Evolutionary Biology of Parasitic Platyhelminths: the Role of Molecular Phylogenetics D

Revmew Evolutionary Biology of Parasitic Platyhelminths: The Role of Molecular Phylogenetics D. Blair, A. Campos, M.R Cummings and J.R Laclette

As our appreciation of the diversity within the flatworms systematic scheme and names of the major groups has grown, so too has our curiosity about the ways in provided in Ehlers 3,4 (Fig. in Box 1). which these varied creatures are related to one another. In Three rnain questions have emerged from the particular, the parasitic groups (trentatodes, cestodes attd literature. (1) Do the major groups of parasitic flat- monogeneans' have been the focus of enquiry. Llntil worms lie. Trernatoda (including Digenea and recently, morphohk~y, anatomy and lift" histories have pro- Aspidobothrea), Monogenea and Cestoda (including vided the raw data for building hypotheses on ivlationships. Gyrocotylidea, Amphilinidea and Eucestoda)l form a Now, ultrastructural evhtence, and most recently, mol- single clade? The name Neodermata was recent!v ecular &Tta front nucleic acid seqttences, have been brought given to this proposed clade ~. (2) How are these para- to beat" on the topic. Here, David Blair, Andrds Campos, sitic groups related to one another? (3) What are the Michael Cummings attd Juan Pedro Laclette discttss the relationships of the major parasitic groups to other ways in which molecular data, in particular, are helping tts flatworm taxa? recognize the various lineages of flatworms. Do the major parasitic groups form an exclusive The phylum Platyhelnlinthes (flatworms) is a large lineage? (>15000 species) and diverse one, and appears to be Members of the rnain parasitic groups differ con- an early diverging lineage within the Metazoa. Flat- siderablv from one another in structure, habits and worms show great diversity in anatomy, habitat, life life cycles. As a result, several proposals to explain history and size, varying from the 20m long euces- their origins assumed that they did not form a mono- tode Diphylh~bothrium latunt, to species barely visible phyletic group. For example, it has been suggested to the naked eye. They also exhibit considerable gen- that trematodes, monogeneans and cestodes all origi- etic diversity among 18S rRNA gene sequences when nated independently from parasitic rhabdocoelan compared to higher taxa of vertebrates~. The phylum ancestors '',7, or that the monogeneans, from which includes free-living, commensal, mutualist and para- de,~cencled tile cestodes, originated from a free-living sitic species, of which many are significant for medi- rhabdocoelan ancestor and had separate origins from cal and economic reasons. All parasitologists k~ow the trematodes". ot tile threat to human health posed by trematodes Ultrastructural studies were the first to plovide such as alembers of the genus St'hislos(una, or cestodes evidence contrary to such ar~pal~nents. All the major sucla as Echino('(~cc'lts granllloslts. Despite its impor- parasitic groups resemble one another in structural tance, many controversies exist concerning the sys- and dew.,Iopmental aspects of epidermal cilia, sensory tematics of the phylum and, in particular, the origin receptors, flame bulbs and sperm. In addition, in and interrelationships of the major parasitic groups. these taxa the epidermis of tile larva is replaced in tile The evolutionary history of flatworms is still uaclear adul[ by a syncitial 'l~eodernlis'J,~,", a feature that led because of the fragmentary information or, their mor- Ehters~ to propose tile name Neoderrnata for the phology, physiology and lift, cycles, and because group. Studies based on molecular data from the these soft-bodied worms have left few fossil remains. nuclear 18S rRNA gene also strongly support a single A robust phylogeny will also provide insights into origin for these major parasitic groups "-t2. the origins of parasitism in the phylum and the shared evolutionary history of parasitic flatworms Relationships among the main groups of Neodermata and their hostsL Two main clades are recognized within the Neo- In this review, we explore recent findings about dermata oil the basis of ultrastructure and aspects phylogenetic relationships in the flatworms, especially of ontogeny. The Trematoda (Aspidobothrea and Di- the parasitic o~rouF-.~s We emphasize conclusions drawn genea) forms one lineage, and the Cercomeromorphae from DNA sequence data (see Boxes 1,2) and focus on (Monogenea and all groups allied with tile Cestoda) a number of questions that have emerged from the the other. The Cercomeromorphae is characterized by literature. For consistency, we will mostly follow the common possession of a cercornere, ie. a posterior attachment structure bearing hooks that is present at some stage in the life cycle. David Blair is at the Department of Zoology, James Cool< Monophyletic Trematoda. On the basis of morpho- University, Townsville, Queensland 481 I, Australia. Andr6s Campos and Juan Pedro Laclette are at the Department of Immunology, logical and life cycle characteristics2.3,13-% the Aspido- Instib Jto de Investigaciones Blot, ~6dicas, Universidad Nacional bothrea (Fig. la) has been proposed as the sister Aut6noma de M6xico, A.P. 70228, M6xico 04510 D.F., M~xico. group of the digeneans. Molecular studies, using 18S Michael Cummings is at the Department of Botany and Plant rR',JA sequences, have also supported this view 9,u. Sciences, Univel~sityof California, Riverside, CA 92521-0124, USA. However, the possibility that Aspidobothrea is the Tel: +61 77 814322, fax: e61 77 251570, sister group to the remaining Neodermata has not e-mail: [email protected] been ruled out 1LI7.

