Comparative analysis of the oocytes and early development of two species of curimatidae teleost fish.

Curimatella lepidura and Steindachnerina elegans are small forage fish, constituting an important link in the food chain, serving as food for larger commercial fish. In this study, characteristics of the eggs, of the oocyte's surface ultrastructure and of the embryogenesis are first described for these species. Absolute fecundity was 40864 ± 8769 oocytes for C. lepidura and 22089 ± 8710 oocytes for S. elegans. Oocytes of both species are yellowish, weakly adhesive and with a post-fertilization diameter of 1019.5 ± 20.6 µm and 978.75 ± 29.16 µm for C. lepidura and S. elegans, respectively. The ultrastructural analysis, using scanning electron microscopy, showed that the oocyte's surface of both species has pore canals over the entire surface and a funnel-shaped micropyle. At 24°C, the embryonic development of C. lepidura was completed 25 h after fertilization, and blastopore closure occurred in 7 h 30 min. In S. elegans, larvae hatched 20 h after fertilization, and blastopore closure occurred in 7 h 15 min. The fertilization rate was 74.5 ± 7.96 and 71.2 ± 10.8% for C. lepidura and S. elegans, respectively. This study provides important support for clarifying phylogenetic relationships and in ecological and zoological understanding of Neotropical Curimatidae fish.

Reproduction and embryogenesis of the mandi-amarelo catfish, Pimelodus maculatus (Pisces, Pimelodidae), in captivity.

To study reproduction and embryogenesis, Pimelodus maculatus specimens were kept in captivity and captured bimonthly during 1 year. Gonads samples (211 specimens) were collected and submitted to routine histological techniques. Pimelodus maculatus prepared to reproduce when water temperature was high, and even reached advanced maturation but did not spawn in captivity. Spent fish gonads were not documented, and atretic follicles were frequent (60%) in late maturation females. When then submitted to hypophysation, 70% of the females responded positively to hormonal treatment. Oocyte extrusion occurred 8 h after a second hormonal injection at 26°C. The fertilisation rate was 65.1 ± 9.2% at 24°C. Recently spawned oocytes of P. maculatus were spherical, non-adhesive, yellow in colour, with an average diameter of 1113.92 ± 37.02 µm and covered by a thick gelatinous layer. Blastopore closure occurred 7 h and 30 min after fertilisation. Embryonic development was completed within 18 h after fertilisation. The results of this work provide important knowledge for the handling and cultivation of not only P. maculatus, but other species of potential value for fish culture.

Apoptosis, cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish.

Aiming to better understand folliculogenesis, this study evaluated cell death and proliferation of ovarian cells, besides cathepsin-D expression in Prochilodus argenteus captured in two sites of the São Francisco River downstream from the Três Marias Dam, Brazil. In the site immediately following the Dam (S1), low levels of dissolved oxygen were registered in the rainy period. The water temperature was higher in the São Francisco River immediately after the confluence with the Abaeté River (S2), regardless of the period. In S1, the ovaries showed smaller oocytes, high caspase-3 enzymatic activity and apoptosis, lower cells in proliferation and GSI, as well as a lesser quantity of cathepsin-D when compared to females captured from S2. Regarding relative frequency of ovarian structures, in the dry period, only oogonia and perinucleolar oocytes were found in fish ovaries from both sites. On the other hand, in the rainy period, the relative frequency of oogonia and perinucleolar oocytes decreased and the vitellogenic oocytes increased in S2. Postovulatory follicles were observed only in S2, whereas atretic follicles occurred at a higher frequency in S1. Our results showed that apoptosis, cell proliferation and cathepsina-D evaluation can be used as biomarkers of environmental impact.

Eggs ultrastructure and early development of Franciscodoras marmoratus (Pisces: Doradidae).

This study presents, for the first time, information on the eggs and early development of Franciscodoras marmoratus, fish of São Francisco river, Brazil. To analyse the egg ultrastructure and morphological events of embryogenesis, a total of 36 F. marmoratus specimens (18 males and 18 females) were captured and subjected to spawning induction. Gametes were collected by manual extrusion, and fertilization was conducted using the dry method. After fertilization, eggs were kept in incubators with water temperature of 24°C. The embryonic development was monitored using a stereomicroscope until hatching. There was a 67% positive response to hypophysation by the females and the fertilization rate was 73.8 ± 6.2%. The oocytes are discoid, yellow, adhesive and covered by a thick jelly coat. Under the electron scanning microscope, the oocytes presented a surface with pore canals and funnel-shaped micropyle with a smooth vestibule. Recently extruded oocytes had a mean diameter of 1.27 ± 0.4 mm and after hydration was 1.91 ± 0.05 mm. The jelly coat was 0.34 ± 0.03 mm thickness, and the perivitelline space was 0.19 ± 0.04 mm. Eight phases of the embryonic development were identified, and embryogenesis was completed at 47 h after fertilization, at 24°C water temperature. The recently hatched larvae had 2.76 ± 0.57 mm of total length. These results provide useful information for the successful breeding and reproductive strategies of fishes.

