Melanoma da íris em idade pediátrica

Apresentado como poster no 57º Congresso Português de Oftalmologia, Algarve, Portugal, Dezembro de 2014

DIFFERENTIATION BETWEEN Nocardia spp. AND Mycobacterium spp.: CRITICAL ASPECTS FOR BACTERIOLOGICAL DIAGNOSIS

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

Nevos Recorrentes. Uma Revisão de 5 Anos

A persistência de uma lesão melanocítica, em local onde tenha sido previamente excisado um nevo melanocítico pode colocar, clínica e histologicamente, problemas no diagnóstico diferencial com melanoma, designado por alguns autores como ‘pseudomelanoma’. Neste estudo, os autores pretendem realizar uma análise comparativa entre os achados clínicos e histopatológicos das lesões melanocíticas primárias e dos nevos recorrentes. Procura-se também avaliar eventuais factores predisponentes para este fenómeno.

Exposição profissional ao chumbo : análise da indução de efeitos genotóxicos

RESUMO - A exposição contínua a substâncias químicas tem consequências para a saúde humana, algumas das quais não estão ainda totalmente estabelecidas. A toxicologia ocupacional é uma área interdisciplinar que envolve conhecimentos de higiene e de medicina ocupacional, de epidemiologia e de toxicologia e que tem por principal objectivo prevenir a ocorrência de efeitos adversos decorrentes do ambiente ocupacional sendo um dos seus principais papéis fornecer o máximo de dados que possam contribuir para o conhecimento dos potenciais efeitos na saúde. O chumbo é um tóxico de características cumulativas que provoca na saúde efeitos principalmente sistémicos, ou seja, o efeito tóxico manifesta-se em locais afastados do contacto inicial que resultam essencialmente de exposições crónicas, resultantes de períodos de exposição mais ou menos longos ao metal (entre meses e anos). Pode interagir com diferentes órgãos e tecidos, ligando-se a moléculas e constituintes celulares. Uma vez que não possui qualquer função fisiológica, a presença do chumbo no organismo humano resulta numa série de efeitos prejudiciais que afectam diversos órgãos e sistemas. A toxicidade do chumbo manifesta-se em diversos órgãos e tecidos, nomeadamente no sistema hematopoiético, no sistema nervoso, no rim, no aparelho reprodutor, no sistema cardiovascular, no sistema endócrino e no sistema imunitário. Da interferência do chumbo com o funcionamento de alguns sistemas biológicos resultam um conjunto de alterações fundamentais ao nível dos processos de transporte através das membranas, da integridade estrutural e funcional das enzimas e de várias vias metabólicas, em especial da fosforilação oxidativa e da síntese do heme sendo os primeiros efeitos bioquímicos do chumbo detectados a partir de valores de plumbémia inferiores a 10 μg/dL. As medidas de higiene e segurança actualmente em vigor nos países desenvolvidos asseguram que os casos de intoxicação grave são cada vez menos frequentes. No entanto, o risco de exposição a nível ocupacional existe em todas as actividades que envolvem materiais que o contenham como as explorações mineiras, as fundições primária e secundária, a produção de baterias de chumbo ácido, a produção de vidro com pigmentos de chumbo, as soldaduras de reparação automóvel e a instrução de tiro. Desde 2006 o chumbo é considerado pela International Agency for Research on Cancer (IARC) uma substância carcinogénica do grupo 2A (provável carcinogénio para o ser humano). Considera-se, assim, que o chumbo tem, inequivocamente, capacidade de induzir cancro em animais experimentais mas que, embora haja fortes indícios de que os mecanismos que medeiam a carcinogénese desses compostos ocorrem no ser humano, os dados disponíveis ainda não podem assegurar essa relação. Com este estudo pretendeu-se contribuir para o conhecimento da toxicidade do chumbo através do estudo da exposição ao chumbo e da influência da susceptibilidade individual (em industrias sem co-exposição significativa a outros agentes conhecidos ou suspeitos de serem carcinogénicos). Pretendeu-se estudar o caso através de uma abordagem múltipla que permitisse relacionar diferentes tipos de marcadores biológicos uma vez que a monitorização biológica integra todas as possíveis vias de entrada no organismo (para além da via respiratória), eventuais exposições fora do contexto estritamente profissional assim como uma série de factores intrínsecos individuais (relacionados com modos de via, de natureza fisiológica e comportamentais). Sendo a co-exposição a outros compostos com propriedades genotóxicas e carcinogénicas uma questão difícil de tornear quando se quer avaliar o potencial genotóxico do chumbo em populações expostas, ocupacional ou ambientalmente este estudo tem a vantagem de ter sido efectuado em populações sem co-exposição conhecida a outras substâncias deste tipo, permitindo concluir sobre os efeitos resultantes apenas da exposição a chumbo na população humana, contribuindo para explicar algumas das aparentes inconsistências e contradições entre diferentes estudos sobre este tema. Os indicadores de exposição usados foram: indicadores de dose interna (doseamento de chumbo e de PPZ no sangue), indicadores de efeitos adversos no heme e genotóxicos (actividade da ALAD, teste do cometa e mutação em TCR) e indicadores de susceptibilidade (polimorfismos genéticos de ALAD e VDR) através de uma abordagem estatística de comparação directa de sub-grupos previamente definidos na população e da aplicação de um modelo de regressão múltipla. Este estudo revelou que os níveis de plumbémia na população portuguesa baixaram significativamente nos últimos 10 anos, tanto na população ocupacionalmente exposta como na população em geral e que a presença do genótipo B-B (do gene VDR) é preditiva das variações de plumbémia, quando comparada com o genótipo mais frequente na população, B-b; ao contrário, o genótipo b-b não aparenta ter influência em nenhum dos marcadores estudados. No que diz respeito a efeitos genotóxicos concluiu-se que estes não se manifestaram na população estudada, levando a concluir que nos níveis de exposição estudados, o chumbo não tem capacidade de induzir este tipo de efeitos per si levando ao reforço da hipótese, já levantada por outros autores, de que o mecanismo de genotoxicidade do chumbo seja essencialmente de promoção de processos de genotoxicidade desencadeados por outros agentes. A realização de estudos de efeitos genotóxicos e de stress oxidativo desenhados de forma a comparar grupos de trabalhadores expostos apenas a chumbo com grupos de trabalhadores com o mesmo nível de exposição a chumbo, mas com co-exposição a outros agentes reconhecidamente carcinogénicos poderá ajudar a aumentar o conhecimento deste efeito do chumbo na saúde humana.

