Polyploidy as a chromosomal component of stochastic noise: variable scalar multiples of the diploid chromosome complement in the invertebrate species Girardia schubarti from Brazil / Poliploidia como componente cromossômico de ruído estocástico: variações escalares múltiplas do componente cromossômico diploide do invertebrado Girardia schubarti ocorrente no Brasil

Abstract Chromosome stoichiometry, a form of genetic plasticity, specifically refers to variation in the standard diploid genomic composition of an individual or species. In the present work, freshwater planarians (Girardia schubarti) were analyzed to recognize variations in chromosomal stoichiometry especially of complete ploidal change between specimens, within specimens and between cells within specimens and any relations they might have with selected components of phenotypic plasticity. Homoploid polyploids for the group reached rational scalar multiples (e.g. tetraploids) or irrational scalar multiples (e.g. triploids). Karyotypic mosaics emerged where individual cells presented polyploid multiples in arithmetic and geometric progressions. Ploidal multiplicity, a chromosomal component of stochastic noise, had positive phenotypic effects (increased dimensions) on morphologic criteria of body length, body width and dorsal surface reflecting a significant genotypic plasticity (GP) and robust phenotypic plasticity (PP). Variable but significant association of genotypic plasticity with robust phenotypic variance suggests kinetics of phenotypic homeostasis that is species-specific permitting phenotypic adaptability to environmental variables by means of GP. That association is diminished, deactivated or lost in more advanced and more complex organisms.

Resumo A estequiometria cromossômica, uma forma de plasticidade genotípica, representa variações na composição genômica diploide de um indivíduo ou espécie. Planárias límnicas (Girardia schubarti) foram analisadas para verificar a estequiometria cromossômica, especialmente alterações na ploidia entre espécimes, em cada espécime e entre células do mesmo espécime, além de relações dessas alterações com a plasticidade fenotípica. Espécimes poliploides homoploides apresentaram múltiplos escalares racionais ou irracionais, tais como triploides. Mosaicos cariotípicos ocorreram quando células apresentaram poliploides múltiplos em progressões aritméticas e geométricas. Nas planárias estudadas, a multiplicidade ploidal, um componente cromossômico de ruído estocástico, apresentou efeitos fenotípicos positivos, causando aumento das dimensões dos indivíduos, tais como comprimento corporal, largura do corpo e superfície dorsal, indicando plasticidade genotípica (GP) significativa e plasticidade fenotípica (PP) robusta. Associações significativas da plasticidade genotípica com variâncias fenotípicas robustas, embora variáveis, sugerem que a homeostase fenotípica, a qual é espécie-específica, possibilita adaptações a variáveis ambientais através da GP. Tal associação apresenta-se reduzida, desativada ou perdida em organismos mais complexos.

Polyploidy as a chromosomal component of stochastic noise: variable scalar multiples of the diploid chromosome complement in the invertebrate species Girardia schubarti from Brazil.

Chromosome stoichiometry, a form of genetic plasticity, specifically refers to variation in the standard diploid genomic composition of an individual or species. In the present work, freshwater planarians (Girardia schubarti) were analyzed to recognize variations in chromosomal stoichiometry especially of complete ploidal change between specimens, within specimens and between cells within specimens and any relations they might have with selected components of phenotypic plasticity. Homoploid polyploids for the group reached rational scalar multiples (e.g. tetraploids) or irrational scalar multiples (e.g. triploids). Karyotypic mosaics emerged where individual cells presented polyploid multiples in arithmetic and geometric progressions. Ploidal multiplicity, a chromosomal component of stochastic noise, had positive phenotypic effects (increased dimensions) on morphologic criteria of body length, body width and dorsal surface reflecting a significant genotypic plasticity (GP) and robust phenotypic plasticity (PP). Variable but significant association of genotypic plasticity with robust phenotypic variance suggests kinetics of phenotypic homeostasis that is species-specific permitting phenotypic adaptability to environmental variables by means of GP. That association is diminished, deactivated or lost in more advanced and more complex organisms.

Microturbellarians (Platyhelminthes and Acoelomorpha) in Brazil: invisible organisms? / Microturbelários (Platyhelminthes e Acoelomorpha) no Brasil: organismos invisíveis?

