Temporal relationship between the onset of oestrus, the preovulatory LH surge and ovulation in farmed fallow deer, Dama dama

A study was conducted to determine the timing of ovulation relative to the onset of oestrus and the preovulatory LH surge in fallow deer. Mature fallow does were randomly allocated to two treatments (N = 10 per treatment) designed to synchronize oestrus on or about 17 May. Does assigned to Group 1 (prostaglandin-induced oestrus) each initially received single intravaginal CIDR [Controlled Internal Drug Release] devices for 13 days followed by an i.m. injection of 750 mg cloprostenol on Day 12 (15 May) of the subsequent luteal cycle. Does assigned to Group 2 (progesterone-induced oestrus) each received CIDR devices for 13 days, with withdrawal occurring on 15 May. All does were run with crayon-harnessed bucks (10:1 ratio) from the start of synchronization (18:00 h 15 May). Ten does (5 per group) were blood sampled via indwelling jugular cannulae every 2 h for 72 h from cloprostenol injection or CIDR device withdrawal and the plasma was analysed for concentrations of progesterone and LH by radioimmunoassay. Does within each treatment were randomly allocated to an ovarian examination time of 12, 16, 20 or 24 h after the onset of oestrus. Laparoscopy was repeated at 12-h intervals until ovulation was recorded. The ovaries of does failing to exhibit oestrus were examined 72 and 86 h after cloprostenol injection or CIDR device withdrawal. A total of 17 does were observed to exhibit oestrus at a mean (+/- s.e.m.) interval from treatment of 44.6 +/- 3.6 h for Group 1 (N = 9) and 34.1 +/- 2.5 h for Group 2 (N = 8).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes

Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.Microsporidia are a eukaryotic group highly adapted as obligate intracellular parasites (31, 50). They infect a diverse range of vertebrate and invertebrate animal hosts. In humans they are the cause of a number of diseases (e.g., gastroenteritis, encephalitis, and hepatitis), having their greatest impact on immunocompromised individuals, notably in children with human immunodeficiency virus (14, 31). Microsporidia are most closely related to fungi, although their high level of specialization as intracellular parasites obscured this relationship for a long time (18, 25, 30). Gene phylogenies now firmly connect these two groups, although it remains uncertain whether microsporidia are sisters to the fungi or represent a lineage derived from within fungal diversity (21, 28).A clear adaptive response to parasitism in microsporidia has been a reduction in cellular complexity. This was first recognized at an ultrastructural level with the apparent lack of peroxisomes, flagella, stacked Golgi bodies, and mitochondria (31). This reductive evolution is mirrored at a genomic level, with microsporidia containing the smallest eukaryotic genomes described to date (28, 29). The complete genomic sequence from the human microsporidian parasite Encephalitozoon cuniculi reveals a genome of only ∼2.9 Mb containing approximately 2,000 genes, in contrast to the 6,000 genes found in the genome of the model fungus Saccharomyces cerevisiae. The minimal genome of E. cuniculi has been achieved through three mechanisms in concert: (i) gene loss, resulting in broad loss of biochemical pathways and capabilities, including much basic energy metabolism and numerous anabolic pathways; (ii) gene compaction with an average intergenic space of ∼130 bp; and (iii) gene shortening, with E. cuniculi genes being on average 14% shorter than their homologues in fungi such as S. cerevisiae (28, 45). Thus, microsporidian evolution has apparently been shaped by a very strong trend to eliminate superfluous molecular and biochemical complexity.Despite earlier suppositions that microsporidia lacked mitochondria, genome and expressed sequence tag data from microsporidia suggested the presence of several proteins typically targeted to this organelle (3, 19, 20, 24, 28, 38). Immunolocalization of a mitochondrial Hsp70 to small double membrane-bound organelles in Trachipleistophora hominis provided strong evidence for the existence of a mitochondrion in microsporidia, albeit a simplified organelle that lacks cisternae (48). Annotation of genomic data from E. cuniculi provided compelling matches for only 22 proteins implicated in mitochondrial function, suggesting that the metabolism of this relict mitochondrion (or mitosome) is also significantly reduced compared to that of canonical mitochondria (28). Further, no mitochondrial genome has been retained; thus, biogenesis of this organelle is wholly dependent on nucleus-encoded proteins. Based on these 22 proteins, a major role for the mitosome is iron-sulfur cluster assembly (22, 28). No genes have been found for ATP synthesis via oxidative phosphorylation, suggesting loss of this activity in mitosomes (28, 46). While it is likely that further mitosome-targeted proteins will be identified, it is clear that compared to mitochondria from fungal relatives, which are known to import ∼1,000 proteins (40, 44), microsporidian mitosomes represent organelles with highly reduced proteomes, a feature consistent with other traits of cellular reduction.The highly reduced state of the microsporidian mitosome, requiring only a fraction of the protein diversity of other mitochondria, presents an interesting case for studying organelle biogenesis—particularly the machinery for protein import of nucleus-encoded proteins. Mitochondrial protein import has been best characterized in fungi, and in these systems most proteins are imported via four major import complexes: a TOM (translocase of the outer mitochondrial membrane), a SAM (sorting and assembly machinery), and one of two TIMs (translocase of the inner mitochondrial membrane), TIM23 or TIM22 (see Fig. ​Fig.5A)5A) (5, 36). These complexes are broadly conserved throughout fungi as well as animals (15). Mitochondrial proteins can take one of several routes to the mitochondrion via this apparatus (5, 36). Broadly, soluble matrix proteins are recognized at the TOM complex by the receptor protein Tom20 through the binding of N-terminal presequences with characteristic features (1, 5, 7, 8, 36). These proteins are passed through the pore protein Tom40 of the TOM to the TIM23 complex and then driven into the mitochondrial matrix by way of the presequence translocase-associated motor (PAM) complex, where their presequences are subsequently removed. Some membrane proteins can also be released into the inner membrane from the TIM23 complex. Mitochondrial proteins that apparently lack such an extension, notably including many of the membrane proteins, are recognized by internal sequence elements. Tom70 has a greater role in recognizing these internal signals and thus the import of hydrophobic proteins (4, 11, 32, 39, 47). Such hydrophobic proteins are often bound by cytosolic molecular chaperones (Hsp70 and/or Hsp90) en route to the mitochondrion, and Tom70 is known to independently bind to both the chaperone and the substrate protein (7, 23, 33, 52). While a measure of substrate overlap between Tom20 and Tom70 occurs, the division of responsibility between these two receptors has likely evolved in response to the wide range of substrate proteins that must be imported into mitochondria and the need to handle this complexity.Open in a separate windowFIG. 5.Schematics of the protein import machinery and pathways in yeast mitochondria (A) and E. cuniculi mitosome (B) based on identified homologues of the general fungal/animal pathways. Protein components of the yeast system were all represented by HMMs used to search the microsporidian genomic data and represent the major presequence-dependent and presequence-independent pathways. Homologues identified in E. cuniculi indicate a severely reduced import apparatus utilizing elements of the presequence-independent pathway.For microsporidia little is known of the protein import apparatus for their relict mitochondrion, the mitosome. Has the very reduced organelle proteome, in concert with a genome-wide trend of the loss of redundant or superfluous genes, resulted in a smaller and/or derived import apparatus? In this study we have investigated the microsporidian mitosome protein import apparatus from E. cuniculi in order to evaluate how this apparatus has responded to the reduction in the number of proteins required to be imported and the overall radical reduction in the number and size of proteins encoded in the nuclear genome. A putative homologue of the outer membrane receptor protein Tom70 is of particular interest as the only receptor for the TOM complex and, given the known structure of Tom70 proteins, provides a highly informative example of how proteins can be shortened in the course of genome reduction.     