Consolidation and chromatic reintegration of historical renders with lime-based pozzolanic products

Historical renders are exposed to several degradation processes that can lead to a wide range of anomalies,such as scaling, detachments, and pulverization. Among the common anomalies, the loss of cohesion and of adhesion are usually identified as the most difficult to repair; these anomalies still need to be deeply studied to design compatible, durable, and sustainable conservation treatments. The restitution of render cohesion can be achieved using consolidating products. Nevertheless, repair treatments could induce aesthetic alterations, and, therefore, are usually followed by chromatic reintegration. This work aims to study the effectiveness of mineral products as consolidants for lime-based mortars and simultaneously as chromatic treatments for pigmented renders. The studied consolidating products are prepared by mixing air lime,metakaolin, water, and mineral pigments. The idea of these consolidating and coloring products rises from a traditional lime-based technique, the limewash, widely diffused in southern Europe and in the Mediterranean area. Consolidating products were applied and tested on lime-based mortar specimens with a low binder–aggregate ratio and therefore with reduced cohesion. A physico-mechanical, microstructural, and mineralogical characterization was performed on untreated and treated specimens, in order to evaluate the efficacy and durability of the treatments. Accelerated aging tests were also performed to assess consolidant durability, when subjected to aggressive conditions. Results showed that the consolidants tested are compatible, effective, and possess good durability.

Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Ontogenetic variation of plastral spotting pattern in Phrynops hilarii (Testudines, Chelidae)

The plastral spotting variation in the chelid turtle Phrynops hilarii (Duméril & Bibron, 1835) in relation to sex, size, and geographic procedence of individuals was analyzed. States for qualitative characters were analyzed using non-parametric tests. Quantitative characters (shell and scute measurements) were standardized for body size by linear regression against carapace length, and were subjected to principal components analysis and canonical discriminant function analysis. Results suggest that increased plastral spotting is a polymorphic ontogenetic trait in P. hilarii. Neither hatchlings nor juveniles have plastral pattern moderately or heavily pigmented. The simplest pattern, however, may persist without changes in some adults. There are no differences between sexes. The spatial distribution of the plastral pattern is not ordered latitudinally or longitudinally, showing no relationship with gradients of elevation, temperature, or precipitation. This pattern trait lacks of taxonomic significance. The morphometric analysis failed to reveal any character of diagnostic utility in the plastron to support the possibility that these patterns correspond to different sympatric taxa.

Two new species of Caulleriella (Polychaeta, Cirratulidae) from Argentina

Caulleriella bremecae and C. galeanoi (Polychaeta: Cirratulidae) are newly described from Mar del Plata, Argentina (38ºS, 57ºW). Both species have small, red-pigmented nuchal organs (lateral "eyes") on the prostomium. Caulleriella bremecae sp. nov. is further characterized by a peristomium without annulations except for a narrow anterior dorsal fold, posterior part of peristomium extending posterodorsally over chaetiger 1, latter extending posterodorsally over chaetiger 2 and with slightly separated grooved palps arising middorsally; notochaetae of chaetigers 1-23 all smooth capillaries, thereafter with six bidentate sigmoid hooks and 3-4 capillaries, both types fewer on last 4-5 chaetigers; neurochaetae of chaetigers 1-2 comprised of 8 bidentate hooks and 1-2 capillaries, thereafter only bidentate hooks, decreasing in number posteriorly. The species is frequent and abundant throughout the year in the intertidal mussel beds situated 200-700m from the sewage outfall of Mar del Plata city. Caulleriella galeanoi sp. nov. is characterized by a peristomium with three annulations dorsally, with grooved palps arising from annulus 3, and by smooth capillary chaetae in noto- and neuropodia of chaetigers 1-2. Bidentate hooks are present in neuropodia from chaetiger 3, numbering 5-6 in anterior and middle chaetigers, 1-3 in posterior ones. Notopodia have 3-6 pairs of smooth capillary chaetae, decreasing in number posteriorly. From chaetiger 13-20, notopodia with 1-2 capillary chaetae and 1-2 bidentate hooks. Specimens held in aquaria supplied with water and the green alga Vaucheria sp. (Vaucheriaceae) from the intertidal zone reproduced asexually by fission. Gametes were not observed in any specimens.

Contribuição ao conhecimento das larvas dos Sarcophagidae com especial referência ao esqueleto cefálico (Diptera)

The author publishes a comparative study of eleven species of Sarcophagid flies and gives a redescription of the larval stages of Musca domestica L. as a model. The work was made upon material obtained from Sarcophagidae reared in the Laboratory. Some of them were parasitic flies from Insects and other invertebrates. Protodexia was reared using the domestic cockroach (Periplaneta americana) instead of the Orthroptera or Mantodea its true hosts. The larvae obtained by dissection of female abdomen was reared in flesh or agarhorse serum. The last proceeding is very good since the skins of all larval stages can be conserved for study and it is possible to observe the ecdyses. Some of the larvae prefer dead snails (Bulimulus and Fruticicola) and is able to destroy larvae of other species found in the same molluscs. The first stage maggot can be obtained by dissections of dried female specimens and furnishes very good characters to determine the species and establish the philogenetical relationship of the genera in the family. Th pseudocephalon presents very curious ornaments or grooves in some species (Oxysarcodexia). Sometimes there is a pigmented capsule covering a great part of the pseudocephalon (Titanogrypa). The cephaloskeletal sclerietes have a peculiar shape and constitution for every species, mainly in the first stage maggot.