{.)I~ "~ 4758/96/$15.00 Parasitology Today, vol. 12, no. 2, 1996 Reviews

Digenea. In the Digenea (Fig. lb), Box 1. Gene Regions Used to Infer Relationships most groupings at low levels There are several different classes of genes that can provide data about phyla- (species, genus) are widely accepted. genetic relationships. Nuclear ribosomal RNA genes, which encode structural Unfortunately, there is still consider- RNA of ribosomes, tend to occur in eukaryotes as a series of tandemly able dispute concerning the higher- repeated units, each contaming three coding regions (5.8S, -150bp; 18S, level (family, order) classification. ~2000bp; 28S, ~4500bp) separated by various spacers. The rate of evolution Information gleaned from morphol- differs both among and within these genes and spacers, providing infor- ogy (including ultrastructure) and mation that is useful at a range of divergence levels. The internal transcribed spacers, located between the 18S and 28S genes and separated by the 5.8S from life cycles has been the main- gene, are the least-conserved regions used m studies, to date, on flatworms. stay of digenean systematics 2. In the Mainly for historical reasons, the gene encoding the 18S subunit (the most view of some workers ~(',~s, the conserved of the ribosomal genes), has been widely used to study relation- amount of homoplasy in the exist- ships of Platyhelminthes (see Fig. b,:iow). A short 5S ribosomal RNA gene ing data sets is such as to render occurs elsewhere in the genome. Studies using this gene for phylogenet~c impossible the accurate reconstruc- inference have been reviewed and dismissed 3". tion of the phylogenetic history of One useful feature of ribosomal repeat uI~its is that they t,~nd to be 'hom- the trematodes. Such clairns l~,ave ogenized' within an individual and among individuals within a species or spurred molecular studies on the population; a process called 'concerted evolution'. As a result, intraspecific variation among ribosomal genes is very low compared to interspecific vari- group: currently, more rnolecular ation. A sequence from one individual should therefore be representative of data are available for Digenea than its species and sample sizes need not be large~.4s. for any other flatworm taxon (see Another source of phylogenetic information is the mitochondrial genome. Fig. in Box 1). This clonally inherited circular molecule usually encodes 37 genes, and is typi- The entire 18S rRNA gene cally less than 20000 bp long in animals 4". Mitochondrial genes often accumu- (1970bp) was evaluated as a source late changes at higher rates than the nuclear genome, and therefore data from of phylogenetic information within this genome are expected to be more useful for relatively recent divergences. the Digenea using sequences from Among the genes encoded by the mitochondrial genome are those for ribo- eight taxa m. Very little phylogenetic somal subunits, which are smaller than their nuclear counterparts and are information was found in the con- derived from the eubacterial ancestor of the mitochondrion. Nuclear protein coding genes have rarely been used in systematic studies served regions of the molecule, of Platyhehninthes (see Fig. belo,v). which make up over half of its length. Most information came from ,e- A the variable domains and especially O4 A A ,e- from the V4 domain 2", which com- o o o o o o o ~:. ¢G prises only about 16% of the mol- ¢5 ¢5 ¢5 e~ 6 6 0 ,- ¢5 o~ 6 d ecule. Data from this region provide N ~N ~ N d d g 6 g c~ ¢5 0; ¢5 c~ c~ (5 c~ d ~d g d o c5 c5 N c5 o c:f c:5 c5 o o ,-- strong support for a number of ei ~ e4 ,-: br familial groupings. For example, lepocreadiids and gyliauchenids are "O closely related to the exclusion of the paramphistomes despite the similar anatomy of the last two"L Although the V4 region is likely to be the target of many future O Z ,~ studies, it (and perhaps the 18S gene as a whole) does not seem to be informative about the earliest di- 