The complete genome sequence of the protogynous hermaphrodite North American annual killifish Millerichthys robustus (Rivulidae, Cyprinodontiformes).

The Mexican Rivulus, Millerichthys robustus, is an enigmatic species of seasonal killifish endemic of the Southeast Mexico that has changed paradigms on the evolution of annualism in killifishes. This species survives in ephemeral environments that experience a period of seasonal drought that causes the death of all adult fish. However, populations persist due to their drought-resistant embryos capable of arresting their development in diapause until the next the rainy season. Millerichthys evolved unique characteristics within annual killifishes as a functional sequential hermaphroditism, in which females change to males (protogynous) under perceived conditions of mate competition. Also, M. robustus express different color phenotypes in both sexes: five-color phenotypes continuously distributed in various perceptual units between yellow and red in males, and different number of ocelli disposed in caudal peduncle in females. The phylogenetic relationships of M. robustus revealed that it is a sister clade to two non-annual species found exclusively in Cuba (Rivulus cylindraceus and Rivulus berovidesi), indicating that the annual life cycle, through the acquisition of embryonic diapause, has evolved independently in this species. Here, we present the complete genome sequences for the North American annual killifish Millerichthys robustus. The raw data and assembled genome are available in GeneBank.

Morphofunctional analysis of the gastrointestinal tract of an annual killifish: In search of structural traits related to the annual life history.

Annual killifishes have active and voracious rates in acquisition of food resources with display of behaviors that allow them to maintain high metabolic rates to deal with the harsh and unpredictable conditions prevalent in temporary pools where they inhabit. The objective of this research was to describe histologically and ultrastructurally, the digestive system of the annual killifish Millerichthys robustus to identify morphological traits related to its annual life history and digestive physiology. Also, we quantify food items along the intestine as a proxy for rates of digestion. Millerichthys has a short digestive system, associated with a carnivorous diet, with no evidence of a stomach. Instead, the presence of pharyngeal jaws with caniform teeth was documented, related to the breakdown of invertebrate exoskeletons, allowing prey fluids to be tasted by taste corpuscles related to selection of food items, and that digestive enzymes penetrate once in the intestine. The histological morphology of the intestine showed four different regions, associated with its digestive rates: (i) reception of food from the esophagus with intact pray; (ii) digestion with enzymes from the pancreas and liver/gallbladder of simple exoskeleton prey (Entognatha), and beginning of absorption; (iii) absorption of nutrients, and digestion of large-complex exoskeleton prey (Hexanauplia, Brachiopoda, and Ostracoda); and (iv) probable absorption of intact macromolecules. The second region of the intestine presented two anatomical loops and the highest thickness that may be related to reducing the speed of food transit, allowing for more efficient digestion given the large amount of food ingested by this species.

Protogynous functional hermaphroditism in the North American annual killifish, Millerichthys robustus.

Sex change (sequential hermaphroditism) has evolved repeatedly in teleost fishes when demographic conditions mediate fundamentally different sex-specific returns for individuals of particular age and size. We investigated the conditions for potential sex change in an annual killifish (Millerichthys robustus) from temporary pools in Mexico. In natural populations, we detected adults with intersex colouration and gonads. Therefore, we experimentally tested whether this apparent sex change can be generated by manipulation of ecological and social conditions, rather than being caused by environmental disturbance. We demonstrated functional protogynous (female-to-male) sex change in 60% replicates, when groups of five females interacted and had a visual and olfactory cue of a male. Only one female changed sex in any given replicate. The sex change never occurred in isolated females. Protandrous (male-to-female) hermaphroditism was not recorded. We characterized gradual changes in behaviour, colouration and gonad structure during the sex change process. The first behavioural signs of sex change were observed after 23 days. Secondary males spawned successfully after 75 days. We discuss the adaptive potential of sex change in short-lived annual fishes through the seasonal decline of males, and during colonization of new habitats. This is the first observation of functional hermaphroditism in an annual killifish.

Agonistic interactions with asymmetric body size in two adult-age groups of the annual killifish Millerichthys robustus (Miller & Hubbs, 1974).

