Spermatozoon ultrastructure of hangingflies, Bittacus strigosus and Bittacus stigmaterus.

In the present study, spermatozoon ultrastructure was documented in two species of hangingflies, Bittacus strigosus Hagen (Mecoptera: Bittacidae) and B. stigmaterus Say. Structures considered important to phylogenetic assessment that were observed in B. strigosus and B. stigmaterus included a short bilayered acrosome, elongated nucleus, tube-like glycocalyx, centriole adjunct material, accessory bodies, two mitochondrial derivatives, extra axonemal rods, globular units, and 9+2 arrangement of microtubules in the axoneme. Comparisons were made to Bittacus planus Cheng, which was previously examined by electron microscopy (Xie and Hua 2010). Similarities among the ultrastructural characteristics of the three Bittacus species support the monophyly of this genus. Displacement of a mitochondrial derivative by an accessory body was documented for the first time. This paper includes clarifications on differences between accessory bodies and extra axonemal rods, which are issues important to phylogenetic placement.

Novel protein genes in animal mtDNA: a new sex determination system in freshwater mussels (Bivalvia: Unionoida)?

Mitochondrial (mt) function depends critically on optimal interactions between components encoded by mt and nuclear DNAs. mitochondrial DNA (mtDNA) inheritance (SMI) is thought to have evolved in animal species to maintain mito-nuclear complementarity by preventing the spread of selfish mt elements thus typically rendering mtDNA heteroplasmy evolutionarily ephemeral. Here, we show that mtDNA intraorganismal heteroplasmy can have deterministic underpinnings and persist for hundreds of millions of years. We demonstrate that the only exception to SMI in the animal kingdom, that is, the doubly uniparental mtDNA inheritance system in bivalves, with its three-way interactions among egg mt-, sperm mt- and nucleus-encoded gene products, is tightly associated with the maintenance of separate male and female sexes (dioecy) in freshwater mussels. Specifically, this mother-through-daughter and father-through-son mtDNA inheritance system, containing highly differentiated mt genomes, is found in all dioecious freshwater mussel species. Conversely, all hermaphroditic species lack the paternally transmitted mtDNA (=possess SMI) and have heterogeneous macromutations in the recently discovered, novel protein-coding gene (F-orf) in their maternally transmitted mt genomes. Using immunoelectron microscopy, we have localized the F-open reading frame (ORF) protein, likely involved in specifying separate sexes, in mitochondria and in the nucleus. Our results support the hypothesis that proteins coded by the highly divergent maternally and paternally transmitted mt genomes could be directly involved in sex determination in freshwater mussels. Concomitantly, our study demonstrates novel features for animal mt genomes: the existence of additional, lineage-specific, mtDNA-encoded proteins with functional significance and the involvement of mtDNA-encoded proteins in extra-mt functions. Our results open new avenues for the identification, characterization, and functional analyses of ORFs in the intergenic regions, previously defined as "noncoding," found in a large proportion of animal mt genomes.

AhR2-mediated, CYP1A-independent cardiovascular toxicity in zebrafish (Danio rerio) embryos exposed to retene.

In the embryo-larval stages of fish, alkylphenanthrenes such as retene (7-isopropyl-1-methylphenanthrene) produce a suite of developmental abnormalities typical of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including pericardial and yolk sac edema, cardiovascular dysfunction, and skeletal deformities. To investigate the mechanism and target tissue of retene toxicity, we used observational, histological, and protein knockdown techniques in zebrafish (Danio rerio) embryos. The primary overt signs of toxicity are pericardial edema and reduced blood flow, first observed at 36 h post-fertilization (hpf). The most pronounced effects at this stage are a reduced layer of cardiac jelly in the atrium and reduced diastolic filling. Conversely, an increased layer of cardiac jelly is observed at 72 hpf in retene-exposed embryos. Induction of cytochrome P4501A (CYP1A) is apparent in a subset of cardiomyocytes by 48 hpf suggesting that early cardiac effects may be due to AhR activation in the myocardium. Myocardial CYP1A induction is transient, with only endocardial induction observed at 72 hpf. Knockdown of cyp1a by morpholino oligonucleotides does not affect retene toxicity; however, ahr2 knockdown prevents toxicity. Thus, the mechanism of retene cardiotoxicity is AhR2-mediated and CYP1A-independent, similar to TCDD; however, the onset and proximate signs of retene toxicity differ from those of TCDD. Retene cardiotoxicity also differs mechanistically from the cardiac effects of non-alkylated phenanthrane, illustrating that alkyl groups can alter toxic action. These findings have implications for understanding the toxicity of complex mixtures containing alkylated and non-alkylated polycyclic aromatic hydrocarbons.

Extra-mitochondrial localization and likely reproductive function of a female-transmitted cytochrome c oxidase subunit II protein.

Our previous study documented a reproductive function for the male-transmitted mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit II (MCOX2) protein in a unionoid bivalve. Here, immunoblotting, immunohistochemistry and immunoelectron microscopy analyses demonstrate that the female-transmitted protein (FCOX2) is: (i) expressed in both male and female gonads; (ii) maximally expressed in ovaries just prior to the time of the annual fertilization event; (iii) displayed in the cytoplasm and more strongly in the plasma membrane (microvilli), vitelline matrix and vitelline envelope of mature ovarian eggs; and (iv) strongly localized to the vitelline matrix of some eggs just prior to fertilization. These findings represent evidence for the extra-mitochondrial localization of an mtDNA-encoded gene product and are consistent with multifunctionality for FCOX2 in eggs.