1/ vergences among the digeneans. Rhabd0c0ela MonOg ~ neas'° a This is unfortunate because there has been lively debate on this topicN,'. ~. Several families have been proposed as the sister group to f the rest of the digeneans (reviewed I in Ref. 16). This question of the sister family has also been ~latyhelminthes approached by molecular analysis, but with the data available so far, no Phylogeny of the I~latyhelminthes according to the widely accepted schen{e of EhlersL Modified after Ref. 9. Numbers in parentheses following single family emerges as sister to the each taxon name are numbers of species in each taxon for which partial remaining Digenea 2~. or complete sequences of the following genes are available: 18S nuclear The 5' end of the 28S rRNA gene, rRNAI, q-12"1t~,21.27'-~4-('l,28S nuclear rRNA 22'2~'27'35"('2, nuclear ribosomal internal including the D1 variable domain, transcribed spacers 24-2"'~Z''3-"',nuclear protein-coding genes 4''"7 and mitochon- has been the subject of a number of drial genes2"~-2".:~4.-~5'"4"('s'%respectively. Note that many nuclear protein coding studies looking at deep branches genes have been sequenced for members of the Neodermata, but are not enu- among metazoans (see, for example, merated here because they are not available for sufficient numbers of taxa to Ref. 22). Analysis of the D1 region permit phylogenetic analysis. The udonellids (no sequences available) and the from 13 digeneans was found to be fecampiids (1,0,0,0,0) are not placed in the tree (see text). Temnocephalids and suitable for inferring relationships pterastericolids belong with the Daiyellioid rhabdocoels. among species, genera and closely 67 Porasi~.ology Today, vol. 12. no. 2, 1996 Review

Box 2. Analysis of Molecular Data and its Problems The affinities of problematical taxa that are mor- For each taxon, raw sequence data comprise a string of phologically distinct can be investigated using DNA nucleotides (A,C,G,T/U), which are aligned to permit sequence data. An example from the Monogenea is r( cognition of homologous sites. The alignment process the genus Anoplodiscus. Members of this genus are is crucial; if homology is not properly established for found on the external surfaces of fish. They lack each position, incorrect trees can be inferred. Alignment hooks on the opisthaptor as adults but otherwise can be done either 'by eye' (if the data set contains few resemble monogeneans (two pairs of eyes and a typi- taxa), or by using computer programs. However, even cal neodermis in the adults). However, Anoplodiscus computer-based methods frequently produce results exhibits spermatogenesis unlike other monogeneans which the human eye finds unacceptable and that even though the mature sperm resembles that seen in require adjustment. Alignment is progressively wore difficult as phylogenetic distance increases among taxa, monopisthocotylean monogeneans 3~. It also exhibits and sequences become more dissimilar. Ribosomal atypical ultrastructure of the flame bulbs and capil- sequences often pose particular alignment problems laries 32. Molecular phylogenetic studies, using 18S because homologous regions may differ in both length rRNA sequences, strongly support the placement of and nucleotide frequencies among taxa. Two recent Anoplodiscus in the Monopisthocotylea ". studies differed concerning relationships between the Another interesting genus that may be close to the Monogenea and Gyrocotylidea using similar ribosomal Monogenea is Udonella (Fig. ld). Members of this sequences but different alignments l°,ll. Consideration of genus are found on the external surface of caligid putative secondary structure features may help with copepods parasitizing marine fish. The genus may be alignment s~,s~, but these techniques are relatively untried as yet. Protein encoding genes are often much easier to allied with the MoI:3genea and Cestoda, or may be align because of their organization into codons. Insertions quite