Reproductive physiology of Arapaima gigas (Schinz, 1822) and development of tools for broodstock management

Arapaima gigas is the largest scaled freshwater fish in the world reaching over 250 kg. With growth rates of 10 kg+ within 12 months, A. gigas is considered as a promising candidate species for aquaculture development in South America. However, the lack of reproductive control in captivity is hindering the industry expansion. The work carried out in this doctoral thesis therefore aimed to better understand the species’ reproductive physiology, develop tools to identify gender and monitor gonad development, test hormonal therapies to induce ovulation and spawning and characterise the cephalic secretion for its potential roles in pheromone release and during parental care. Initially, a genomic study investigated the overall extent of polymorphism in A. gigas, which was found to be surprisingly low, with only 2.3 % of identified RAD-tags (135 bases long) containing SNPs. Then, a panel with 293 single nucleotide polymorphism (SNP) was used to characterise the genetic diversity and structure of a range of Amazon populations. Results revealed populations from the Amazon and Solimões appeared to be genetically different from the Araguaia population, while Tocantins population comprised individuals from both stocks. This data provided a tool for broodstock identification and future management. The PhD then aimed to evaluate the effects of slow-release mGnRH implants and different broodstock size pairings on maturation and spawning. Results showed that the implants stimulated the brain-pituitary-gonad axis resulting in increased plasma levels of testosterone (females) and 11-ketotestosterone in males, respectively regardless of pairing sizes. However, no spawning was observed. Results also showed the release of sex steroids with potential pheromonal action through the cephalic secretion, a biological fluid released from the adult head along the reproductive period. Thereafter, a non-surgical field endoscopy method was developed and validated for ovarian assessment and gender identification. The method was then used to describe the female gonopore and obtain biopsy of the ovary through cannulation which allowed the description of oogenesis in A. gigas. Importantly, oocytes obtained by cannulation confirmed that adult females under investigation were maturing with oocytes in final maturation stage but failed to ovulate/spawn. Another hormonal induction trial was therefore performed in which a combination of GnRHa (mGnRHa/sGnRHa) was used by injection to induce ovulation and spawning in selected maturing females with effects on oocyte maturation monitored post-induction through biopsy. However, this trial appeared to not be successful at inducing ovulation or spawning. Finally, the peptidome and proteome of the cephalic secretion was further characterised through the comparison between parental and non-parental fish. Results highlighted the complex role of this biological fluid including potential roles on the developing offspring during the parental care period. Overall, this doctoral thesis provided new basic and applied data on A. gigas reproduction and tools that can be used in future studies to better understand the environmental and hormonal control of oogenesis and spawning

Endoscopy and Cannulation as Non-Invasive Tools to Identify Sex and Monitor Reproductive Development in Arapaima gigas

The lack of tools for sex identification and assessment of gonadal development are hindering our ability to study the reproductive dysfunction of Arapaima gigas in captivity. This study initially aimed to validate a non-surgical endoscopy procedure to identify sex in juveniles and assess stage of ovary development in female broodstock under field operational conditions. Cannulation, assisted through the description of the genital anatomy, made ovarian biopsy possible to describe oocyte development from primary growth to pre-ovulation, providing a first classification scheme for oogenesis in the species including description of the micropyle morphology using scanning electron microscopy. Cannulation was also successfully performed without endoscopic guidance, which allowed monitoring of ovarian development along the reproductive season together with profiling of plasma sex steroids (17β-estradiol [E2] and 11-ketotestosterone [11-KT] in females and males, respectively). The monitoring of our study population showed females paired with males in earthen ponds sexually matured and reached oocyte maturation during the spawning season. However, since no spawning was recorded, eggs had either been resorbed or released and not fertilized by the male. Plasma E2 levels remained high in females, as expected in an asynchronous species during the spawning season with multiple batches of oocytes being recruited. Plasma 11-KT showed a tendency to decrease, suggesting a male reproductive dysfunction or the end of the reproductive season with a lack of synchronization between sexes. In conclusion, endoscopy and cannulation are tools that can be promptly applied to aid sex identification, assessment of reproductive function, and overall broodstock management in wild and captive stocks. These tools will greatly help future studies looking at the effects of environmental, social, and hormonal cues on reproductive development with the aim of developing a spawning induction protocol for the species

Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins

Background Arapaima gigas (Schinz, 1822) is the largest freshwater scaled fish in the world, and an emerging species for tropical aquaculture development. Conservation of the species, and the expansion of aquaculture requires the development of genetic tools to study polymorphism, differentiation, and stock structure. This study aimed to investigate genomic polymorphism through ddRAD sequencing, in order to identify a panel of single nucleotide polymorphisms (SNPs) and to simultaneously assess genetic diversity and structure in wild (from rivers Amazon, Solimões, Tocantins and Araguaia) and captive populations. Results Compared to many other teleosts, the degree of polymorphism in A. gigas was low with only 2.3% of identified RAD-tags (135 bases long) containing SNPs. A panel of 393 informative SNPs was identified and screened across the five populations. Higher genetic diversity indices (number of polymorphic loci and private alleles, Shannon’s Index and HO) were found in populations from the Amazon and Solimões, intermediate levels in Tocantins and Captive, and very low levels in the Araguaia population. These results likely reflect larger population sizes from less urbanized environments in the Amazon basin compared to Araguaia. Populations were significantly differentiated with pairwise FST values ranging from 0.086 (Amazon × Solimões) to 0.556 (Amazon × Araguaia). Mean pairwise relatedness among individuals was significant in all populations (P

