AES BI OFLUX
Ad va n ce s in En vir on m e n t a l Scie n ce s -
I n t e r n a t ion a l Jou r n a l of t h e Bioflu x Socie t y
D ie t a n d e ndopa ra sit e s of
Ra n a gr a n docu la
( Am phibia , Ra n ida e ) a n d
Lim n one ct e s m a gn u s
( Am phibia , D icr oglossida e )
in M t . Sa m bilik a n ,
D iw a t a Ra nge , Agu sa n de l Su r , Ph ilippine s
Karyl Marie B. Fabricant e and Olga M. Nuñeza
Depart m ent of Biological Sciences, College of Science and Mat hem at ics, Mindanao St at e University – I ligan I nst it ute of Technology, 9200 I ligan Cit y, Lanao del Nort e, Philippines.
Corresponding aut hor: K. M. B. Fabricant e, karylfab@gm ail.com
Abst r a ct. Anurans are recognized as one of t he m ost t hreat ened fauna on t he planet , yet poor knowledge on t he basic biology of t hese species is said t o im pede proper conservat ion effort s. The Philippines m ay have one of t he m ost unique anuran faunas in t he world, however st udies on t he feeding habit s and endoparasit es of anuran species are st ill scarce. This st udy exam ined t he diet and endoparasit es of Rana gr andocula and Lim nonect es m agnus using gut cont ent analysis on available voucher specim ens: L. m agnus ( 21) and R. gr andocula ( 16), collect ed in t he forest of Mt . Sam bilikan, Diwat a Range, Agusan del Sur, Philippines. Nine food it em s were ident ified including plant debris observed in L. m agnus. Result s on diet exam inat ion indicat ed the frogs’ high dependence on eight orders of invert ebrat e fauna dom inat ed by Ort hopt era. Niche overlap bet ween t he t wo species was dem onst rat ed as well as new records on being int erm ediat e host s t o t he cest ode, Diphyllobot hr ium lat um and nem at odes. L. m agnus showed higher int ensit y of infect ion for cest ode while nem at ode infect ion was higher for R. gr andocula. Sex and body size were non- det erm ining fact ors for infect ion in bot h species except for t he cest ode, D. lat um which showed a significant associat ion wit h sex ( p= 0.049) in R. gr andocula, wit h m ale frogs showing higher infect ion rat es t han fem ales despit e t heir significant ly sm aller size ( p= 0.013) . Diet and endoparasit es were found t o be int errelat ed, as well as t o t he habit at s of t he t wo species.
Ke y W or ds: anurans, conservat ion, feeding habit s, food it em s, infect ion.
I n t r odu ct ion. Anurans, being a highly sensitive t axa, are crucial indicat ors of
especially for species t hat inhabit endangered areas, and as an indicat or of t he qualit y of t he environm ent in which t he am phibians live (Kovacs et al 2007; Sant os et al 2004) . On t he ot her hand, endoparasit es affect survival and healt h and m ay also be sources of pat hogens ( Johnson & Hoverm an 2012; Blaust ein et al 2012). While t he Philippines m ay have one of t he m ost unique anuran fauna in t he world, st udies on t he feeding habit s and endoparasit es of anuran species are still scarce. The only existing record for endoparasite in Lim nonect es m agnus ( St ej neger, 1909) is Parapy lost om a crooki ( Van de Vusse 1976)
while no records exist for Rana grandocula ( Taylor, 1920) ( Nat ional Hist ory Museum
2012) ( Figure 1). I n t erm s of t heir diet , alt hough som e anurans can be considered specialist s, t hey are usually report ed as exhibiting generalist and opport unistic feeding habit s ( Piat ti & Souza 2011) . Alcala & Brown ( 1998) recorded invert ebrat es as prim ary com ponent s in t he diet of bot h L. m agnus and R. grandocula. At es et al ( 2007) in t heir
st udy on t he diet s of six anuran species found t hat Ort hopt era is bot h t he const antly frequent occurring and preferential food item in R. grandocula. L. m agnus however, was
not included in t he st udy.
Figure 1. Rana grandocula (A) and Lim nonect es m agnus (B).