distinct from other neodermatans 33. No DNA and deletions usually occur in multiples of three, and the sequences are yet available for this taxon. inferred amino acid sequences can aid al,gnment. Cestoda. Like rnonogeneans, cestodes (Fig. le) have Several methods exist for inferring trees, all of which received relatively little attention to date from mol- embody implicit and explicit assumptions. Some meth- ecular systematists. Relationships witLin some genera ods, such as parsimony and maximum likelihood, evalu- in the Taeniidae have been exarnin~.l using nuclear ate the character states (nucleotides present) at each ribosomal and mitochondrial sequences34. 3~. The only position. Another class of methods require:; reduction of other studies have concentrated on relationships the data to a matrix of genetic distances antong all pairs among higher taxa of cestode:;: Gyrocotylidea, Amphi- of taxa. Any of a number of algorithms (eg. the neigh- bout-joining method) is then used to infer a tree. (For a linidea a~:d Eucestoda. The Gyrocotylidea emerged as major review of these and other methods, see Ref. 52.) the sister group of the rest of the Cestoda'U~, which is Computer programs will produce a tree from virtually consistent with several recent proposals based on any data set, but often give no indication of how well the morphological data2. The first molecular study to dai:a fit the inferred tree. Advances have been made in examine the position of Gl/Ir'oCOtyh, I° placed this taxon methods for testing the robustness of playlogenetic in the Monogenea, but this may have been an artifact trees 47,~2, It is possible to assess the degree of support for of the aligmnent used. particular groupings using various tests of which the bootstl'ap ~ is the best kl'~t)Wla and Ollt' ol: the Int,~t What is the sister group to the Neodermata? controversial. Schemes t'esenlbling that shown in Box I, placing rhabdocoel groups as sisters to the Net~dermata, have related families, but at deeper levels relationships been increasingly accepted in recent years (but see were poorly resolved a3, Refs 36-38 for an alternative phylogeny that sepa- The internal transcribed spacers are less conserved rates the rhabdocoels and the neodermatans). It is not than the coding regions, and therefore informative at surprising, therefore, that recent theories on the affin- a lower (species/genus) level. For the schistosomes, ities of the Neodermata have focused on parasitic or including the human blood flukes, sequences from commensal rhabdocoels. In particular, the temno- these spacers yield phylogenies that are very robust cephalids ~s, the fecampiids3" and the pterastericolids 3'' and consistent with conclusions based on anatomical are among the taxa that have recently been proposed data 24-2". Mitochondrial sequences have also been as sisters to the Neodermata. used to infer relationships within ~chistosoma24-2., and The family Fecampiidae (Fig. lf) consists of species the results are congruent with those found using data parasitic in crustaceans, but which leave their host to from the nuclear ribosomal region2S.27. spawn inside a secreted coccoon. On ultrastructural Mtmogenea. The Monogenea (Fig. lc) has long been grounds, this group initially appeared close to the Neo- considered a monophyletic group. The most corn- dermata 4",4~. However, a study using partial 18S rRNA monly cited shared features of the grou F are the pos- sequences from 19 flatworms found the Fecampiidae to session of two pairs of eyes and three rows of epider- be remote from both the Neodermata and the Rhabdo- mal ciliav 7 bands in the oncomiracidium. However, coe!a, where they are customarily placed ~2. Parallelisms recent ultrastructural studies of sperm have been and convergences must have produced the sin'filar persistent in pointing out that no relevant synapo- ultrastructural features observed in the fecampiids and morphies exist for the Monogenea as a whole 2s-3", neodermatans. Certainly, the