Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Karyotype analysis of Eucinostomus argenteus, E-gula, E-harengulus, and Eugerres plumieri (Teleostei, Gerreidae) from Florida and Puerto Rico

The karyotypes of four gerreids of the western Atlantic Ocean are documented. A diploid chromosome complement of 48 telocentric chromosomes was found in the four species (2N = 48t, fundamental number FN = 48). No differences were detected either in the number of chromosomes of the standard karyotype, in their karyotype size, or between the karyotypes derived from male or female specimens of any of the species. Chromosome length decreased progressively and slightly from pair 1 to pair 24. The Ag - NOR karyotypes of E. argenteus and E. harengulus were characterized by the position of the nucleolar organizer regions next to the centromere in chromosome pair 1, whereas in E. gula and E. plumieri Ag - NORs were detected in pair 4. The other 46 chromosomes showed a light staining of the centromere with no terminal or intermediate heterochromatic blocks. All Eucinostomus species showed Ag - NORs of similar size, while Eugerres plumieri showed Ag - NORs 10 - 20% larger than Eucinostomus species. A combination of size and position of the Ag - NORs identified E. gula, while size alone identified E. plumieri. However, the ancestral state for size and position of Ag - NORs could not be established. There was no differential staining of the chromosomes by G-banding. The karyotype of the gerreids appears similar to the hypothetical ancestral karyotype of fish. The phylogenetic relationships among these species could not be established because of the lack of chromosome G-bands. Most likely this indicates a homogeneous distribution of GC nucleotides in the chromosomes

Phylogenetic assessment of Eucinostomus gula, Eugerres plumieri, and Diapterus auratus (Pisces : Gerreidae) based on allozyme and mtDNA analyses

The phylogeny of three gerreid species was evaluated through allozymes and mtDNA analyses. Genetic similarities were estimated with data from 18 protein-coding loci and from mtDNA fragments produced by 3 restriction enzymes. The allozyme and mtDNA dendograms had identical topology. Genetic distance, genetic similarity, and mtDNA sequence divergence indicated that E. plumieri and D. auratus are closer than any other combination of the three taxa. Eugerres and Diapterus are clearly sister groups within the dendogram. Allozyme and mtDNA analyses support the common taxonomic practice of grouping the mojarras that possess a serrated preoperculum

Assessment of the population genetic structure of Sphyrna lewini to identify conservation units in the Mexican Pacific

Due to the current status of the scalloped hammerhead shark (Sphyrna lewini) as a threatened species, the assessment of genetic diversity, divergence, and demographic parameters of populations in the eastern Pacific Ocean may assist in developing appropriate strategies for sustainable fisheries and species conservation. We analyzed samples collected from 2001 to 2003 from seven locations in the Mexican Pacific Ocean and two in the southwestern Gulf of Mexico, using single-stranded conformation polymorphism of a mitochondrial control region fragment and five microsatellite loci. The mitochondrial data did not show population divergence among locations from the Mexican Pacific Ocean; however, the microsatellite data showed a divergent population in Baja California. Additional differences were also observed between the northern and central locations of the Mexican Pacific. The historical demography analysis revealed spatial expansion events, possibly related to glacial-interglacial cycles that occurred approximately 450,000 years ago. The divergence found should be considered in the formulation of fisheries management and conservation policies of the species in the region

Lack of genetic differentiation among size groups of jumbo squid (Dosidicus gigas)

The population structure of the jumbo squid (Dosidicus gigas) is complex, containing several cohorts and three groups defined by their size (small, medium, and large) and by differences in maturation, growth, and life span. Several authors have indicated the possibility of such groups representing discrete genetic units even at level subspecies or species in statu nascendi. Genetic divergence was tested in samples from the Gulf of California (Mexico) and Peruvian Sea by estimation of population divergence statistics, an exact test of homogeneity of allele frequencies, analysis of molecular variance, and genealogical trees applied to data obtained with two molecular markers: RAPDs and mtDNA sequences identified by SSCPs. Neither significant values of θ (FST) nor significant heterogeneity in allele frequencies were detected. Lack of evidence does not imply complete lack of differentiation among the groups but supports the fact that a geographically spread population can have different size groups without relevant genetic differentiation, implying that the hypothetical genetically differentiated groups can occur in different ecological niches.

Electrophoretic variation between the central and southern populations of the northern anchovy Engraulis mordax Girard 1854 (Engraulidae, Pisces) from Baja California, Mexico

Distinctive genetic markers of the central and southern populations of the northern anchovy Engraulis mordax were sought by electrophoretic techniques. Three samples of the central population were collected in Ensenada, Baja California, and one of the southern population in Puerto San Carlos, Baja California Sur. Although the electrophoretic separation of the hemoglobins in polyacrilamide gels were identical in both populations, the FST of the esterases loci showed significant differences between them, and the proteins of the muscle extract as well as the parvalbumins yielded some bands that were exclusive to one population or the other. These bands might prove useful as genetic markers in studies of population dynamics on which the management of this valuable fishery resource can be based

Spatial and temporal mitochondrial DNA genetic homogeneity of dolphinfish populations (Coryphaena hippurus) in the eastern central Pacific

Dolphinfish (Coryphaena hippurus) samples were collected over four consecutive years from four locations in the eastern central Pacific to evaluate the genetic variation in a 751 bp segment of the mitochondrial NADH subunit 1 (ND1) to test for the presence of genetic population structure. Sequence analyses revealed no significant differences among collections from the same location in the different years sampled nor between locations. Mismatch distributions, estimations of population expansion parameters, and neutrality tests revealed significant fluctuations in population size in coincidence with past glacial and interglacial periods during the late Pleistocene. The low levels of nucleotide diversities and shallow coalescence of mtDNA genealogies observed were coincident with the estimated demographic parameters and neutrality tests, in suggesting the presence of important past population size fluctuations or range expansion. The prevention of the accumulated of deep lineages independantly on how it was originated, probably delayed the emergence of a population divergence process which might account for the lack of genetic differences detected. (c) 2006 Elsevier B.V. All rights reserved