R. grandocula is an endem ic least concern species but loss of t he rainforest and t he
pollution of m ount ain st ream s and rivers seem t o t hreat en t he populat ion ( Am phibiaWeb 2012) . L. m agnus on t he ot her hand is a near t hreat ened species of t he fam ily
Dicroglossidae found t o be dist ribut ed in t he Philippines as well as on Sulawesi, I ndonesia. However, hum an exploit ation and rapid habit at loss had t hreat ened t he population t o a significant decline ( Diesm os et al 2004) . Conservat ion effort s for bot h species include only a few prot ect ed areas covering it s range. This st udy seeks t o provide inform ation on t he diet and endoparasit es of t hese t wo species in t he hope t o supplem ent dat a vit al for t heir conservation and m anagem ent as t hese fact ors crucially affect t he survival of a species.
M a t e r ials a n d Me t h ods
An u r a n sa m plin g. Sam pling was done in Mt . Sam bilikan, which is a part of t he Diwat a
Figure 2. The st udy area locat ed at Sit io I nagawan, Brgy. La purisim a, Prosperidad, Agusan del Sur bet ween 8° 34′56″N, 125°53′34″E. (A-B) - Locat ion in t he Philippines
(www.agusandelsur.gov.ph), (C) - t opographical m ap (www.googleeart h.com ) and (D) - Panoram ic view of Mt . Sam bilikan, Diwat a range.
D ie t a n d e n dopa r a site e x a m in a t ion. Am ong t he 21 frog species found in t he area t he
t wo m ost abundant species, R. grandocula and L. m agnus were select ed for t he diet and
endoparasit e exam ination. Available voucher specim ens of L. m agnus ( 21) and R. grandocula ( 16) were used. The whole digestive t ract of each specim en, st arting from t he
species were det erm ined. Chi- square t ests were done t o check whet her t here exists a correlation bet ween sex and size t o t he presence or absence of t he endoparasit es.
Re su lt s a n d D iscu ssion
D ie t com posit ion. Diet exam ination revealed nine food it em s which include plant s and
t he following invertebrat es: Crust acea, Arachnida, Blat t odea, Coleopt era, Dipt era, Hym enopt era, Odonat a, and Ort hopt era ( Figure 3) . Plant debris were observed and described t o resem ble gram inae seeds and undigest ed dicot yledon leaves. Percent age com position values and percent age frequency of occurrence of prey it em s in L. m agnus
and R. grandocula is shown in Figure 4. Grasshoppers ( Ort hopt era) com prised t he largest
port ion consum ed and t he m ost frequent prey item select ed for bot h species.
Figure 3. Food it em s of L. m agnus and R. grandocula showing: (A-B) Hym enopt era,
(C) m ixed Crust acea and Plant ae, and (D-E) m ixed insect debris.
Am phibians are recognized as t he prim ary vertebrat e predat ors of invert ebrat es, m aj ority insects, in m any freshwat er and m oist t errest rial environm ent s, however, som e species are also found t o occasionally scavenge or rarely feed on plant m at erials and ot her nonm oving food it em s ( Sant os et al 2004; St ebbins & Cohen 1997; Zug 1993) .
Results in t his st udy indicate t hat L. m agnus and R. grandocula feed m ainly on
invertebrat es as recorded by Alcala & Brown ( 1998) . Dat a on R. grandocula also confirm s
t he st udy of At es et al ( 2007) t hat Ort hopt era dom inat es as t he m ost frequent and preferred food it em for t he species. However, since t he abundance of each prey it em in Mt . Sam bilikan was not t aken, it is difficult t o relat e whet her grasshoppers ( Ort hopt era) were greatly consum ed because t hey really were preferred by bot h L. m agnus and R. grandocula or because t hey were t he m ost readily available in t he area. Abundance and
availabilit y of prey is said t o greatly influence what is ingest ed and m ay t end t o conceal preferences ( Piat ti & Souza 2011; Van Sluys et al 2006; Bull 2003) . Alt hough it often m ay appear t hat m any am phibians have a low level of discrim ination in feeding, it m ust not be assum ed t hat t hey are wholly lacking in food preferences ( St ebbins & Cohen 1997) . Furt herm ore, plant debris were observed as t he least frequent food it em s observed in L. m agnus m aybe due t o t he frog’s difficult y in digesting fiber ( Zug 1993) or
Lept odact y lus nat alensis, and Phy salaem us cuv ieri) . Plant cont ent s m ay help in t he
elim ination of int estinal parasit es, provide roughage t o assist in grinding up art hropod exoskelet ons, or provide nut rient s and an additional source of wat er. Report s on herbivory am ong t ropical frogs is said t o be increasing. I n t he present st udy, this inform ation on plant consum pt ion m ay cont ribut e t o t he underst anding of t he species’ behavioural pat t erns as veget ation m ay be used by t hese frogs not only as a reproductive site, but also as foraging t errit ory. Moreover, variety in prey item s m ay also suggest differences in habit at occupation indicating t hat dat a on diet com position can support bot h ecological and behavioural st udies.