same features used to suggesting that the group may not be monophyletic. infer a close relationship between these groups also Indeed, the Monogenea, and especially the appear, in part, in totally unrelated species scattered Monopisthocotylea, differ from other groups of r,eo- throughout the phyluml2,3s. A new class, Fecarnpiida, dermatans in exhibiting a considerable degree of has been proposed for the family 12. ultrastructural variation. Molecular data also suggest The temnocephalids (Fig. lg) are quaint creatures non-monophyly of the Mono,oenea. with a posterior sucker used to attach to the external 611 Parasitolog~y Today, vol. 12, no. 2, 1996 Reviews

surface of a host (usually a freshwater decapod crus- Conclusions and prospects tacean), and the anterior of the body bears two or When congruent, robust trees are inferred from more tentacles (except in Didymorchis). In at least several different data sets, we can have a considerable one major systematic scheme for the parasitic flat- amount of faith in the conclusions. The most extensive worms 2,1,~, the temnocephalids were placed as sister molecular data sets to date for any flatworm group group to the Neodermata, but recent anatomicaP ~. and are from digeneans of the family Schistosomatidae molecular m-12 studies do not support this view. Mol- and come from several regions of the nuclear and ecular data suggest that the temnocephalids are close mitochondrial genomes. Phylogenies inferred have to the dalyelliids and the pterastericolids'L Ultra- been robust, congruent with one another and congru- structure of protonephridia also suggests that the ent with those inferred from other kinds of data. This temnocephalids are close to other rhabdocoels 37. demonstrates that robust phylogel~ies can be obtained The pterastericolids (Fig. lh) are common inhab- for flatworms using molecular daf:a. itants of the digestive tract of echinoderms 6. On the For deep-level phylogenies of flatworms, the situ- basis of sperm ultrastructure and other characteris- ation is not quite as clear. In 1988, at a time when tics, it has been proposed that this group, together much ultrastructural data had ah"eady been brought with the fecampiids, could be sister to the Neoder- to bear on the problem, but before the advent of DNA mata 39,42. H~,wever, additional study 43,~ led to the sequence data, Rohde 4-~ could say '... the sister group conclusion that the pterastericolids are not, after all, of the Neodermata cannot be identified at present ...' close to the Neodermata; a result also supported by By 1994, following several studies using 18S rRNA molecular data ~. sequences, he was scarcely more definite~7: '... the Porasitolo~/ Today, vol. 12, no. 2, 1996 69 ~m

sister group of the Neodermata [may be] a ... taxon 12 Rohde, K. et al. (1994) The phylogenetic relationships of comprised of the Proseriata, Rhabdocoela and Tricla- Kronborgia (Platy~.elminthes, Fecampiida) based on compari- dida ...' Scenarios of relationships among the rest of son of 188 ribosomal DNA sequences. Int. J. Parasitol. 24, 657--669 . the non-parasitic groups are, of course, much more 13 Brooks, D.R. et al. (1985) The phylogeny of the Cercomeria, ' ~'or,fused. Brooks, 1982 (Platyhelminthes). Prec. Hehninth. Sac. Wash. 52, ' The 18S data sets used to date have given clear 1-20 ~mswers (outlined earlier) about some relationships 14 Brooks, D.R. ct al. (1989) Aspects of the phylogeny of the within the flatworms, but have provided only weak Trematoda Rudolphi, 1808 (P!atyhelminthes: CercomeriaY. Can. J. Zeal. 67, 2609-2624 suggestions about others. There could be many rea- 15 Brooks, D.R. et al. (1985) Phyiogenetic analysis of the Digenea sons for this. Some are outlined in Box 2 and others (Platyhelminthes: Cercomeria) with comments on their adap- include: lack of data for key taxa and the possibility tive radiation. Can. J. Zeal 63, 411-443 that all flatworm taxa arose almost simultaneously 16 Gibson, D.I. (1987) Questions in digenean systematics and evolution. Parasitology 95, 429-460 during an explosive ancient radiation. Additional 17 Rohde, K. (1994) The minor groups of parasitic data, hopefully involving the entire 18S gene, will cer- Platyhelminthes. Adv. Parasitol. 