In this study, the author evaluated two adult age groups of the Mexican rivulus Millerichthys robustus with body size asymmetries to determine the strategies used by an annual killifish during agonistic interactions of different ontogenetic stages. To achieve this goal, the author first characterized the ethogram of agonistic interactions of M. robustus composed of seven behavioural units in males and five behavioural units in females. The author then analysed agonistic interaction strategies used by males and females with body size asymmetries in two groups of different adult ages that represent different ontogenetic stages: (a) just after sexual maturity was reached, at 5 weeks of age, and (b) near natural death, at 24 weeks of age. The agonistic behaviour patterns of M. robustus were compatible with the logic of mutual assessment. Large males had an advantage during their interactions in both age groups, winning all of the encounters. Nonetheless, there was more aggression in 5-week-old fish encounters. In addition, small 24-week-old fish were more aggressive than small 5-week-old fish. These changing strategies may be because of the cost-benefits required during a fight at each ontogenetic stage. In the female encounters, size did not predict winners, as both small and large fish won a similar number of encounters, and some contests remained unresolved regardless of age group. There was a tendency for small females of any age to risk more than males in fights to maintain reproductive fitness.

Morphological patterns of cell death in ovarian follicles of primary and secondary growth and postovulatory follicle complex of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae).

The dynamics of cellular development and homeostasis of the ovary depend on the balance between proliferation and cell death throughout the reproductive cycle. Millerichthys robustus is an annual fish whose ovarian follicles develop asynchronously, allowing daily reproduction from sexual maturity until death. The objective of this research is to describe, histologically, the processes of follicular atresia and regression of postovulatory follicular complexes (POC) throughout a reproductive cycle of M. robustus. Patterns of cell death were documented by apoptosis in atretic follicles and POC, and necrosis in the POC after ovulation with an associated inflammatory response. Atretic follicles were seen from the onset of sexual maturity, during week three post-hatching (PH), both in primary growth (from the Cortical alveoli step, with folliculogenesis completed) and secondary growth Stages, with a higher prevalence in the latter. POCs were observed in different stages of regression from week four PH until the death of the fish. The apoptotic characteristics found were: (i) fragmentation of the nuclear membrane and zona pellucida, and liquefaction of the cortical alveoli and yolk; (ii) follicular cells becoming phagocytic, increasing their size, and migrating within the oocyte; and (iii) formation of an intrafollicular lumen, a product of phagocytosis of the oocyte constituents and dispersed pigments that remain after the digestion of yolk and cortical alveoli. The morphological changes of the follicular cells of the POC, from a squamous morphology after ovulation to columnar during its regression with PAS+ contents, was documented, suggesting a secretory activity.

Ovarian structure, folliculogenesis and oogenesis of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae).

Cellular aspects of oocyte development of the Mexican rivulus Millerichthys robustus were morphologically described in order to analyze ovarian function and the cellular recruitment dynamics associating it with life history strategies of annual killifishes. Millerichthys is an iteroparous batch spawner with continuous oocyte recruitment and indeterminate fecundity with asynchronous development of the follicles. It has two ovaries of cystovarian type, with a central lumen, which communicates with the outside through the caudal region of the ovary, that is, the gonoduct. From the walls of the ovary, irregular lamellae composed of germinal epithelium and vascularized stroma project. Oogenesis starts with oogonial proliferation, found alone or in nests within the germinal epithelium. The oogonia come into meiosis becoming oocytes and advancing to the chromatin nucleolus stage and to early primary growth stage. Folliculogenesis is completed in the primary growth stage and cortical alveoli step. Follicles moves toward the stroma, but they continue to be attached to the germinal epithelium through the basement membrane until ovulation. The inclusion of fluid yolk in the follicles during the secondary growth stage was observed. During ovulation, the follicle collapsed, the oocyte was released into the lumen, and the constitutive elements of the post-ovulatory follicle complex remained in the stroma.

Developmental ecology of annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae).

Populations of annual killifishes persist in temporary water bodies over the dry season through the expression of diapause in their drought-resistant embryos. Environmental cues may influence expression of the diapause phenotype during embryonic incubation. Millerichthys robustus is the only annual killifish distributed in North America. The aim of this review is to analyze the ecology of M. robustus development and contrast this with that of annual killifishes in austral locations. The temporary water bodies inhabited by M. robustus present the following environmental conditions: flood, drought, and humidity. During the flooding period, the environment presents the lowest temperatures, shortest photoperiod, and highest precipitation, and embryos were found in diapause I. The drought period features the highest temperatures and lowest precipitation, and embryos were found in diapause II. In contrast, during the humid period at the beginning of the rainy season, embryos were found in diapause I, II, and III, associated with the longer photoperiod and high temperatures. These dynamics of the diapause phenotypes can be explained by a combination of the strategies of phenotypic plasticity during flood and drought periods, and bet-hedging during the humid period. Moreover, the microenvironmental conditions in which embryos were buried could influence developmental trajectories. Developmental Dynamics 246:802-806, 2017. © 2017 Wiley Periodicals, Inc.