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes).

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g., granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.

Relationship among follicular apoptosis, integrin beta1 and collagen type IV during early ovarian regression in the teleost Prochilodus argenteus after induced spawning.

Early ovarian regression was analyzed in the neotropical freshwater teleost, curimatã-pacu (Prochilodus argenteus), in order to evaluate follicular apoptosis, basement membrane morphology, and integrin beta1 and collagen type IV immunostainning in postovulatory follicles. Mature females were induced to spawn by using carp pituitary extract for study of ovarian regression up to 5 days post-spawning. Morphometric analyses showed that the postovulatory follicle area decreased progressively after spawning and was coupled to the gonadosomatic index (r=0.92). During ovarian regression, follicular cells detached from the neighboring cells and basement membrane and then died by apoptosis. The follicular basement membrane became thicker and diffuse and was breached during regression of the postovulatory follicles. Follicular apoptosis was detected by TUNEL, histology, and electron microscopy. The ladder pattern of apoptotic DNA was revealed by agarose gel electrophoresis. The apoptotic index for the follicular cells increased until 3 days post-spawning and decreased thereafter. Immunohistochemistry reactions detected caspase 3, integrin beta1, and collagen type IV in the follicular layer of the postovulatory follicles. Labeling for integrin beta1 and collagen type IV decreased significantly, whereas a peak in cell death occurred 3 days post-spawning. At 4-5 days post-spawning, the connective theca was more thickened and vascularized. Simultaneously, granulocytes migrated toward the follicular lumen. Thus, follicular apoptosis contributes to early ovarian regression in P. argenteus. Additionally, our findings suggest integrin beta1 and collagen type IV as possible survival factors for follicular cells in teleost ovary.

Continuous gametogenesis in the neotropical freshwater teleost, Bryconops affinis (Pisces:Characidae).

The gametogenesis of Bryconops affinis was studied by light, transmission and scanning electron microscopy. The spermatogenesis is semi-cystic and spermatids are released into the lumen of seminiferous tubules, where spermiogenesis is completed. Spermatozoa have an ovoid head, a rudimentary middle piece with a small number of mitochondria and long flagellum (primitive spermatozoa). The Sertoli and Leydig cells show secretory activity during spermatogenesis. By the end of this phenomenon, the Sertoli cells phagocytize the residual spermatozoa, while the Leydig cells show involuted characteristics. With regard to the oogenesis process, the oocyte development was divided into four stages based on the cytological characteristics of the oocyte and its surrounding layers. Ultrastructural analysis revealed that the zona pellucida is formed during the previtellogenic stage. Specializations associated to the outer layer of the zona pellucida may be related to the egg's adherence to the substrata.

Postovulatory follicle: a model for experimental studies of programmed cell death or apoptosis in teleosts.

This is the first evidence of programmed cell death, or apoptosis, occurring in the postovulatory follicle (POF) of teleost fish. Females of Astyanax bimaculatus lacustris were submitted to induced ovulation through injecting pituitary extract. Ultrastructural analyses of POFs at time intervals varying from zero to four days postspawning showed several characteristic events of the apoptosis. Typical apoptotic figures, such as nucleus with chromatin condensation underlying the nuclear envelope in a crescent pattern and apoptotic bodies at different stages of formation and reabsorption, were observed in the follicular cells a few days after the onset of the postovulatory period. The results indicated that apoptosis is the major mechanism responsible for the elimination of the follicular cells in the POFs of A. bimaculatus lacustris during ovarian recovery postspawning. It is suggested that POFs might be used as an experimental model in dynamic studies involving cell death in teleosts.

A new species of Calomys (Rodentia, Sigmodontinae) from Central Brazil identified by its karyotype.