Abstract Microturbellarians typically belong to the benthos and may occur in a wide variety of environments. They are abundant in freshwater and marine ecosystems and may occur in moist terrestrial habitats. However, turbellarians are seldom taken into account in studies of biodiversity. Most studies on Brazilian microturbellarians had taxonomical purposes and were done in the years 1940-1950. Thus, information on their occurrence and ecological aspects are dispersed throughout several papers. We intend here to summarize the biogeographical distribution and ecological aspects of microturbellarians recorded for Brazil, indicating the main gaps in their knowledge and possible actions to enhance studies on this group. There are 239 species of microturbellarians registered for Brazil, with records distributed in 12 states. However, just three states located in southern Brazil have records of 94% of microturbellarian species. Thus, knowledge on the systematics and geographical distribution of Brazilian microturbellarians clearly reflect the scientific activity over many years or decades in two states of southeastern and southern Brazil. Considering the scant information on this group in Brazil, which is also the situation of the Neotropical microturbellarians in general, some actions should be proposed. First, it would be necessary to sample in the diverse biomes, as well as in the various river and sea basins, based on standardized sampling protocols. Second, it would be necessary to encourage diverse research groups to include microturbellarians and/or turbellarians in general into biodiversity inventories and studies on community structure of invertebrates. Third, it is necessary to increase the number of research groups on microturbellarians, in order to augment the studies on their morphology, systematics, and ecology. Considering their abundance, species richness and ecological importance in aquatic environments, despite some peculiarities regarding their sampling, sorting and identification procedures, the challenge to study microturbellarians and enhance knowledge about them in Brazilian ecosystems should be faced.

Resumo Microturbelários são tipicamente bentônicos e podem ocorrer em uma ampla variedade de ambientes. São abundantes em ecossistemas marinhos e de água doce, podendo ocorrer em ambientes terrestres úmidos. Entretanto, turbelários raramente são considerados em estudos de diversidade. A maioria dos estudos sobre microturbelários brasileiros tiveram propósito taxonômico e foram realizados nos anos 1940-1950. Assim, informações sobre ocorrência e aspectos ecológicos estão dispersos em diversos artigos. O objetivo deste trabalho é sumarizar a distribuição biogeográfica e aspectos ecológicos dos microturbelários registrados para o Brasil, indicando as principais lacunas do conhecimento e possíveis ações para ampliar estudos sobre esse grupo. Há 239 espécies de microturbelários registradas no Brasil, com registros distribuídos em 12 estados. No entanto, 94% das espécies de microturbelários foram registradas em apenas três estados localizados no sul e sudeste do Brasil. Assim, o conhecimento sobre a sistemática e distribuição geográfica dos microturbelários claramente reflete as atividades científicas realizadas por muitos anos ou mesmo décadas em dois estados do sudeste e sul do Brasil. Considerando as escassas informações existentes sobre esse grupo no Brasil, assim como a situação dos microturbelários neotropicais em geral, algumas ações devem ser propostas. Primeiramente, é necessário realizar amostragens em diversos biomas, assim como nas várias bacias e regiões hidrográficas marinhas, baseadas em protocolos de amostragem padronizados. Em segundo lugar, faz-se necessário incentivar diversos grupos de pesquisa a incluir microturbelários e/ou turbelários em geral em inventários da biodiversidade e estudos de estrutura de comunidades de invertebrados. Em terceiro lugar, é necessário ampliar o número de grupos de pesquisa em microturbelários, para aumentar os estudos sobre sua morfologia, sistemática e ecologia. Considerando sua abundância, riqueza de espécies e importância ecológica em ambientes aquáticos, apesar de suas peculiaridades de amostragem, triagem e identificação, o desafio de estudar e ampliar o conhecimento sobre microturbelários em ecossistemas brasileiros deve ser enfrentado.

Microturbellarians (Platyhelminthes and Acoelomorpha) in Brazil: invisible organisms?

