Comparative local analysis of metabolites, lipids and proteins in intact fish tissues by LAESI mass spectrometry.

Direct mass spectrometric analysis of animal tissues is an emerging field enabled by recent developments in ambient ion sources. Label-free in situ analysis of metabolites, lipids, and peptides/proteins from intact tissues in whole fish specimens of different gender and age were performed by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Hypertrophied glandular tissue (gill gland) of adult male Aphyocharax anisitsi (bloodfin tetra) was compared with gill tissues in females of the same species. Comparison of a large number of sample-specific ions was aided by a multivariate statistical method based on orthogonal projections to latent structures discriminant analysis. More than 200 different ions were detected in the mass spectra corresponding to primary metabolites, hormones, lipids and peptides/proteins. The gill tissues of the sexually mature males exhibited multiply charged ions in the 6+ to 10+ charge states corresponding to a protein with a molecular weight of 11 380 Da. This protein was present only in the mature male gill glands but absent in the corresponding area of the female and immature male specimens. An additional nine proteins were detected by LAESI-MS in both the male and female gill tissues.

Relationships among major lineages of characid fishes (Teleostei: Ostariophysi: Characiformes), based on molecular sequence data.

The family Characidae is a group of freshwater bony fishes that exhibits high species-level diversity and whose members inhabit parts of Texas, Mexico, and Central and South America. Thus far, morphological data have been of limited use in discerning relationships among subfamilies and incertae sedis genera of the family Characidae. In this study, DNA sequence data from GenBank were combined with new sequences for analyses under Bayesian and parsimony schemes. Sequences fell into four gene partitions, with three genes in the mitochondrial subset (12S, 16S, COI genes) and one gene in the nuclear subset (RAG2 gene). Inferred Bayesian and parsimony-based phylogenies reject the monophyly of certain groups (e.g., Astyanax, Hyphessobrycon, and Bryconamericus), do not reject the monophyly of others (e.g., Cheirodontinae and "clade A" of previous authors), and present new sister-group hypotheses (e.g., Brittanichthys sister to Paracheirodon). Sister to clade A is a lineage referred to herein as "clade B" which includes Exodon and exemplars from Cheirodontinae (the most basal lineage within clade B), Aphyocharacinae, Tetragonopterinae, and Characinae (excluding Gnathocharax). "Clade C" is sister to A+B and contains representatives of large incertae sedis genera (e.g., Hyphessobrycon, Hemigrammus), as well as members of Stethaprioninae. Unless certain other subfamilial names are to be disregarded, the use of Tetragonopterinae should continue to be restricted to species of Tetragonopterus because other genera previously referred to this subfamily grouped in clades A or C, quite distant from Tetragonopterus.

Reproductive morphology of Brittanichthys axelrodi (Teleostei: Characidae), a miniature inseminating fish from South America.

Light and electron microscopy were used to investigate the morphology of reproductive characters in a characid fish, Brittanichthys axelrodi. Spermatozoa were found in ovaries of females, thereby confirming insemination in this species. Bony hooks can be found on the fourth unbranched ray and branched rays 1-4 of the anal fin and the unique sigmoidally-curved ray of the caudal fin in mature males. Testes have three distinct regions: an anterior spermatogenic region, an aspermatogenic middle region lined with a simple squamous epithelium and used for storage of mature spermatozoa, and a posterior region of coiled chambers lined with a high simple cuboidal epithelium. The most posterior region appears to be instrumental in the formation and storage of spermatozeugmata, unencapsulated sperm packets. Thus far, this tripartite testis morphology is unique among characids. The mature spermatozoon has an elongate nucleus ( approximately 5 microm in length). A striated rootlet originates at the anterior end of the distal centriole and continues to the anterior tip of the cell. The striated rootlet wraps around the entire ventral area of the anterior part of the nucleus and appears to continue around the anterior tip of the nucleus and down the dorsal side as electron-dense material. Several large, spherical mitochondria ( approximately 0.6 microm in diameter) with lamellar cristae overlap the posterior end of the nucleus and continue beyond together with the cytoplasmic collar that contains the flagellum which lacks axonemal fins. Each spermatozeugma is lanceolate in shape when sectioned mid-sagitally, with the core staining positively for mucopolysaccharides. In both sexes, the gonopore opens posterior to the anus, with the urinary pore having a separate opening posterior to the gonopore. Bands of skeletal muscle were found in the area of the male gonopore. These morphological features are likely linked to the reproductive mode of insemination, a trait that is so far as known, relatively rare among teleost fishes, but is proving increasingly frequent among certain groups of characid fishes.