33,145-234 tainly provide more answers: our knowledge has 18 Pearson, J.C. (1992) On the position of the digenean family already been increased greatly thanks to this gene. Heronimidae: an enquiry into a cladistic classification of the Protein-encoding genes, especially from the Digenea. Syst. Parasitol. 21, 81-1{~6 19 Blair, D. and Barker, S.C. (1993) Affinities of the nuclear genome, are likely to be the focus of future Gyliauchenidae: utility of the 188 rRNA gene for phylogen- phylogenetic studies on flatworms at all levels. Such etic inference in the Digenea (Platyhelminthes). Int. J. ParasitoL genes are far easier to align, even at great phylogen- 23, 527-532 etic depths, than are ribosomal genes. However, care 20 Nelles, L. et al. (1984) Nucleotide sequence of a crustacean 188 is required in selection of an appropriate candidate ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs. Nucleic Acids Res. 12, gene. Only single-copy genes should be used, other- 8749-8768 wise it might be hard to confirm that gene regions 21 Barker, S.C. et al. (1993) Phylogenetic position of Heronimus involved are strictly orthologous. Many nuclear genes mollis (Digenea): evidence from 188 ribosomal RNA. htt. J. occur as families, members of which may differ Pro'astral. 23, 533-536 among themselves (eg. eggshell protein genes of the 22 Qu, L-H. et al. (1988) Phylogenetic calibration of the 5' termi- nal domain of large rRNA achieved by determining twenty trematodes4~'). Suitable genes for phylogenetic studies eucaryotic species. ]. Mol. Eval. 28, 113-124 on flatworms remain to be identified. 23 Barker, S.C. et al. (1993) Utility of the D1 domain of nuclear While continuing to expand molecular data sets for 288 rRNA for phylogenetic inference in the Digenea. Syst. flatworms, every effort should be made to enhance Parasitol. 26, 181-188 ti~e quality and quantity of morphological data sets. 24 Bowles, J. et al. (1993) Nuclear and mitochondrial genetic markers highly conserved between Chinese and Philippine Hypotheses generated from one type of data can be Schistosoma japonicum. Acta Tropica 55, 217-229 tested using the other. Perhaps in this way the one 25 Bowles, J. et al. (1995) A molecular phylogeny of the human true tree of platyhelminth ew~lution will eventually schistosomes. Mol. Phyl. Evol. 4, 103-109 be traced! 26 Despr6s, L. et al. (19~2) Molecular evidence linking hominoid evolution to recent radiation of schistosomes (Platyhehninthes: Trematoda}. Mol, Phvl. End, 1,295-304 Acknowledgements 27 Barker, S.C. ct al. Molecular phylogen)) of Schistosoma species 1his work was supportecl ,~ p,wt by gntnts f?om I::und,.:iOn lvllguol recognises "traditional" groupings within the genus. ], Para- Alem,in ,ind CONACYT I I 31 -N9202 and 510]N (JPL) and from the silol. (in press) Australian Researctl Council (DB). We wish to thank Klaus Rohde, 28 Justine, J-L. (1993) Phylog6nie des monoghnes bas6e sur une Nikki Watson and Steve Badger for comments on the manuscript and analyse de parcimonie des charact~res de I'ultrastructure de la tot allowin~ us to cite papers in press, spermiog6n/zse et des spermatozo'ides incluant les r4suitats r6cents. Bull. Ft. P&'he Piscic. 328, 137-155 References 29 Justine, J-L. (1991) Phylogeny of parasitic Platyhelminthes: a I Riutort, M. t,t al. (1993) 18S rRNA sequences and phylogeny of critical study of synapomorphies proposed on the basis of the ultrastructure of spermiogenesis and spermatozoa. Can. ]. Zeal. Platyhelminthes. t~iochelll. S)/sL Ecol. 21, 71-77 69, 1421-1440 2 Brooks, D.R. and McLennan, D.A. (1993) Parascript: pan,sites told the langttt~e o]'evohttion, Smithsonian Institution Press 30 Justine, I-L. (1991) Cladistic study in the Monogenea 3 Ehlers, U. (1985) Das phylogenetische St/stem dcr Phltyhehninlhes. (Platyheiminthes), based upon a parsimony analysis of G, Fischer spermiogenetic and spermatozoal ultrastructurai characters. lat. ]. Parasitol. 