Microturbellarians typically belong to the benthos and may occur in a wide variety of environments. They are abundant in freshwater and marine ecosystems and may occur in moist terrestrial habitats. However, turbellarians are seldom taken into account in studies of biodiversity. Most studies on Brazilian microturbellarians had taxonomical purposes and were done in the years 1940-1950. Thus, information on their occurrence and ecological aspects are dispersed throughout several papers. We intend here to summarize the biogeographical distribution and ecological aspects of microturbellarians recorded for Brazil, indicating the main gaps in their knowledge and possible actions to enhance studies on this group. There are 239 species of microturbellarians registered for Brazil, with records distributed in 12 states. However, just three states located in southern Brazil have records of 94% of microturbellarian species. Thus, knowledge on the systematics and geographical distribution of Brazilian microturbellarians clearly reflect the scientific activity over many years or decades in two states of southeastern and southern Brazil. Considering the scant information on this group in Brazil, which is also the situation of the Neotropical microturbellarians in general, some actions should be proposed. First, it would be necessary to sample in the diverse biomes, as well as in the various river and sea basins, based on standardized sampling protocols. Second, it would be necessary to encourage diverse research groups to include microturbellarians and/or turbellarians in general into biodiversity inventories and studies on community structure of invertebrates. Third, it is necessary to increase the number of research groups on microturbellarians, in order to augment the studies on their morphology, systematics, and ecology. Considering their abundance, species richness and ecological importance in aquatic environments, despite some peculiarities regarding their sampling, sorting and identification procedures, the challenge to study microturbellarians and enhance knowledge about them in Brazilian ecosystems should be faced.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola) / Desenvolvimento embrionário de Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola)

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

O desenvolvimento embrionário dos tricladidos é conhecido, fundamentalmente, por estudos realizados em espécies de Dendrocoelum, Planaria, Polycelis e, mais recentemente, Schmidtea. O presente estudo descreve o desenvolvimento embrionário de Girardia tigrina (Girard, 1850), a partir de análises realizadas em cortes histológicos seriados e semifinos de glicol-metacrilato, ao microscópio óptico. Noventa e quatro casulos foram coletados no período entre a postura e a eclosão, em intervalos de até vinte e quatro horas. A seqüência das modificações morfológicas no embrião permitiu a identificação de nove estágios embrionários. Na postura dos casulos, envoltos por uma cápsula rígida, observam-se numerosos embriões dispersos entre grande quantidade de células vitelinas. No estágio I (aproximadamente até 6 horas após a postura), as células vitelinas e as embrionárias mostram uma distribuição aleatória. O estágio II (entre 12 e 24 horas após a postura) caracteriza-se pela formação de agrupamentos de blastômeros, os quais posteriormente formam uma enteroblástula. Aproximadamente dois dias após a postura (estágio III), ocorre a formação da epiderme e do sistema digestivo embrionário, sendo que este último degenera no estágio seguinte. O estágio V (até o quarto dia após a postura) caracteriza-se pela formação da epiderme definitiva. Entre o quarto e o sexto dia posteriores à postura, começa a organogênese dos órgãos internos definitivos (estágio VI). Aproximadamente catorze dias após a postura (estágio IX), completa-se a formação do sistema nervoso. Neste estágio, o embrião já apresenta características similares aos espécimes juvenis. A eclosão de Girardia tigrina ocorre entre doze e vinte e dois dias após a postura dos casulos.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola).

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

Chromosome polymorphism and complements in populations of Girardia species (Platyhelminthes, Tricladida, Paludicola) from southern Brazil

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n = 9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Os cariótipos de quatro espécies de planárias de água doce do gênero Girardia (Platyhelminthes), a saber, G. schubarti, G. tigrina, G. anderlani e G. biapertura, de populações ocorrentes em diferentes locais do estado do Rio Grande do Sul, na região sul do Brasil, foram analisados. O cariótipo de G. biapertura é apresentado pela primeira vez. Foram observados três complementos básicos, de 4, 8 e 9 cromossomos. Espécimes diplóides, triplóides e mixoplóides (2n/3n) foram observados freqüentemente nessas populações. O complemento cromossômico básico de n = 9 foi verificado em duas espécies (G. biapertura e G. anderlani), apresentando um grande cromossomo acrocêntrico que é raro na família Dugesiidae. Também foi observada certa variabilidade cromossômica, tanto intra- como interespecífica, cujas implicações evolutivas são discutidas.

Chromosome polymorphism and complements in populations of Girardia species (Platyhelminthes, Tricladida, Paludicola) from southern Brazil.

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n=9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Histological and histochemical characterization of the secretory cells of Choeradoplana iheringi Graff, 1899 (Platyhelminthes: Tricladida: Terricola).

