger M Kielgast J 2013b A molecular phylogeny ofAfrican Dainty Frogs with the description of four new species (AnuraPyxicephalidae Cacosternum) Zootaxa 3701 518ndash550

Combes C 1996 Parasites biodiversity and ecosystem stability Biodivers Conserv5 953ndash962

Conradie W 2014 The King of the Dwarves a new cryptic species of Dainty Frog(Anura Pyxicephalidae Cacosternum) from the eastern Great Escarpment ofSouth Africa Zootaxa 3785 438ndash452

Cook CA Smit NJ Davies AJ 2009 A redescription of Haemogregarina fitzsimonsiDias 1953 and some comments on Haemogregarina parvula Dias 1953(Adeleorina Haemogregarinidae) from southern African tortoises (CryptodiraTestudinidae) with new host data and distribution records Folia Parasitol 56173ndash179

du Preez L Carruthers V 2009 A Complete Guide to the Frogs of Southern AfricaStruik Nature Cape Town

Davies AJ Johnston MRL 2000 The biology of some intraerythrocytic parasitesof fishes amphibians and reptiles Adv Parasitol 45 1ndash107

Davis AK Devore JL Milanovich JR Cecala K Maerz JC Yabsley MJ 2009New findings from an old pathogen intraerythrocytic bacteria (familyAnaplasmatacea) in red-backed salamanders Plethodon cinereus EcoHealth 6219ndash228

Desser SS 1987 Aegyptianella ranarum sp n (Rickettsiales Anaplasmataceae)ultrastructure and prevalence in frogs from Ontario J Wildl Dis 23 52ndash59

Desser SS Hong H Martin DS 1995 The life history ultrastructure andexperimental transmission of Hepatozoon catesbianae n comb an apicomplexanparasite of the bullfrog Rana catesbeiana and the mosquito Culex territans inAlgonquin Park Ontario J Parasitol 81 212ndash222

Dobson A Lafferty KD Kuris AM Hechinger RF Jetz W 2008 Homage toLinnaeus how many parasites How many hosts Proc Natl Acad Sci U S A105 11482ndash11489

Fantham HB Porter A Richardson LR 1942 Some haematozoa observed invertebrates in eastern Canada Parasitology 34 199ndash226

Haddad CR 2003 Fruit chafers (Coleoptera Scarabaeidae Cetoniini) of the NdumoGame Reserve and Tembe Elephant Park KwaZulu-Natal Afr Entomol 11130ndash133

Kruger N Du Preez LH 2015 Reproductive strategies of the Kangaroo LeechMarsupiobdella africana (Glossiphoniidae) Int J Parasitol Parasites Wildldoi101016jijppaw201501005

Mohammed AHH Mansour NS 1959 A general survey of blood protozoa in someEgyptian amphibians and reptiles Bull Zool Soc Egypt 14 21ndash26

Netherlands EC Cook CA Smit NJ du Preez LH 2014a Redescription andmolecular diagnosis of Hepatozoon theileri (Laveran 1905) (ApicomplexaAdeleorina Hepatozoidae) infecting Amietia quecketti (Anura Pyxicephalidae)Folia Parasitol 61 293ndash300

Netherlands EC Cook CA Smit NJ 2014b Hepatozoon species (AdeleorinaHepatozoidae) of African bufonids with morphological description and moleculardiagnosis of Hepatozoon ixoxo sp nov parasitising three Amietophrynus species(Anura Bufonidae) Parasit Vectors 7 552

Nordstokke DW Zumbo BD 2007 A cautionary tale about Levenersquos tests forequality of variances J Edu Res Policy Stud 7 1ndash14

Nordstokke DW Zumbo BD 2010 A new nonparametric Levene test for equalvariances Psicologica 31 401ndash430

Readel AM Goldberg TL 2010 Blood parasites of frogs from an equatorial Africanmontane forest in western Uganda J Parasitol 96 448ndash450

SPSS 2013 IBM SPSS Statistics for Windows Version 220 IBM Corp Armonk NYIBM Corp Released

Stuart SN Chanson JS Cox NA Young BE Rodrigues AS Fischman DL et al2004 Status and trends of amphibian declines and extinctions worldwide Science306 1783ndash1786

Telford SR 2009 Hemoparasites of the Reptilia Color Atlas and Text CRC PressNew York

Werner JK 1993 Blood parasites of amphibians from Sichuan Province PeoplersquosRepublic of China J Parasitol 79 356ndash363

Wesołowska W Haddad CR 2009 Jumping spiders (Araneae Salticidae) of theNdumo Game Reserve Maputaland South Africa Afr Invert 50 13ndash103


Biodiversity of frog haemoparasites from sub-tropical northern KwaZulu-Natal South Africa

Introduction

Materials and methods

Study area and frog collection

Preparation and screening of frog blood

Statistical analysis

Results

Discussion

Acknowledgements


References

et al 2014a 2014b) Yet such diversity of knowledge regarding theseparasites is necessary before further studies can be done on eluci-dating the effects that these parasites may have on their natural hostsand the role these parasites may have in amphibian conservation