Morphological development of the structures related to annualism in the ovarian follicle of the killifish Millerichthys robustus (Costa,1995) (Teleostei: Cyprinodontiformes).

Ovaries of five females of the annual fish teleost species Millerichthys robustus were processed, and the development of the cortical alveoli, zona pellucida and secondary envelope during oogenesis were described. We also documented the origin of the cortical alveoli in time and space similar to the Balbiani body; the synthesis of three generations of cortical alveoli and an active zona pellucida prior to vitellogenesis, which is implicated in the entry of oils to the interior of the oocyte. We found that in this species, the diameter of the alveoli is greater than in the other teleost fish species reported in the literature, except for Fundulus heteroclitus, in which the diameter is similar. The thickness of the zona pellucida recorded in M. robustus is the greatest reported to date. Likewise, two periods of secondary envelope deposition were documented: filaments during pre-vitellogenesis and, subsequently, trapeze-shaped projections during the maturation of the oocytes. We report about development of structures that are considered key for the survival of embryos in annual fish during the long periods of diapause in their extreme habitats. The development of peripheral structures described here probably reflects the changes in the physiology of the oocytes in M. robustus. J. Morphol. 277:1219-1230, 2016. © 2016 Wiley Periodicals, Inc.

Cambios morfológicos en gametos del barbo tigre Puntius tetrazona (Cypriniformes: Cyprinidae) e implementación de la fertilización in vitro / Morphological changes in gametes of tiger barb Puntius tetrazona (Cypriniformes: Cyprinidae) and the implementation of in vitro fertilization

The production of ornamental fishes represents an economic activity of a growing number of Mexican families. Nevertheless, the reproduction of fish in captivity is one of the complications faced by farmers. This study was set up to: (i) evaluate the morphological and functional changes induced by hydration in the gametes of fish tiger barb (Puntius tetrazona; 240 samples) at tree times after hydration (10, 20 and 30s) with classic spermograms (volume, sperm concentration, viability, motility, and normal morphology); and (ii) evaluate the implementation of in vitro fertilization based on the ovulation rate, the percentage of fertilization and hatching, and the larval numbers obtained after 72 hours. The average volume of milt was 3.0±0.7μL, and the minimum, maximum and average concentration of sperm was 44.4x10(6) spz/mL, 52.3x10(6)spz/mL, and 48.1±5.9x10(6)spz/mL, respectively. The viability and motility of the sperm was 84.6±3.2% and 81.5±2.2%, respectively. The diameter of the sperm with/without water contact was 2.1±0.6μm and 3.8±1.0μm (p<0.05); the largest diameter was recorded 30 seconds after the contact with water. For oocytes, the smaller and larger diameters were recorded at 10 and 30s, respectively (both with/without water contact); the oocytes diameters after 10 and 30 seconds of contact with water were 1.11 and 1.55mm, respectively. A higher ovulation rate was recorded using the in vitro fertilization: 250±50 oocytes versus 28±09 oocytes (during natural fertilization; p<0.05). Nevertheless, fertilization and hatching rates were higher for the natural fertilization (80 and 60%, respectively). Considering the number of larvae obtained after 72 hours, our results showed a higher value for the in vitro fertilization (75±18 compared to 13.4±12 of the natural fertilization; p<0.05). We propose this fish as a model for other ornamental fishes of commercial interest. Our results demonstrate that the in vitro fertilization is a very high viable option to optimize and maximize resources; besides, the reproduction management optimization under controlled conditions may enhance wild fish stocks preservation. Rev. Biol. Trop. 62 (4): 1353-1363. Epub 2014 December 01.