Ten species of small rodents of genus Calomys are found in South America. Three of these ten species are known to occur in Brazil: C. tener, C. laucha and C. expulsus (= C. callosus expulsus). Almost all Calomys karyotypes are made up of acrocentric pairs. In this paper we describe a new karyotype with 2n = 46 (FN = 66), including 11 meta/submetacentric and 11 acrocentric autosomal pairs. This is not related to any described Calomys karyotype. The X chromosome is a medium submetacentric and the Y is a small acrocentric. This new karyotype is briefly compared to karyotype of the sympatric species C. tener (2n = 66, FN = 66). The reduced diploid number and small amount of pericentromeric heterochromatin observed in the biarmed chromosomes that contrasts to large blocks seen in acrocentrics seem to indicate that centric fusion and loss of constitutive heterochromatin have led to the new karyotype. Cytogenetic evidence suggests strongly that a new species with 2n = 46 from Central Brazil should be described in the genus Calomys.

Ovarian follicular atresia in two teleost species: a histological and ultrastructural study.

Follicular atresia is a common phenomenon in vertebrate ovaries involving the oocyte and the follicular wall degeneration. Female Astyanax bimaculatus lacustris and Leporinus reinhardti were kept in aquaculture cages inside tanks from February 1994 to January 1995 for the study of the characteristics of different stages of follicular atresia. Histological and ultrastructural analysis demonstrated similarities in the degenerative events and in the resorption of oocytes in both species. Degradation of organelles, such as mitochondria, cortical alveoli, and annulate lamellae occurred in the peripheral ooplasm during the initial stage of the process. Follicle cells showed marked phagocytic activity with digestive vacuoles, myelin figures, and lipofuscin granules during the intermediate and advanced stages of follicular atresia. Granulocytes were in activity during the final stage of follicle resorption. The duration of follicular atresia was 4 months in Leporinus reinhardti and 7 months in Astyanax bimaculatus lacustris. When submitted to induced reproduction in December 1995, 50-60% of the females of both species responded to induced spawning, indicating the recovery of gonadal activity. It is suggested that, in captive conditions, follicular atresia is shorter in total-spawning fishes when compared to those showing partial spawning, and that it has no apparent deleterious effects on induced reproduction in the subsequent cycle.

Studies on prevalence of Strongyloides infection in Holambra and Maceió, Brazil, by the agar plate faecal culture method.

Prevalence of Strongyloides stercoralis infection in three areas of Brazil was surveyed by a recently developed faecal culture method (an agar plate culture). The Strongyloides infection was confirmed in 11.3% of 432 subjects examined. The diagnostic efficacy of the agar plate culture was as high as 93.9% compared to only 28.5% and 26.5% by the Harada-Mori filter paper culture and faecal concentration methods, when faecal samples were examined simultaneously by these three methods. Among the 49 positive samples, about 60% were confirmed to be positive only by the agar plate culture. These results indicate that the agar plate culture is a sensitive new tool for the correct diagnosis of chronic Strongyloides infection.

Tamanho de primeira maturaçäo sexual do Schizodon knerii e do Leporinus piau (Teleostei, Anostomidae) na represa de Três Marias, MG / First sexual maturation of Schizodon knerii and of Leporinus piau (Teleostei, Anostomidae) from Três Marias reservoir, MG - Brazil

Determinaram-se os tamanhos de primeira maturaçäo sexual (L50) para S. knerii, piau-branco e para L. piau, piau-gordura. Diferenciaram-se peixes juvenis de peixes adultos por critérios microscópicos. O comprimento padräo correspondente à primeira maturaçäo sexual (L50) foi estimado em: S. knerii 14,5 cm para fêmeas e 2,3 cm para machos; L. piau 10,2 cm para fêmeas e 9,6 cm para machos. Todos os peixes estavam aptos a reproduzirem-se (L100) a partir de: S. knerii 17,5cm para fêmeas e 14,7cm para machos; L. piau 12,0cm para fêmeas e 10,8cm para machos. O coeficiente de correlaçäo (r) entre frequência de adultos e classes de comprimento padräo foi superior a 0,97 em cada caso indicando eficiência do método

Hipofisaçäo do peixe Prochilodus affinis da bacia do Rio Säo Francisco / Hypophysation of the fish Prochilodus affinis from the Rio Sao Francisco basin, Brazil

