Research note Reproductive interference in concurrent infections of two Protopolystoma species (Monogenea: Polystomatidae)

International Journal for Parasitology 17 "0887# 0190Ð0193

Research note

Reproductive interference in concurrent infections of two Protopolystoma species "Monogenea] Polystomatidae# J[A[ Jackson\ R[C[ Tinsley School of Biological Sciences\ University of Bristol\ Bristol BS7 0UG\ U[K[ Received 06 December 0886^ received in revised form 09 March 0887^ accepted 09 March 0887

Abstract The prevention of interspeci_c reproductive interference is one possible explanation for spatial niche divergence between congeneric monogeneans[ However\ there is little direct evidence that reproductive interactions with other species are potentially deleterious to the majority of parasitic platyhelminths[ Xenopus fraseri!like clawed toads from lowland rainforest in eastern Democratic Republic of Congo are infected by two species of polystomatid monogenean\ Protopolystoma _ssilis and Protopolystoma ramulosus[ Both occur as adults in the host urinary bladder\ and exhibit identical copulatory structures and similar body sizes[ The small area of the habitat in relation to parasite body size makes close proximity inevitable in concurrent infections[ Eggs were collected from _ve naturally infected hosts] two of these harboured concurrent infections\ and three were infected with P[ _ssilis only[ Eggs from concurrent infections showed reduced viability "46[5) embryonation\ n  302# compared with those from P[ _ssilis!only infections "74[1)\ n  328#[ This e}ect may be due to some form of reproductive interference\ possibly failure to develop following interspeci_c cross!fertilisation[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Keywords] Reproductive interference^ Egg viability^ Concurrent infections^ Protopolystoma species^ Monogenea^ Xenopus

Where related parasitic organisms share spatially restricted microhabitats\ the chance of encounters between potential heterospeci_c mates may be increased[ Amongst platyhelminth parasites\ lab! oratory studies have demonstrated cross!insemi! nation in a pair of Philophthalmus ð0Ł\ and a pair of Echinostoma species ð1Ł[ Hybridisation is known in Schistosoma ð2\ 3Ł "an unusual dioecious genus# and Paragonimus ð4Ł\ and is suspected from morpho! logical intermediates between some other digeneans ð5Ł[

Corresponding author[ Fax] 33 006 814 6263^ e!mail] R[C[TinsleyÝBristol[ac[uk[

The reinforcement of reproductive barriers "pre! venting waste of reproductive potential# has been suggested as a factor in~uencing the evolution of spatial niche segregation in co!occurring congeneric parasites ð6\ 7Ł[ However\ there is little direct evi! dence that reproductive interference is of potential signi_cance amongst the majority of parasitic pla! tyhelminths\ and to our knowledge no examples of interspeci_c reproductive interactions have been recorded in the Monogenea[ Recent studies of polystomatid monogeneans from clawed toads in central Africa ð8\ 09Ł show that concurrent infections with adults of two species sometimes occur in Xenopus fraseri!like hosts[ Both parasites "Protopolystoma _ssilis and Proto!

S9919!6408:87 ,08[99¦9[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Printed in Great Britain PII] S9919!6408"87#99958!0

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J[A[ Jackson\ R[C[ Tinsley : International Journal for Parasitolo`y 17 "0887# 0190Ð0193

polystoma ramulosus# infect the host urinary blad! der\ which presents a simple sac!like surface of small area in relation to parasite size "Fig[ 0#[ As close contact would be inevitable in concurrent infec! tions\ and there is no morphological barrier to inter! speci_c insemination ð8Ł\ reproductive interactions between these species are a possibility[ Data on Protopolystoma spp[ egg viability\ a quantity which might re~ect negative reproductive interactions of some kinds\ have been collected in our laboratory "as part of separate studies#[ Some of these\ from isolated X[ fraseri!like hosts\ were subsequently dis! covered to have originated from toads infected with two Protopolystoma species[ This study reports variation in egg viability associated with eggs from concurrent infections[ Specimens of a tetraploid "1n  25 chromo! somes# X[ fraseri!like clawed toad "here termed X[ fraseri a}[# were collected in November 0880 at Ebisha\ near Irangi research station\ Kivu district\ eastern Democratic Republic of Congo\ by Dr H[ Hinkel[ Of _ve X[ fraseri a}[ studied\ three were infected with P[ _ssilis\ and two with both P[ _ssilis and P[

ramulosus "see Table 0# "the parasite specimens on which this study is based were recorded by Tinsley and Jackson ð8Ł#[ Hosts had been maintained in the laboratory "under transmission!free conditions ð00Ł# for 4 months before the collection of eggs and all parasite specimens were gravid[ It was not poss! ible to distinguish the eggs of the two Proto! polystoma species[ Toads were isolated in 1!L jars two!thirds _lled with dechlorinated tap!water which was changed every 1Ð2 days[ Parasite eggs were collected "by the method of Jackson and Tinsley ð01Ł# from sediment accumulated during this time[ Each batch "n  25Ð 074 eggs# was transferred to a 099!ml crystallising dish two!thirds full of a commercial brand of natu! ral mineral water and covered with a non!airtight lid[ These were maintained in a Gallenkamp cooled incubator at 1421>C "photoperiod\ 01 h L]01 h D# and water loss due to evaporation was periodically replaced[ Each dish was removed from the incu! bator for approximately 04 min every 37 h so that development of the eggs could be monitored[ Embryonated eggs were separated from the remain! der\ and the process was continued until all eggs