Fish community changes after minimum flow increase: testing quantitative predictions in the Rhône River at Pierre-Bénite, France

1. Many aspects of the flow regime influence the structure of stream communities, among which the minimum discharge left in rivers has received particular attention. However, instream habitat models predicting the ecological impacts of discharge management often lack biological validation and spatial generality, particularly for large rivers with many fish species. 2. The minimum flow at Pierre‐Bénite, a reach of the Rhône river bypassed by artificial channels, was increased from 10 to 100 m³ s^?1 in August 2000 (natural mean discharge 1030 m³ s^?1), resulting in a fivefold increase in average velocity at minimum flow. Fish were electrofished in several habitat units on 12 surveys between 1995 and 2004. 3. Principal components analysis revealed a significant change in the relative abundance of fish species. The relative abundance of species preferring fast‐flowing and/or deep microhabitats increased from two‐ to fourfold after minimum flow increase. A change in community structure confirmed independent quantitative predictions of an instream habitat model. This change was significantly linked to minimum flow increase, but not to any other environmental variables describing high flows or temperature at key periods of fish life cycle. The rapidity of the fish response compared with the lifespan of individual species can be explained by a differential response of specific size classes. 4. The fish community at Pierre‐Bénite is in a transitional stage and only continued monitoring will indicate if the observed shift in community structure is perennial. We expect that our case study will be compared with other predictive tests of the impacts of flow restoration in large rivers, in the Rhône catchment and elsewhere.     

Laying the foundations for a new classification of Agaonidae (Hymenoptera: Chalcidoidea), a multilocus phylogenetic approach

A phylogeny of the Agaonidae (Chalcidoidea) in their restricted sense, pollinators of Ficus species (Moraceae), is estimated using 4182 nucleotides from six genes, obtained from 101 species representing 19 of the 20 recognized genera, and four outgroups. Data analysed by parsimony and Bayesian inference methods demonstrate that Agaonidae are monophyletic and that the previous classification is not supported. Agaonidae are partitioned into four groups: (i) Tetrapus, (ii) Ceratosolen + Kradibia, (iii) some Blastophaga + Wiebesia species, and (iv) all genera associated with monoecious figs and a few Blastophaga and Wiebesia. The latter group is subdivided into subgroups: (i) Pleistodontes, (ii) Blastophaga psenes and neocaledonian Dolichoris, (iii) some Blastophaga and Wiebesia species, and (iv) Platyscapa, all afrotropical genera and all genera associated with section Conosycea. Eleven genera were recovered as monophyletic, six were para‐ or polyphyletic, and two cannot be tested with our data set. Based on our phylogeny we propose a new classification for the Agaonidae. Two new subfamilies are proposed: Tetrapusiinae for the genus Tetrapus, and Kradibiinae for Ceratosolen + Kradibia. Liporrhopalum is synonymized with Kradibia and the subgenus Valisia of Blastophaga is elevated to generic rank. These changes resulted in 36 new combinations. Finally, we discuss the hypothesis of co‐speciation between the pollinators and their host species by comparing the two phylogenies. © The Willi Hennig Society 2009.     

Micropropagation by tissue culture triggers differential expression of infectious endogenous Banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus ) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.     

Caspase-2 is resistant to inhibition by inhibitor of apoptosis proteins (IAPs) and can activate caspase-7

Caspases are a family of cysteine proteases with roles in cytokine maturation or apoptosis. Caspase-2 was the first pro-apoptotic caspase identified, but its functions in apoptotic signal transduction are still being elucidated. This study examined the regulation of the activity of caspase-2 using recombinant proteins and a yeast-based system. Our data suggest that for human caspase-2 to be active its large and small subunits must be separated. For maximal activity its prodomain must also be removed. Consistent with its proposed identity as an upstream caspase, caspase-2 could provoke the activation of caspase-7. Caspase-2 was not subject to inhibition by members of the IAP family of apoptosis inhibitors.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.