21,821-838 4 Ehlers, U. (1986) Comments on a phylogenetic system of the Platyhelminthes. Hy,h'obioh}gia 132, 1-12 31 Watson, N.A. and Rohde, K. (1992) Ultrastructure of sperm and 5 Ehlers, U. (1984) Phylogenetisches System der Platyhehninthes. spermaLogea.,.:sis of Anoplodiscus cirrusspiralis (Platy- Ver/r Natm'wiss. Vet. Hambg. 27, 291-294 helminthes, Monogenea, Monopisthocotylea). Aml. Parasitol. Hma. Corot,. 07, I )i i4~; 6 Cannon, L.R.G. (1986) in Parasite Lives (Cremin, M., Dobson, C. and Moorhouse, D.E., eds), pp 15-32, University of Queensland 32 Rohde, K. et a,. (1992) UIUastructure of the protonephridial Press system of Anoplodiscus cirrusspiralis (Monogenea, Mono- pisthocotylea), l,t. ]. Parasitol. 22, ,143-457 7 Hyman, L.H. (1951) The lm,ertcbrates. Vol. II, Platyhehninthes and Rhym'hocoela - The Acoeh}mate Bilateria, McGraw-Hill 33 Ronde, K. and Watson, N.A. (1993) Spermatogenesis in 8 Llewelyn, J. (1965) in Evolution in Parasites (Taylor, A.E., ed.), Udoneila (Platyhelminthes, Udonellidea) and the phylogen- pp 47-78, British Society for Parasitology etic position of the genus. Int. ]. Pm'asitoi. 23, 725-735 34 Bowles, J. and McManus, D.P. (1993) NADH dehydrogenase 1 9 Rohde, K, et al. (19':)3) Contributions to the phylogeny of Platyhelminthes based on partial sequencing of 18S ribo- gene sequences compared for species and strains of the genus somal DNA. hit. J. ParasitoL 23, 705-724 Echinococcus. Int. J. Parasitoi. 23, 969-~72 10 Baverstock, P.R. et al (1991) Conflicting phylogenetic hypoth- 35 Bowles, J. and McManus, D.P. (1993) Genetic characterization eses for the parasitic platyhelminths tested by partial of the Asian Taenia, a newly described taeniid cestode of humans. Am. J. Trap. Mat. Hyg. 50, 33-44 sequencing of 188 ~ibosomal RNA. Int. ]. Parasitol. 21,329-339 11 Blair,D. (1993) The phylr~genetic position of the Aspidobothrea 36 Rohde, K. (1990) Phylogeny of Platyhelminthes, with special within the parasitic flatworms inferred from ribosomal RNA reference to parasitic groups. Int. ]. Parasitol. 20, 979-1007 sequence data. Int. J. Parasitol. 23,169-178 37 Rohde, K. (1991) The evolution of protonephridia of the Platyhelminthes. Hydrobiologia 227, 315-321 7~ Parasitology Today, vol. 12, no. 2, 1996 Reviews I

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In Search of the Most Effective Ways of Implementing Insecticide-treated Bednets: TDR Invites Operational Research Proposals Insectici6e-treated (bed)nets (ITNs) have been considered one of the most promising malaria concroi interventions. Results of large-scale trials evaluating the efficacy of ITNs in reducing morbidity and mortality in well-defined epi- demiological settings in Africa will become available early in 1996. Preliminary results from these trials are very encouraging. In order to match the ongoing field work on efficacy assessment, the UNDP/WB/WHO Special Programme for Research and Trainin~ in Tropical Diseases (TDR) and the International Development Research Center of Canada (IDRC) have developed a joint initiative to encourage and support operational research on the most efficient means of promoting, implementing and sustaining ITN interventions in different settings. A first round of proposals has been approved in September 1995 by the TDR Task Force on bednets. A second round of funding is planned for September 1996. Research groups are invited to submit detailed letters of intent for work that falls within the topic~ of the initiative. Further details can be obtained from Dr J. Cattani, Secretary, Bednet Task Force, WHO/TDR, 1211 Geneva 27, Switzerland. Tel: +41 22 791 3737; Fax: +41 22 791 48541. Letters of intent should be sent by 15 May 1996 to the same address.

Parasitology Today, vol. 12, no. 2, 1996 11