El objetivo del presente estudio fue conocer las características de los gametos de Puntius tetrazona (n=240), los cambios morfológicos a partir de su activación mediante espermogramas cásicos y por otro lado, se evaluó la implementación de la fertilización in vitro a partir de la tasa ovulatoria, el % de fertilización y eclosión y el número de larvas vivas a las 72h. El volumen promedio de semen fue de 3.0±0.7µL. La concentración espermática mínima, máxima y promedio fue 44.48x10(6)spz/mL, 52.3x10(6)spz/mL y 48.1±5.9x10(6)spz/mL, respectivamente. La viabilidad promedio fue de 84.68±3.27%. La motilidad promedio fue 81.53±2.28%. El diámetro de los espermatozoides fluctuó entre 2.16±0.2 y 2.79±0.3µm; 3.84±0.3 y 4.86±0.31µm sin y con contacto con el agua respectivamente, con diferencias significativas. El diámetro mayor fue a los 30s en contacto con el agua. Los ovocitos de menor y mayor diámetro se registraron a los diez y 30s sin y con contacto con el agua respectivamente. Los diámetros de los ovocitos en diez y 30s en contacto con el agua fluctuaron entre 1.11 y 1.55mm respectivamente. La mayor tasa ovulatoria fue en la fertilización in vitro con 250±50 ovocitos frente a 28±09 de la natural, con diferencias significativas. Los porcentajes de fertilización y eclosión fueron más elevados en la fertilización natural con 80% y 60% respectivamente. Se registraron 75±18 larvas a las 72 horas en el grupo in vitro comparado con 13.4±12 larvas de la fertilización natural. Con lo anterior, la técnica que permitió mayor cantidad de larvas fue la de fertilización in vitro.

[Morphological changes in gametes of tiger barb Puntius tetrazona (Cypriniformes: Cyprinidae) and the implementation of in vitro fertilization]. / Cambios morfológicos en gametos del barbo tigre Puntius tetrazona (Cypriniformes: Cyprinidae) e implementación de la fertilización in vitro.

The production of ornamental fishes represents an economic activity of a growing number of Mexican families. Nevertheless, the reproduction of fish in captivity is one of the complications faced by farmers. This study was set up to: (i) evaluate the morphological and functional changes induced by hydration in the gametes of fish tiger barb (Puntius tetrazona; 240 samples) at tree times after hydration (10, 20 and 30s) with classic spermograms (volume, sperm concentration, viability, motility, and normal morphology); and (ii) evaluate the implementation of in vitro fertilization based on the ovulation rate, the percentage of fertilization and hatching; and the larval numbers obtained after 72 hours. The average volume of milt was 3.0 ± 0.7 µL, and the minimum, maximum and average concentration of sperm was 44.4 x 10(6) spz/mL, 52.3 x 10(6) spz/mL, and 48.1 ± 5.9 x 10(6) spz/mL, respectively. The viability and motility of the sperm was 84.6 ± 3.2% and 81.5 ± 2.2%, respectively. The diameter of the sperm with/without water contact was 2.10 ± 6 µm and 3.8 ± 1.0 µm (p < 0.05); the largest diameter was recorded 30 seconds after the contact with water. For oocytes, the smaller and larger diameters were recorded at 10 and 30s, respectively (both with/without water contact); the oocytes diameters after 10 and 30 seconds of contact with water were 1.11 and 1.55 mm, respectively. A higher ovulation rate was recorded using the in vitro fertilization: 250 ± 50 oocytes versus 28 ± 09 oocytes (during natural fertilization; p < 0.05). Nevertheless, fertilization and hatching rates were higher for the natural fertilization (80 and 60%, respectively). Considering the number of larvae obtained after 72 hours, our results showed a higher value for the in vitro fertilization (75 ± 18 compared to 13.4 ± 12 of the natural fertilization; p < 0.05). We propose this fish as a model for other ornamental fishes of commercial interest. Our results demonstrate that the in vitro fertilization is a very high viable option to optimize and maximize resources; besides, the reproduction management optimization under controlled conditions may enhance wild fish stocks preservation.

[Performance of recirculating aquaculture systems in the intensive farming of Pacú Piaractus mesopotamicus (Characiformes: Characidae)]. / Desempeño de los sistemas acuícolas de recirculación en el cultivo intensivo del Pacú Piaractus mesopotamicus (Characiformes: Characidae).