Aspectos parasitários observados no local inoculado com esporozoitos de Plasmodium Gallinaceum

The following is a summary of the studies made on the development of Plasmodium gallinaceum sporozoites inoculated into normal chicks. Initially large numbers of laboratory reared Aëdes aegypti were fed on pullets heavily infected with gametocytes. Following the infectious meal the mosquitoes were kept on a diet of sugar and water syrup until the appearance of the sporozoites in the salivary glands. Normal chicks kept in hematophagous arthropod proof cages were then inoculated either by bite of the infected mosquitoes or by subcutaneous inoculations of salivary gland suspensions. By the first method ten mosquitoes fed to engorgement on each normal chick and were then sacrificed immediately afterwards to determine the sporozoite count. By the second method five pairs of salivary glands were dissected out at room temperature, triturated in physiological saline and inoculated subcutaneously. The epidermis and dermis at the site of inoculation were excised from six hours after inoculation to forty eight hours after appearance of the parasites in the blood stream and stretched out on filter paper with the epithelial surface downward. The dermis was then curretted. Slides were made of the scrapings consisting of connective tissue and epithelial cells of the basal layers which were fixed by metyl alcohol and stained with Giemsa for examination under the oil immersion lens. Skin fragments removed from normal chicks and from regions other than the site of inoculation in the infected chicks were used as controls. In these, only the normal histological aspect was ever encountered. In the biopsy made at the earliest period following inoculation clearly defined elongated forms with eight or more chromatin granules arranged in rosary formation were found. The author believes these to be products of the sporozoite evolution. Search for transition stages between these forms and sporozoites is planned in biopsies to be taken immediately following inoculation and at given intervals up to the six hour period. 1.) 6 and 12 hour periods. The bodies referred to above found in the first period in great abundance, apparently in proportion to the large numbers of sporozoites inoculated, were perceptibly reduced in numbers in the second period. 2.) 18 hour period. Only one biopsy was examined. This presented a binuclear body shown in Fig. 1, having a more or less hyaline protoplasm staining an intense blue and a narrow vacuole delimiting the cell boundaries. The two chromatin grains were quite large presenting a clearly defined nuclear texture. 3.) 24 hour period. A similar body to that above (Fig. 2) was seen in the only preparation examined. 4.) 60 hour period. The exoerythrocytic schizonts were found more frequently from this period onward. Several such were found no longer to contain the previously described vacuoles (Fig. 3). 5.) 84 hour period. Cells bearing eight or more schizonts were frequently encountered here. That these are apparently not bodies in process of division may be seen in Fig. 4. From this time onward small violet granules similar to volutine grains appeared constantly in the schizont nucleus and protoplasm. These are definitely not hemozoin. The above observations fell within the incubation period as repeated examinations of the peripheral and visceral blood were negative. Exoery-throcytic parasites also were never encountered in the viscera at this time. Exoerythrocytic schizonts searched for at site of inoculation 1, 24 and 48 hours after the incubation period were present in large number at all three times with apparent tendency to diminish as the number within the blood stream increased. Many of them presented the violet granules mentioned above. The appearance of the chromatin and the intensity of staining of the protoplasm varied from body to body which doubtless corresponds to the evolutionary stage of each. This diversity of aspect may frequently be seen in the parasites of the same host cell (Fig. 5.). These findings lend substance to the theory that the exoerythrocytic forms are the link between the sporozoites and the pigmented parasites of the red blood corpuscles. The explanation of their continued presence in the organism after infection of the blood stream takes place and their presence in cases infected by the inoculation blood does not come within the scope of this work. Large scale observations shortly to be undertaken will be reported in more detail particularly observations on the first evolutionary phases of the sporozoite within the organism of the vertebrate host.