P. affinis é um peixa da bacia do Rio Säo Francisco de importância na pesca comercial. Ele é iliófago, de piracema e pode atingir cerca de 6 kg de peso corporal (PC). Os dados aqui apresentados referem-se à hipofisaçäo de 154 fêmeas e 225 machos na Estaçäo de Hidrobiologia e Piscicultura de Três Marias, CODEVASF, Três Marias, MG, Brasil. Utilizou-se extrato bruto de hipófise de carpa (EBHC) em duas doses para as fêmeas (0,8 mais ou menos 0,2 mg de EBHC/kg de PC e 5,7 mais ou menos 0,4 mg de EBHC/kg de PC), com intervalo de 16,5 mais ou menos 2,3 h entre doses, e uma dose para os machos (2,8 mais ou menos 0,4 mg de EBHC/kg de PC). Cerca de 76 por cento das fêmeas tratadas liberaram ovos viáveis a 219 mais ou menos 12 horas-grau, à temperatura da água de 23,9 mais ou menos 0,8§C. Os ovos eram livres, demersais e de cor cinza. Eles mediram 1,4 mais ou menos 0,1 mm de diâmetro e aumentaram cerca 9,8 vezes seu volume após a hidrataçäo. A razäo entre peso da ova: peso corporal = 22,1 mais ou menos 4,1 por cento, o número de ovos/g de ova = 1222 mais ou menos 119 e a taxa de fertilizaçäo (estimada por ocasiäo do fechamento do blastóporo) = 71 mais ou menos 16 por cento. A fertilidade inicial relativa (número de ovos extruídos/kg PC) = 269,9 x 103 mais ou menos 53,7 x 10 ao cubo e a fertilidade final relativa (número de ovos viáveis por ocasiäo do fechamento do blastóporo/kg PC) = 201,3 x 10 elevado ao cubo mais ou menos 52,9 x 10 elevado ao cubo. As relaçöes entre PC e fertilidades inicial (FI) e final (FF) foram expressas, respectivamente, por: FI = 33.085 + 227.231 PC e FF = 25.852 + 169.987 PC. Quando estes valores de FI, FF e PC foram acumulados (a), as equaçöes foram: Fla = 162.066 + 269.042 PCa e FFa = 95.567 + 204.173 PCa

Hipofisaçäo do peixe anostomídeo piau-branco Schizodon knerii da bacia do Rio Säo Francisco / Hypophysation of the anostomid fish white-piau Schizodon knerii from the Rio Sao Francisco basin

Schizodon knerii é um peixe importante na pesca profissional na represa de Três Marias onde ele pode atingir cerca de 1,2kg de peso corporal (PC). Quatorze fêmeas e 14 machos foram hipofisados com extrato bruto de hipófise de carpa (EBHC). As fêmeas receberam 2 doses de EBHC (1a. dose = 0,7 mais ou menos 0,2mg de EBHC/kg de PC; segunda dose = 5,6 mais ou menos 0,5mg de EBHC/kg de PC; intervalo entre doses = 18,6 mais ou menos 0,5h) e os machos receberam dose única de EBHC (2,6 mais ou menos 0,2 mg de EBHC/kg de PC). Nove fêmeas produziram ovos viáveis sob massagem abdominal 210 mais ou menos 7,9 horas-grau após a 2a. dose, estando a temperatura da água a 23,9 mais ou menos 1§C. Os ovos eram opacos, cinza ou marrom-claro e adesivos. Eles mediram 1,1 mais ou menos 0,1 mm antes de se hidratarem e 1,3 mais ou menos 0,1 mm após hidrataçäo. A razäo peso da ova: peso corporal (porcentagem) = 8,5 mais ou menos 1,4, o número de ovos/g de ova = 2448 mais ou menos 59 e a taxa de fertilizaçäo (estimada por ocasiäo do fechamento do blastóporo, em porcentagem) = 69 mais ou menos 9,4. Fertilidade inicial (i.e., o número de ovos extruídos x 10 elevado ao cubo) = 118,9 mais ou menos 33,5 enquanto que fertilidade final (i.e., o número de ovos viáveis por ocasiäo do fechamento do blastóporo x 10 elevado ao cubo) = 82,8 mais ou menos 28,6. As relaçöes entre PC e fertilidade inicial (FI) e final (FF) foram expressas, respectivamente, por: FI = -72010 + 340 PC (r ao quadrado = .88; n=9) e FF = -71442 + 274 PC (r ao quadrado = .79; n=9)

Aspectos reprodutivos da piranha, Pygocentrus piraya (Teleostei, Characiformes), espécie nativa da bacia do Rio Säo Francisco / Reproductive aspects of piranha Pygocentrus piraya (Teleostei, Characiformes), a native species from the Rio Sao Francisco basin, Brazil

Analisaram-se histologicamente gônadas de 305 exemplares de Pygocentrus piraya, capturados no reservatório de Três Marias, MG, no período de julho/85 a junho/86. Determinaram-se os estádios de desenvolvimento dos ovócitos e das gônadas. De desova parcelada, aparentemente essa espécie de piranha reproduz-se no próprio reservatório, durante os meses de janeiro a abril, quando o nível de água está em elevaçäo. Existem indicaçöes que os machos acham-se aptos a reproduzirem-se durante todo o ano. Näo foram observados machos esgotados