A high-density linkage map and sex-linked markers for the Amazon Tambaqui Colossoma macropomum

Tambaqui (\textit{Colossoma macropomum}, Cuvier, 1818) is the most economically important native freshwater fish species in Brazil. It can reach a total length of over 1~m and a weight of over 40~kg. The species displays a clear sex dimorphism in growth performance with females reaching larger sizes at harvest. In aquaculture, the production of monosex populations in selective breeding programmes has been therefore identified as a key priority. In the present study, a genetic linkage map was generated by double digest restriction-site associated DNA (ddRAD) sequencing from 248 individuals sampled from two F1 families. The map was constructed using 14,805 informative SNPs and spanned 27 linkage groups. From this, the tambaqui draft genome was improved, by ordering the scaffolds into chromosomes, and sex-linked markers were identified. A total of 235 markers on linkage group 26 showed a significant association with the phenotypic sex supporting an XX/XY sex determination system in the species. The four most informative sex-linked markers were validated on another 206 sexed individuals demonstrating an accuracy in predicting sex ranging from 90.0\% to 96.7\%. The genetic mapping and novel sex-linked DNA markers identified and validated offer new tools for rapid progeny sexing, thus supporting the development of monosex female production in the industry while also supporting breeding programs of the species

Ultrastructure description of the spermatozoa of the Amazon bonytongue Arapaima gigas (Schinz, 1822)

Dataset containing micrographs obtained through scanning and transmission electron microscopy (SEM and TEM) depicting the ultrastructure of the spermatozoa of Arapaima gigas (Schinz, 1822

Ontogenetic and evolutionary change of external morphology of the neotropical shrimp potimirim (Holthuis, 1954) explained by a molecular phylogeny of the genus

A phylogenetic analysis of a fragment of the mitochondrial gene 16S was used to test the monophyletic status of Potimirim. Existing doubts on the taxonomic status of brasiliana (once P glabra) and P potimirim (once P mexicana) were clarified. Potimirim mexicana and P potimirim are distinct species according to molecular data and appendix masculina morphology. A new species (Potimirim sp. 1) from Puerto Rico was revealed with molecular data, and it is evolutionarily related to P potimirim and P mexicana according to our analysis. We found out three distinct species under the name P glabra. Then, we recommend the application of the name P glabra for the populations of the Pacific slope of Central America and revalidation of P brasiliana for the Brazilian ones. The need for a new name to those "P glabra" of the Caribbean is highlighted, and it was provisionally referred as Potimirim sp. 2. The ontogenetic (juveniles to adults) development of the appendix masculina of P brasiliana was observed and compared to the other species of Potimirim (adults). In the light of our phylogenetic hypothesis, we postulate a pattern of character addition for the evolution of the appendix masculina of Potimirim. This hypothesis is plausible for two key reasons. First. Potimirim is a monophyletic group according to our hypothesis. Second, the shape of appendix masculina found in adults of P. americana is similar and comparable to those found in the earliest juvenile stages of P brasiliana, a derived species according to our phylogeny (P americana, ((P mexicana, Potimirim sp. 1. P potimirim), (P glabra, (brasiliana, Potimirim sp. 2)))). As so, the basal P americana retain the ancestral morphological state of the appendix masculina when compared to the other species of Potimirim. In our interpretation the ontogeny of the appendix masculina recapitulated the proposed phylogeny, giving further support to it.FAPESP [2007/51018-6, 2002/08178-9, 2009/54931-0, 2010/50188-8]FAPESPCNPq [472746/2004-9, 473050/2007-2, 491490/2004-6, 490353/2007-0, 471011/2011-8, PQ 301261/2004-0, 302748/2010-5]CNP

Inclusion of Spirulina sp. (Arthospira) in the feed of Arapaima gigas (Schinz, 1822) broodstocks: effects on gonadal maturation

Dataset containing information on gonadal development from a trial which tested the Inclusion of Spirulina sp. (Arthospira) in the feed of Arapaima gigas (Schinz, 1822) broodstocks

Development of a pressure shock protocol to induce triploidy in tambaqui Colossoma macropomum (Cuvier, 1816)

Dataset on triploidization, fertilization, larval survival and growth rates from trials used to develop a pressure shock protocol to induce triploidy in tambaqui Colossoma macropomum (Cuvier, 1816