Observation on the morphology of Australorbis glabratus

The present morphological study of A. glabratus was based on the observation of shell, radula, renal region and genitalia of 50 specimens having a shell diameter of 18 mm. In this summary we record the data pertaining to the chracteristics that can be used in systematics. The numerals refere to the mean and their standard deviation; no special reference being made, they correspond to length measurements. Shell: 18 mm in diameter, 5.59 ± 0.24 mm in greatest width, 5 to 6 whorls. Right side umbilicated, left one weakly depressed. Last whorl about thrice as tall as the penultimate one at the aperture, the measurements being taken on the right side. Aperture perpendicular or a little oblique. Body, extended: 47.06 ± 3.31 mm. Renal tube: Narrow and elongated, 23.84 ± 1.90 mm, showing a pigmented ridge along its ventral surface. Ovotestis: 12.78 ± 1.50 mm. Mainly trifurcate diverticula attaching in fan-like manner to the collecting canal (this arrangement is seen to best advantage in the cephalic middle of the ovotestis). The collecting canal greatly swells at the cephalic end, narrowing suddenly as it leaves the ovotestis. Ovisperm duct: 13.70 ± 1.68 mm, including the non-unwound seminal vesicle. The latter, situated about 1 mm from the beginning af the ovisperm duct, was 1.14 ± 0.29 mm in greatest diameter, and is beset by numerous short diverticula. Sperm duct: 14.16 ± 1.27 mm, pursuing a sinous course along the oviduct. Prostate: Prostate duct 5.53 ± 0.74 mm, collecting a row of long diverticula, the latter 21.6 ± 3.5 in number. Last diverticulum generally simple or bifurcate, penultimate generally arborescent, bifurcate or simple, antepenultimate nearly always arborescent, the remaining ones arborescent. The arborescent diverticula frequently give off secondary branches. Vas deferens: 17.50 ± 2.05 mm. The ratio vas deferens/vergic sac was 4.7 ± 0.6. Verge: 3.70 ± 0.54 mm long, 0.12 ± 0.03 mm wide. Free end tapering to a point where the sperm canal opens. No penial stylet. Vergic sac: 3.77 ± 0.50 mm long, 0.19 ± 0.01 mm wide. The length ratio vergic sac/preputium was 1 ± 0.02. Preputium: Deeply pigmented, 3.79 ± 0.40 mm long, 0.89 ± 0.12 mm wide in the middle. Muscular diaphragm between it and the vergic sac. Two muscular pilasters along its lateral walls. Oviduct: 10.24 ± 1.29 mm, suddenly swollen at the cephalic end so that it forms a folded pouch capping the beginning of the uterus. Uterus: 10.58 ± 1.18 mm. Vagina: 2.06 ± 0.15 mm long, 0.32 ± 0.05 mm wide, showing a swelling at its caudal portion, just above the opening of the spermathecal duct. Spermatheca: 1.57 ± 0.41 mm long, 0.92 ± 0.23 mm wide. Spermathecal duct 1.15 ± 0.23 mm. Radula: 125 to 163 rows of teeth (mean 141.4 ± 9.8). Radula formula 27-1-27 to 34-1-34 (mean 30.9 ± 1.7).

Observations on the morphology of Australorbis nigricans

A morphological study was done on A. nigricans, based on the observation of shell, radula, renal region and genitalia of 50 specimens measuring 18 mm in diameter. The data obtained are to be compared with those recorded in our previous paper (PARAENSE & DESLANDES, 1955) on A. glabratus. The characteristics common to both species will not be mentioned here. The numerals refere to the means and their standard deviations: no special reference being done, they correspond to length measurementes. Shell - 18 mm in diameter, 6.37 ± 0.29 mm in greatest width, 6 whorls. Prevailing colur ferruginous sepia, a minority of olivaceous, ochreous, nigrescent and deeply black specimens being found. Right side variously depressed, umbilicated, 1.5 to 3.5 mm deep from the bottom of the umblicus to the highest level of the last whorl. Left side more depressed than the right one, broadly concave, 1.5 to 3.5 mm deep. Both sides show a varously distinct keel, that looks sharper at the left. Aperture deltoid, varying in outline and width. Body, extended - 60.26 ± 3.62 mm, less pigmented than in glabratus. Renal tube - 30.68 ± 1.69 mm, showing neither ridge nor pigmented line along its ventral surface, this negative character affording a sure means of separation from glabratus. Ovotestis - 14.48 ± 1.93 mm. Ovisperm duct - 13.04 ± 1.60 mm, including the non-unwound seminal vesicle. The latter was 0.97 ± 0,21 mm in greatest width. Carrefour - Resembling that of glabratus. Sperm duct - 21.36 ± 1.53 mm. Prostate - Prostate duct 7.14 ± 0.74 mm, collecting a row of long diverticula numbering 19.6 ± 3.1 and more separate than in glabratus. Last diverticulum generally bifurcate or arborescent, the remaining ones arborescent. Vas deferens - 28.68 ± 1.38. Ratio vas deferens/vergic sac = 6.8±0.8. Verge - 3.08 ± 0.28 mm long, 0.11 ± 0.02 mm wide. Vergic sac - 3.07 ± 0.28 mm long, about 0.20 mm wide. Ratio vergic sac/preputium = 0.84 ± 0.12. Preputium - 3.69 ± 0.47 mm long, 0.85 ± 0.10 mm wide. Albumen gland - Resembling taht of glabratus. Oviduct - 16.26 ± 1.41 mm, swollen at the cephalic end. Uterus - 13.24 ± 1.19 mm. Vagina - 1.70 ± 0.22 mm, swolen at the caudal portion. Spermatheca - 2.78 ± 0.40 mm long, 0.86 ± 0.16 mm wide. Spermathecal duct 1.11 ± 0.20 mm. Radula - 125 to 168 horizontal rows of teeth (mean 153.9 ± 8.4). Radula formula 28-1-28 to 36-1-36 (mean 31.8 ± 1.9). Mode formula 31-1-31. The morphological characteristics of the renal region and shell, and the great body length in the same condition of shell diameter, distinguish A. nigricans from the most related species A. glabratus, giving support to considering it a good species from a txonomic or phenotypic standpoint (morphospecies).