18.57 33.30 6.19 9.52 22.86 33.30 29.76 38.09 7.86 9.58 4.76 4.76 0.00 0.00 1.19 4.76 1.43 4.76 0% 20% 40% 60% 80% 100% P e rc e n ta g e Cole opte ra Arac hnida Hym enop tera Orth opte ra Blatt odea Odon ata Dipt era Plan tae Crus tace a Food Item
A Percentage composition Percentage Occurrence
28.44 31.25 4.69 12.50 1.88 12.50 47.81 56.25 0.00 0.00 4.06 18.75 0.63 6.25 0.00
0.00 0.000.00
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% P e rc e n ta g e Coleo ptera Arach nida Hym enop tera Orth opte ra Blatto dea Odon ata Dipter a Plant ae Crus tace a Food Item
B Percentage composition Percentage Occurrence
Figure 4. Percent age com posit ion and occurrence of prey it em s in (A) L. m agnus and (B) R. grandocula.
have bot h im port ant conservation concerns with L. m agnus being a near t hreat ened
species while t he R. grandocula is endem ic. Further st udies on t he feeding ecology and
int eractions bet ween anurans wit hin t he area are recom m ended as dat a could help in fut ure conservat ion applications. Furt herm ore, bot h species were observed t o be locally popular as a delicacy, highly prized for t heir m eat . This form of resource ut ilization m ay becom e a m aj or t hreat if done in large num bers. Hunt ing of bot h species m ust be furt her investigat ed t o determ ine whet her t his needs t o be m anaged sust ainably.
En dopa r a sit e e x a m in a t ion. Endoparasit e exam ination on t he t wo species showed
organism s under phylum Plat yhelm int hes, subphylum Cest oda and phylum Nem at oda ( Figure 5) . For Cest oda, t he organism was classified as Diphy llobot hrium lat um,
ot herwise known as t he broad fish t apeworm ident ified t o be in t he plerocercoid st age. The frog being an aquat ic organism m ay be one of t he t ransport host s of D. lat um. Frogs
eat copepods in t he form of sm all crust aceans. This was confirm ed in t his st udy t hrough t he diet analysis of L. m agnus which showed rem nant s of crabs in it s st om ach. This m ay
be t he ent ry point of t he organism . Being non- definitive host s, t he endoparasite rem ains as a plerocercoid in it s body (Levine 1978) . The ot her endoparasit e was identified up t o t he phylum level only and were classified under phylum Nem at oda generally belonging t o a group known as t he roundworm s. Roundworm s are highly diverse wit h up t o 10,000 nam ed species living in all sort s of habit ats from t he soil.
Figure 5. Endoparasit es of L. m agnus and R. grandocula showing: Cest oda (A-C) and
Nem at oda (D–E).
A st udy by Goldberg et al ( 2009) exam ining eleven species of ranid frogs from Papua New Guinea also revealed one species of Cest oda and 18 species of Nem at oda am ong 24 endoparasit e species observed. Given t heir im port ance as prey or as predat ors in food webs, herps can be host t o a range variet y of larval and adult helm int h parasit e species. Host habit at and diet ary preferences play critical roles in t he t ransm ission of helm int h parasites of herps ( Robert s & Janovy 2005) .
Of t he t wo species, m agnus showed higher int ensit y of infection for cest odes while
nem at ode infection was higher in R. grandocula ( Table 1) . This can be at t ribut ed t o t he
insects and crust aceans as prey it em s which m ay be one of t he cest ode’s point of ent ry. Ent ry of t he endoparasit e t o it s host predom inant ly is t hrough t he ingestion of eggs or larvae int o it s syst em while in wat er ( Levine 1978) . The preference of L. m agnus t o t ype
I V m icrohabit at s ( Gonzales & Dans 1994) which are m ainly st ream s or vicinit y of st ream s, also provides great er opport unit y for t he cest odes t o infect t his host . R. grandocula in t his st udy was not observed t o have crust aceans in it s diet but a m ore
com prehensive st udy could yield conclusive result s. R. grandocula was observed t o
inhabit t ype I I I m icrohabit at ( Gonzales & Dans 1994) in t ree holes, and t ype I V m icrohabit at allowing t he species t o be exposed to a great er variet y of possible habit at s for nem at odes which could also lead t o infection. Nem atodes greatly abound in a high variet y of habit at s such as in soil, m arine, and freshwat er bodies or decom posing m at t er. I n addit ion, m any kinds of insects harbor at least one species of parasitic nem at odes and t hese organism s are also parasitic t o t he rest of t he anim al kingdom which does not leave frogs as an exception ( Robert s & Janovy 2005) .