The present study aims at providing a detailed description of the histology, as well as the first histochemical characterization, of the secretory cells of the epidermis, pharynx, and copulatory organs of Choeradoplana iheringi, in order to give further support to studies on the physiology of these organs. The secretory cells are distinguished on the basis of secretion morphology and its staining properties, using trichrome methods and histochemical reactions. Four cell types open through the epidermis of Ch. iheringi, three of them secreting basic protein and a fourth containing glycosaminoglycan mucins. The epidermal lining cells store glycogen. In the pharynx, four secretory cell types were distinguished. Two types produce glycoprotein, a third type secretes basic protein, and another one produces glycosaminoglycan mucins. In the male copulatory organs, the prostatic vesicle receives four secretory cell types containing basic protein, except for one type which produces glycoprotein. The two secretory cell types opening into the male atrium secrete, respectively, glycoprotein, and glycosaminoglycan mucins. In the female copulatory organs, the female atrium and its proximal diverticulum, the vagina, receive two types of secretory cells producing, respectively, basic protein and glycosaminoglycan mucins. Another secretory cell type constitutes the so-called shell glands which open into the common glandular duct, secreting basic protein. The lining cells of the male and female atria produce a mucous secretion containing glycosaminoglycans. In addition, the lining epithelium of the female atrium presents an apical secretion of a proteic nature. The occurrence of a kind of spermatophore is reported for the first time for a species of Choeradoplana. This structure is located in the male or female atria in different specimens, and characterized by erythrophil, xanthophil, and/or mixed secretions associated with sperm.

Histological and histochemical characterization of the secretory cells of Choeradoplana iheringi Graff, 1899 (Platyhelminthes: Tricladida: Terricola)

O presente estudo tem por objetivo fornecer detalhada descrição da histologia e a primeira caracterização histoquímica das células secretoras da epiderme, da faringe e do aparelho reprodutor de Ch. iheringi, visando a propor estudos da fisiologia desses órgãos. As células secretoras foram diferenciadas com base na morfologia da secreção e em sua coloração, com métodos tricrômicos e reações histoquímicas. Quatro tipos de células secretoras desembocam na epiderme de Ch. iheringi, sendo três com secreção protéica básica e uma do tipo mucoso, contendo glicosaminoglicanas. As células de revestimento que compõem a epiderme armazenam glicogênio. Na faringe, quatro tipos de células secretoras são observadas: duas produzem secreção de natureza glicoprotéica, uma apresenta secreção protéica básica e uma secreta glicosaminoglicanas. No aparelho copulador masculino, em sua vesícula prostática desembocam quatro tipos de células secretoras, as quais contêm secreção protéica básica, excetuando uma cuja secreção é de natureza glicoprotéica. O átrio masculino recebe a desembocadura de dois tipos de células secretoras, um tipo contendo secreção glicoprotéica e outro, glicosaminoglicanas. No aparelho copulador feminino, o átrio feminino e seu divertículo ental, a vagina, recebem a desembocadura de dois tipos de células secretoras, que produzem, respectivamente, proteína básica e glicosaminoglicanas. Um outro tipo de célula secretora constitui as chamadas glândulas da casca, que desembocam no ducto glandular comum e secretam proteína básica. As células de revestimento dos átrios masculino e feminino produzem secreções mucosas constituídas por glicosaminoglicanas. O epitélio de revestimento do átrio feminino apresenta, ainda, um tipo diferenciado de secreção protéica acumulada apicalmente. A ocorrência de um tipo de espermatóforo é registrada pela primeira vez para uma espécie de Choeradoplana. Tal estrutura foi observada em três espécimes, ocorrendo no átrio masculino ou feminino, sendo caracterizada pela associação de espermatozóides com secreções eritrófilas, xantófilas e/ou mistas.

Histological and histochemical aspects of the penial glands of Girardia biapertura Sluys, 1997 (Platyhelminthes, Tricladida, Paludicola)

Girardia biapertura was described with sperm ducts penetrating the penis bulb, subsequently opening separately at the tip of the penis papilla and receiving the abundant secretion of penial glands. In the present work, the penial glands of this species have been histologically and histochemically analysed, and four types of secretory cells are distinguished. The openings of the penial glands into the intrabulbar and intrapapillar sperm ducts, designated here as intrapenial ducts, allow for the distinction between three histologically differentiated regions. The most proximal region possibly corresponds to the bulbar cavity of other freshwater triclads whereas the median and distal portions correspond to the ejaculatory duct. The proximal region of the intrapenial ducts receives mainly the openings of a secretory cell type (type I) that produces a proteinaceous secretion. A second type of secretory cell (type II) that secretes neutral mucopolyssacharides opens into the median region of the intrapenial ducts. The distal portion of the ducts receives two types of secretory cells (types III and IV) which secret glycoprotein and glycosaminoglycans, respectively. Types III and IV open also directly into the male atrium through the epithelium of the penis papilla. A comparison with the results presented here and those of other authors for species of Girardia is provided and the importance of the study of the penial glands for taxonomic characterisation of freshwater triclads is emphasised

Histological and histochemical aspects of the penial glands of Girardia biapertura Sluys, 1997 (Platyhelminthes, Tricladida, Paludicola).