Physa Marmorata Guilding, 1828 (Pulmonata: Physidae)

A description of Physa marmorata Guilding, 1828, based on material collected at its type-locality, the Caribbean island of Saint Vincent, is presented. The shell is thin, horn-colored, surface very glossy, diaphanous. Spire acute, elevated; protoconch distinct, rounded-conical, reddish-brown; five not shouldered, broadly convex whorls with subobsolete spiral lines and thin growth lines. Aperture elongated, 1.4-2.0 times as long as the remaining shell length, narrow obovate-lunate; upper half acute-angled,lower half oval,narrowly rounded at the base, outer lip sharp, inner lip completely closing the umbilical region; a very distinct callus on the parietal wall; columellar lip with a low ridge gradually merging into the callus. ratios: shell width/shell length = 0.44 - 0.52 (mean 0.47); spire length /shell lenght = 0.33-0.41 (mean 0.39); aperture length/shell lenght = 0.59-0.67 (mean 0.62). Oral lappets laterally mucronate, foot spatulate with deeply pigmented acuminate tail. Mantle reflection with 6-10 short triangular dentations covering nearly half the right surface of the body whorl, and 4-6 covering a part of the ventral wall. Body surface with tiny dots of greenish-yellow pigment besides melanin. Renal tube tightly folded in toa zigzag course. Ovotestis diverticula acinous, laterally pressed against each other around a collecting canal. Ovispermiduct with well-developed seminal vesicle. oviduct highly convoluted, merging into a less convoluted nidamental gland which narrows to a funnel-shaped uterus and a short vagina. Spermathecal body oblong, more or less constricted in the middle and somewhat curved; spermathecal duct uniformly narrow, a little longer than be body. About 20 prostatic diverticula, simple, bifurcate or divided into a few short branches, distalmost ones assembled into a cluster. Penis long, nearly uniformly narrow; penial canal with lateral opening about the junction of its middle and lower thirds. Penial sheath with a bulbous terminal expasion the tip of which isinserted into the caudal end of the prepuce. Prepuce shouldered, much wider than the narrow portion of the penial sheath. Penial sheath/prepuce ratio about 2.08 (1.45-2.75). The main extrinsic muscles of the penial complex are a retractor, with a branch attached to the bulb, and another to the caudal end of the penial sheath; and a protractor, with a branch attached to the shoulder of the prepuce and adjoining area of the penial sheath, and another to the caudal end of the penial sheath. Egg capsule C-shaped, with 10-30 elliptical eggs (snails 10mm long) measuring about 1.10 mm (0.90-1.32) through the long axis and surrounded by an inner and an outer lamellate membranes. Jaw a simple obtusely V-shaped plate. radula will be described separately.