Table 1 Mean int ensity of infection and percent age occurrence of endoparasit es
in L. m agnus and R. grandocula
Endoparasit es
Cest oda Nem at oda Species
Mean intensity
of infection occurrence (% ) Percentage Mean intensity of infection occurrence (% ) Percentage
Lim nonect es m agnus 3 57.00 1 0.10 Rana grandocula 2 31.25 6 37.50
Sex was not a determ ining fact or for t he presence or absence of endoparasit es in L. m agnus wit h p = 0.406 and p = 0.375 for t he cest ode and nem at ode, respectively. Sex
and body size ( p = 0.160) of t he species also showed t o be not significantly associat ed. Sex also did not significantly affect endoparasit e infection in R. grandocula for t he
nem at odes ( p = 0.91) . However, infection of t he cest ode (D. lat um) showed a significant
association wit h sex of t hese species, showing m ales t o be highly infect ed t han fem ales ( p = 0.049) alt hough m ales were found t o be significantly sm aller in term s of size ( p = 0.013) . According t o Van Sluys et al ( 2006) , fem ale frogs have on average m ore parasites t han m ales and t his could be at t ribut ed t o t heir m uch larger size. However, t hey also argued t hat t he host ’s sex influence on endoparasit e infection is variable am ong different host s and parasit e species and appears t o have m ore com plex causes t han sim ply t he body size of host s. This m ay be t he case for t he present st udy as m ales were significantly sm aller t han fem ales for t he R. grandocula species. Parasit es and host s
int eract wit h m em bers of a diverse com m unit y that can influence patterns of infection
( Orlofske et al 2012) . A parasit e’s dist ribution is oft en influenced by m any fact ors such as t he host ’s diet, physiological condition or t he presence of ot her helm int hes ( Robert s & Janovy 2005) . I ncrease in parasit e richness m ay lead t o a decrease in host survival and growt h however, t hese effect s of parasite richness on host growt h and survival were found t o be cont ext dependent em phasizing t he im port ance of com m unit y com position and assem bly ( Johnson & Hoverm an 2012) . Furt her analysis should be done t o t race t he prim ary fact or t hat m ay have influenced t he great er infection in t he m ales of R. grandocula for t he cest ode species. Body size of R. grandocula and infect ion on t he ot her
hand were not significantly associat ed ( p = 0.465 and p = 0.055 for bot h t he cest ode and nem at ode, respectively).
Con clusion. R. grandocula and L. m agnus show ed a generalist diet of nine food it em s
consisting of plant s and invert ebrates wit h Orthopt era dom inating as bot h t he m ost frequent and largely consum ed prey item . Behavioral pat t ern of L. m agnus could be
furt her investigat ed t o see whet her veget ation is used not only as a reproduct ive sit e, but also as foraging t errit ory. Possible niche overlap bet ween L. m agnus and R. grandocula
L. m agnus and R. grandocula are int erm ediat e host s t o t he cest ode (D. lat um) and
nem at odes. The presence of endoparasit es was related t o t he diet and habit ats of t he t wo species indicating t he int errelatedness of t hese basic fact ors for survival.
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Received: 05 August 2012. Accept ed: 14 August 2012. Published online: 17 Sept em ber 2012. Aut hors:
Karyl Marie B. Fabricant e, Depart m ent of Biological Sciences, College of Science and Mat hem at ics, Mindanao St at e Universit y – I ligan I nst it ut e of Technology, 9200 I ligan Cit y,Lanao del Nort e, Philippines, e- m ail: karylfab@gm ail.com
Olga M. Nuñeza, Depart m ent of Biological Sciences, College of Science and Mat hem at ics, Mindanao St at e Universit y – I ligan I nst it ut e of Technology, 9200 I ligan Cit y,Lanao del Nort e, Philippines, e- m ail:
olgam nuneza@yahoo.com
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