Southern Africa currently boasts 159 known species of frogs in33 genera and 13 families (du Preez and Carruthers 2009 Channingand Baptista 2013 Channing et al 2013a 2013b Conradie 2014)This study presents the results of a haemoparasite survey of frogsfrom three localities in KwaZulu-Natal South Africa (see Fig 1) Lo-calities include the formally protected Ndumo Game Reserve (NGR)and the reserversquos anthropogenically impacted surrounds as well asKwa Nyamazane Conservancy (KNC) All three sampling areas fallwithin a sub-tropical region known for its biological richness andas such the province KZN boasts the highest diversity of frog speciesin South Africa (see du Preez and Carruthers 2009) The followingstudy thus aimed to determine and record through a multispecieshaemoparasite survey on frogs if this parasite diversity paralleledthat of its rich frog diversity

2 Materials and methods

21 Study area and frog collection

Ndumo Game Reserve (NGR) (26deg52prime000PrimeS 32deg15prime000PrimeE) issituated in the West of the Maputaland bioregion close to the

borders of South Africa Swaziland and Mozambique The Maputalandbioregion located in northern KwaZulu-Natal (KZN) and crossinginto southern Mozambique is one of the most biologically rich areasin southern Africa (Haddad 2003) and has a sub-tropical climateNdumo is a large reserve holding 10117 ha of diverse habitatsincluding floodplains sub-tropical bush savannah and woodlandto riparian forest (Wesołowska and Haddad 2009) The areadirectly surrounding the NGR (27deg00prime138PrimeS 32deg16prime499PrimeE) isnot formally protected and thus is covered in rural tribal villagescausing the vegetation to be heavily impacted by the villagersrsquolivestock and subsistence farming practices Approximately 80 kmto the south lies the Kwa Nyamazane Conservancy (KNC)(27deg23prime349PrimeS 32deg08prime408PrimeE) a small conservation area runningalong the Phongola River and surrounded by large sects of agricul-tural land most of it utilised for sugar cane farming These localitieswere specifically chosen as all three are located on and are thussupplied by a permanent water source the Phongola River (seeFig 1)

Frogs were collected via active sampling at night in all three lo-calities as mentioned above All these sites were visited during thewarmer and rainy months of February and November 2012 Apriland November 2013 and February 2014 During collection possi-ble invertebrate vectors feeding on frogs were searched for howevernone were observed Captured frogs were held in disposable plasticbags and transported back to a field laboratory either at the NGR

Fig 1 Map displaying the three sampling localities in northern KwaZulu-Natal South Africa Map displaying the three sampling localities at which frogs were surveyed forhaemoparasite biodiversity top to bottom Ndumo Game Reserve (NGR) outside NGR and Kwa Nyamazane Conservancy in northern KwaZulu-Natal South Africa All sam-pling sites were directly or indirectly linked to the Phongolo River








3 Results






4 Discussion









BNGR 2KNC 5






BNGR2010


BNGR 1KNC 3





BNGR 3Total 29 11 3 436




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Results

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3 Results






4 Discussion









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BNGR 3Total 29 11 3 436




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Results

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Acknowledgements


References







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BNGR 3Total 29 11 3 436




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Results

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Acknowledgements


References



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721

ps

(Fig

2Lndash

M)

Am

ieto

phry

nus

gutt

ural

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33

82

4( F

ig2

AndashB

)A

mie

toph

rynu

sm

acul

atus

97

77

195

( F

ig2

AndashB

)3

73

ps

(Fig

2O

ndashP)

Chir

oman

tis

xera

mpe

lina

441

11

26p

s( F

ig2

Q)

Sem

i-aq

uat

icH

emis

usm

arm

orat

us22

22

20

8( F

ig2

B)

Hild

ebra

ndti

aor

nata

61

11

03

( Fig

2E)

Hyp

erol

ius

mar

mor

atus

364

34

205

( F

ig2

D)

14

1p

s(F

ig2

N)

Hyp

erol

ius

tube

rilin

guis

121

11

20p

s( F

ig2

R)

Kass

ina

mac

ulat

a8

11

14

ps

( Fig

2M

ndashS)

Phry

noba

trac

hus

mab

abie

nsis

131

11

03

( Fig

2B

)1

18

ps

(Fig

2O

)Ph

ryno

man

tis

bifa

scia

tus

11

11

6p

s( F

ig2

OndashP

)Pt

ycha

dena

anch

ieta

e78

4731

47

24

( Fig

2C

)13

47

1(F

ig2

FndashG

)4

47

75

(Fig

2I)

294

713

3p

s(F

ig2

LndashP

RT

)1

47

1p

sPt

ycha

dena

mas

care

nien

sis

52

12

02

( Fig

2B

)2

28

8p

s(F

ig2

O)

Ptyc

hade

nam

ossa

mbi

ca19

75

73

( Fig

2B

)2

799

(F

ig2

J)4

76

ps

(Fig

2O

NR

)Sc

hism

ader

ma

care

ns7

21

20

8( F

ig2

AndashB

)1

228

ps

(Fig

2K

)To

tal

1529

487

59

53

13

16

87

46

106

ps

214

5p

s

Prev

alen

ce=

Pin

ten

sity

=I

per

slid

e=

ps

(Fig

ure

refe

ren

ce=

Fig

)









Acknowledgements




References






































Introduction





Results

Discussion

Acknowledgements


References








Acknowledgements




References






































Introduction





Results

Discussion

Acknowledgements


References

Biodiversity of Frog Haemoparasites From Sub-tropical Northern KwaZulu-Natal, South Africa

Documents

Transcript of Biodiversity of Frog Haemoparasites From Sub-tropical Northern KwaZulu-Natal, South Africa