Girardia biapertura was described with sperm ducts penetrating the penis bulb, subsequently opening separately at the tip of the penis papilla and receiving the abundant secretion of penial glands. In the present work, the penial glands of this species have been histologically and histochemically analysed, and four types of secretory cells are distinguished. The openings of the penial glands into the intrabulbar and intrapapillar sperm ducts, designated here as intrapenial ducts, allow for the distinction between three histologically differentiated regions. The most proximal region possibly corresponds to the bulbar cavity of other freshwater triclads whereas the median and distal portions correspond to the ejaculatory duct. The proximal region of the intrapenial ducts receives mainly the openings of a secretory cell type (type I) that produces a proteinaceous secretion. A second type of secretory cell (type II) that secretes neutral mucopolyssacharides opens into the median region of the intrapenial ducts. The distal portion of the ducts receives two types of secretory cells (types III and IV) which secret glycoprotein and glycosaminoglycans, respectively. Types III and IV open also directly into the male atrium through the epithelium of the penis papilla. A comparison with the results presented here and those of other authors for species of Girardia is provided and the importance of the study of the penial glands for taxonomic characterisation of freshwater triclads is emphasised.

A new species of terrestrial planarian (Platyhelminthes: Tricladida: Terricola) from south Brazil.

Geoplana josefi sp. nov. of South Brazil is described herein. The species shows etary polymorphism. Neither youngs nor adults show the characteristic colour pattern of the mature worms. The species has a multilayered lining in the female atrium. The allopatric G. trigueira, which does not have such a lining, is similar externally and internally with adults of G. josefi, its seminal vesicle, however, being unpaired.

Histological processing techniques for the study of Dugesiidae development (Platyhelminthes, Tricladida, Paludicola).

The objective of the present study was to adapt techniques for the histological processing of Dugesiidae cocoons for the study of embryo development. The cocoons were fixed with formalin, SUSA, Bouin or paraformaldehyde/glutaraldehyde and subsequently embedded in Paraplast or glycol methacrylate (Historesin). Paraplast embedding yielded reasonable results only after the cocoon was perforated or fixed for a prolonged period of time using softening techniques with acid solutions. When the SUSA or Bouin fixative and Historesin embedding techniques were used the results were good for light microscopical analysis. Fixation with paraformaldehyde/glutaraldehyde and glycol methacrylate embedding resulted in better tissue preservation, and did not require prolonged fixation or softening techniques. Thus, we suggest this technique for light microscopical analysis of embryo development in Dugesiidae.

A new species of terrestrial planarian (Platyhelminthes: Tricladida: Terricola) from South Brazil

Geoplana josefi sp. nov. of South Brazil is described herein. The species shows etary polymorphism. Neither youngs nor adults show the characteristic colour pattern of the mature worms. The species has a multilayered lining in the female atrium. The allopatric G. trigueira, which does not have such a lining, is similar externally and internally with adults of G. josefi, its seminal vesicle, however, being unpaired

Histological processing techniques for the study of Dugesiidae development (Platyhelminthes, Tricladida, Paludicola)

The objective of the present study was to adapt techniques for the histological processing of Dugesiidae cocoons for the study of embryo development. The cocoons were fixed with formalin, SUSA, Bouin or paraformaldehyde/glutaraldehyde and subsequently embedded in Paraplast or glycol methacrylate (Historesin). Paraplast embedding yielded reasonable results only after the cocoon was perforated or fixed for a prolonged period of time using softening techniques with acid solutions. When the SUSA or Bouin fixative and Historesin embedding techniques were used the results were good for light microscopical analysis. Fixation with paraformaldehyde/glutaraldehyde and glycol methacrylate embedding resulted in better tissue preservation, and did not require prolonged fixation or softening techniques. Thus, we suggest this technique for light microscopical analysis of embryo development in Dugesiidae