Fig[ 0[ Ventral dissection of Xenopus laevis "a larger\ but anatomically similar species to X[ fraseri a}[# to show moderately distended urinary bladder infected with eight sub!adult and adult Protopolystoma xenopodis "host length\ snoutÐvent59 mm#[ Concurrent infections in the present study involved six "three P[ _ssilis] three P[ ramulosus# and 03 "09 P[ _ssilis] four P[ ramulosus# adult Protopolystoma spp[ from X[ fraseri a}[ 20 and 23 mm long\ respectively[ Note] posterior body cavity with ventral internal organs exposed "BC#\ urinary bladder "UB#\ parasite "P#\ junction of urinary bladder with cloaca "J#[

J[A[ Jackson\ R[C[ Tinsley : International Journal for Parasitolo`y 17 "0887# 0190Ð0193

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Table 0 Viability of eggs from Protopolystoma _ssilis and Protopolystoma ramulosus] single species and concurrent infections Worm burden P[ _ssilis]P[ ramulosus

Replicate "egg batch#

Percentage embryonationa

1]9

0

73

02]9

0 1

68 77 73 "6#

093 074

0]9

0 1

89 73 76 "3#

49 45

2]2

0 1

46 33 40 "09#

36 30

09]3

0 1 2 3

38 63 55 36 48 "02#

093 28 035 25

n 33

a

Per host mean "with S[D[ in parentheses# in bold type[

had embryonated or died[ Examination of eggs was carried out under a stereo binocular microscope with _bre!optic illumination[ Overall\ 74[1) of eggs from P[ _ssilis!only infections embryonated "n  328^ 88) con_dence interval] 79[1Ð78[9#\ compared with 46[5) of those from mixed infec! tions with P[ _ssilis and P[ ramulosus "n  302^ 88) con_dence interval] 40[5Ð53[0#[ Per batch per! centage embryonation "see Table 0# from single and mixed infections was signi_cantly di}erent "P ³ 9[990\ ANOVA#[ In both concurrent infec! tions there was extreme crowding of the urinary bladder habitat "for comparison see Fig[ 0#\ with worms in very close physical proximity[ Viability did not show a relationship with parasite density] mean percentage embryonation was relatively high "73)# in the 02!worm P[ _ssilis infection\ whilst both the high "03 worms# and lower density "six# mixed infections showed reduced viability "48 and 40) embryonation\ respectively#[ Natural concurrent infections with di}erent polystomatid species in the urinary bladder of anu! rans are rare ð8\ 02\ 03Ł[ Although Tinsley and Jack! son ð8Ł reported such multiple infections at several

localities\ in most cases juvenile stages of one or both parasites were involved[ Adults of P[ _ssilis and a P[ ramulosus!like species did co!occur in a preserved X[ fraseri!like toad captured at Mieri\ Cameroon ð8Ł[ Apart from this\ the three hosts infected with P[ _ssilis and P[ ramulosus at Ebisha\ Zaire "ð8Ł^ this study# represent the only recorded concurrent adult infections out of thousands of polystomatid!infected clawed toads from through! out sub!Saharan Africa examined over a 14!year period[ Percentage embryonation was signi_cantly lower in parasite eggs from these concurrent infec! tions with P[ _ssilis and P[ ramulosus\ when com! pared with P[ _ssilis!only infections[ It is noted that data are available for only two concurrently infected hosts and it is also possible that P[ ramu! losus eggs display reduced viability irrespective of the concurrent infections "as it was not possible to study single species infestations with this parasite#[ However\ extensive data for single species infec! tions with other Protopolystoma taxa show that percentage embryonation for eggs kept under simi! lar conditions is invariably high "above 74)#[ This is the case for Protopolystoma xenopodis "×83)

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J[A[ Jackson\ R[C[ Tinsley : International Journal for Parasitolo`y 17 "0887# 0190Ð0193

embryonation\ based on hatching success\ n  07 454 ð01Ł#\ P[ _ssilis at localities in Rwanda "86)\ n  1546#\ Protopolystoma simplicis "88)\ n  307#\ Protopolystoma occidentalis "77)\ n  342# and Protopolystoma orientalis "89)\ n  019#[ Parasite density e}ects on egg viability cannot explain variation in the present data[ This is consistent with the study of Jackson and Tinsley ð01Ł\ who found that while density might have a slight e}ect on the percentage hatch of P[ xenopodis eggs\ even at a worm burden of 01 this was above 74)[ Protopolystoma _ssilis and P[ ramulosus have comparable adult body sizes and similar terminal genitalia] both lack vaginae and possess a muscular copulatory organ armed with two circles of eight spines ð8Ł[ It is not known whether insemination occurs via the terminal section of the female repro! ductive system or direct penetration of the tegu! ment\ which has been observed in the monogenean Diclidophora merlangi ð04Ł[ As the urinary bladder habitat is so con_ned "Fig[ 0# that close contact would be inevitable in concurrent infections\ inter! speci_c copulation is a possibility[ However\ mor! phological intermediates between P[ _ssilis "n30# and P[ ramulosus "n03#\ which might suggest hybridisation\ were not found at the study locality ð8Ł[ The unexpectedly low percentage embryonation in eggs from concurrent P[ _ssilis:P[ ramulosus infections suggests that some form of reproductive interference is occurring\ possibly interspeci_c cross! fertilisation with the production of non!viable eggs[ It has been suggested that spatial niche diver! gence and morphological divergence of copulatory structures between co!occurring\ congeneric mon! ogeneans evolved to prevent reproductive inter! ference ð05\ 06Ł^ the present results suggest that such interactions between species are a potentially sig! ni_cant phenomenon[

Acknowledgements We are grateful for research grants from NERC "GR2:5550 and GR8:521# and the Royal Society "to R[C[T[# and The Systematics Association "to J[A[J[#[ We are also very grateful to Dr H[ Hinkel for collecting the hosts on which this study was based[

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