Aspectos da biologia reprodutiva da corvina Pachyurus squamipinnis Agassiz, 1829 (Teleostei, Sciaenidae) na Represa de Três Marias, MG / Reproductive biological aspects of the corvina Pachyurus squamipinnis Agassiz, 1829 (Teleostei, Sciaenidae) in the Tres Marias reservoir, MG, Brazil

Gônadas de 599 exemplares de P. squamipinnis, capturados na represa de Três Marias no período de julho/85 a junho/86, foram analisadas por métodos histológicos. A maioria dos peixes encontravam-se em repouso. Machos em maturaçäo foram observados durante todo o ano. Somente nove fêmeas em maturaçäo avançada foram registradas e fêmeas desovadas näo foram capturadas. Entretanto, há indicaçöes de que a espécie completa seu ciclo reprodutivo na represa. O tamanho de primeira maturaçäo sexual (L50) de fêmeas foi estimado em 21,4cm de comprimento padräo (CP). Todas as fêmeas estäo aptas a reproduzirem-se (L100) a partir de 26,5cm CP

Ciclo reprodutivo do tucunaré Cichla ocellaris (Schneider, 1801) na represa de Três Marias, MG / Reproductive cycle of tucunare Cichla ocellaris (Schneider, 1801) in the Tres Marias reservoir, MG, Brazil

Pela análise microscópica de gônadas constatou-se que a desova de C. ocellaris é do tipo parcelada e que peixe em estádio de maturaçäo ocorrem durante todo o ano. Registrou-se maior frequência de fêmeas desovadas no mêes de janeiro, quando a precipitaçäo pluviométrica na regiäo de Três Marias é alta e o nível de água da represa começa a subir

Efficacy of stool examination for detection of Strongyloides infection.

To determine the efficacy of stool examination for detection of Strongyloides infection, 1,350 stool samples collected in Japan, Brazil, and Thailand were examined by four different methods (direct fecal smear, formalin-ether concentration. Harada-Mori filter paper culture, and agar plate culture). The newly developed agar plate culture method was highly effective; more than 96% of the positive cases were diagnosed by this method. The coprologic examination, however, was not sensitive enough for detecting chronic infections because more than 40% of the positive cases were overlooked even when persons with proven Strongyloides infection were re-examined several months later without intervening treatment. Therefore, it is essential to examine stool samples repeatedly to achieve a correct diagnosis, and even so, it is important to note that a negative result does not necessarily indicate the unequivocal absence of the infection.

Prevalence of intestinal parasitic infection in five farms in Holambra, São Paulo, Brazil.

A parasitological survey was carried out on 222 inhabitants of five farms in Holambra, located 30 km north of Campinas, São Paulo, Brazil, on October 1992. Approximately 70% of the inhabitants were found to be infected with at least one species of intestinal parasite. The positive rates of 6 helminths and 7 protozoan species detected are as follows: 5.4% Ascaris lumbricoides; 8.6% Trichuris trichiura; 19.8% Necator americanus; 10.4% Strongyloides stercoralis; 1.4% Enterobius vermicularis; 0.9% Hymenolepis nana; 3.2% Entamoeba histolytica; 2.7% E. hartmanni; 9.9% E. coli; 14.0% Endolimax nana; 2.3% Iodamoeba butschlii; 10.4% Giardia lamblia; 37.8% Blastocystis hominis. The positive rates of helminth infection were generaly higher in the younger-group under 16 years-old than those in the elder group aged 16 or more, whereas the infection rates of protozoan species were higher in the elder group. The infection rate of Strongyloides was found to be 10.4% by a newly developed sensitive method (an agarplate culture methods).

Serodiagnosis of strongyloidiasis. The application and significance.

Parasitological diagnosis based on the faecal examination is frequently difficult in cases of chronic, low-level S. stercoralis infection. Even when a newly developed sensitive method (an agar plate culture) is applied, it is essential to examine faecal samples repeatedly to achieve a correct diagnosis. Additionally, it is important to note that a negative result does not necessarily indicate the unequivocal absence of the infection. On the other hand, several serological tests which have recently been developed for strongyloidiasis have proven reliable when used to complement parasitological examination. We have developed two serological tests, ELISA and GPAT, to demonstrate Strongyloides infection and possible applications of the serological tests for diagnosis, mass-screening, epidemiological study and postchemotherapy evaluation of strongyloidiasis were reviewed based on our recent studies.