An alternative to intensify fish production, reducing the environmental impact and production costs are recirculating aquaculture systems (RAS). The performance of a RAS was evaluated, as fish growth and water quality conditions, in a culture of Piaractus mesopotamicus reared for ornamental purposes. Two commercial food brands with different protein contents (18%-T1 and 28%-T2), were given to juvenile fishes during an eight weeks period. Growth was measured bi-weekly: standard length (Lp), peak height (A), weight and multiple condition factor (KM). The evaluation of water parameters included: dissolved oxygen, NH3-NH4, pH, NO2, NO3, KH carbonate hardness and PO4. NH3-N rate production was analyzed following Timmons-Ebeling model. Results demonstrated significant differences in weight only, and T2 showed a 7.5% higher value than T1; nevertheless, treatment T1 had a higher KM. In general, water quality values were suitable for growth: OD=T1: 4.23 +/- 1.23; T2: 4.13 +/- 0.86; NH3=T1: 0.02 +/- 0.02; T2: 0.06 +/- 0.10; however, pH was an exception (T1: 6.95 +/- 0.98; T2: 7.11 +/- 1.03), displaying lethal rates (<5) by the fifth week. Systems NH3 removal had a 99.4% to 100% efficiency. Final fish biomass was 22.03kg for T1 and 27.49kg for T2. We concluded that the systems were able to maintain suggested density up to the experimental fifth week. Water quality parameters remained in suitable levels, with the pH exception. Cultured fishes reached their commercial size (10cm) in eight weeks.

Desempeño de los sistemas acuícolas de recirculación en el cultivo intensivo del Pacú Piaractus mesopotamicus(Characiformes: Characidae)

Performance of recirculating aquaculture systems in the intensive farming of Pacú Piaractus mesopotamicus (Characiformes: Characidae). An alternative to intensify fish production, reducing the environmentalimpact and production costs are recirculating aquaculture systems (RAS). The performance of a RASwas evaluated, as fish growth and water quality conditions, in a culture of Piaractus mesopotamicus reared forornamental purposes. Two commercial food brands with different protein contents (18%-TI and 28%-T2), weregiven to juvenile fishes during an eight weeks period. Growth was measured bi-weekly: standard length (Lp),peak height (A), weight and multiple condition factor (KM). The evaluation of water parameters included: dissolvedoxygen, NH3-NH4, pH, NO2, NO3, KH carbonate hardness and PO4. NH3-N rate production was analyzedfollowing Timmons-Ebeling model. Results demonstrated significant differences in weight only, and T2 showeda 7.5% higher value than T1; nevertheless, treatment T1 had a higher KM. In general, water quality valueswere suitable for growth: OD=T1: 4.23±1.23; T2: 4.13±0.86; NH3=T1: 0.02±0.02; T2: 0.06±0.10; however,pH was an exception (T1: 6.95±0.98; T2: 7.11±1.03), displaying lethal rates (<5) by the fifth week. SystemsNH3 removal had a 99.4% to 100% efficiency. Final fish biomass was 22.03kg for T1 and 27.49kg for T2. Weconcluded that the systems were able to maintain suggested density up to the experimental fifth week. Waterquality parameters remained in suitable levels, with the pH exception. Cultured fishes reached their commercialsize (10cm) in eight weeks.

Una alternativa que permite intensificar la producciónacuícola, y reducir el impacto ambiental y los gastosde operación son los sistemas acuícolas de recirculación(SAR). Se evaluó el desempeño de SAR AquaticEcosystems® en el cultivo ornamental de Piaractus mesopotamicus,y se estimó el crecimiento y la calidad del agua,con dos alimentos comerciales de 18 (T1) y 28% (T2) deproteínas. Del crecimiento se midieron quincenalmente:la longitud patrón, altura máxima, peso y factor de condiciónmúltiple (KM). De la calidad de agua se evaluósemanalmente, O.D., NH3-NH4, pH, NO2, NO3, KH y PO4.Con el modelo de Timmons-Ebeling se calculó la tasa deproducción de NH3-N. Los resultados muestran diferenciassignificativas en el peso, donde T2 presentó un incrementode 7.14% más que T1, que mostró un mayor KM. La calidad del agua fue adecuada en los parámetros limitantes(OD=T1: 4.23±1.23; T2: 4.13±0.86; NH3=T1: 0.02±0.02;T2: 0.06±0.10), a excepción del pH (T1: 6.95±0.98; T2:7.11±1.03) que presentó valores letales (<5) a partir de laquinta semana. La remoción de NH3 fue de 99.4 a 100%. La biomasa final fue de T1: 22.03kg y T2: 27.49kg. Se concluye que los sistemas pudieron alojar la densidad propuestasolo hasta la quinta semana de experimentación. Los peces tardaron cuatro quincenas en llegar a talla comercial de ornato.