The Globin Gene Family in Arthropods: Evolution and Functional Diversity.

Globins are small heme-proteins that reversibly bind oxygen. Their most prominent roles in vertebrates are the transport and storage of O2 for oxidative energy metabolism, but recent research has suggested alternative, non-respiratory globin functions. In the species-rich and ecologically highly diverse taxon of arthropods, the copper-containing hemocyanin is considered the main respiratory protein. However, recent studies have suggested the presence of globin genes and their proteins in arthropod taxa, including model species like Drosophila. To systematically assess the taxonomic distribution, evolution and diversity of globins in arthropods, we systematically searched transcriptome and genome sequence data and found a conserved, widespread occurrence of three globin classes in arthropods: hemoglobin-like (HbL), globin X (GbX), and globin X-like (GbXL) protein lineages. These globin types were previously identified in protostome and deuterostome animals including vertebrates, suggesting their early ancestry in Metazoa. The HbL genes show multiple, lineage-specific gene duplications in all major arthropod clades. Some HbL genes (e.g., Glob2 and 3 of Drosophila) display particularly fast substitution rates, possibly indicating the evolution of novel functions, e.g., in spermatogenesis. In contrast, arthropod GbX and GbXL globin genes show high evolutionary stability: GbXL is represented by a single-copy gene in all arthropod groups except Brachycera, and representatives of the GbX clade are present in all examined taxa except holometabolan insects. GbX and GbXL both show a brain-specific expression. Most arthropod GbX and GbXL proteins, but also some HbL variants, include sequence motifs indicative of potential N-terminal acylation (i.e., N-myristoylation, 3C-palmitoylation). All arthropods except for the brachyceran Diptera harbor at least one such potentially acylated globin copy, confirming the hypothesis of an essential, conserved globin function associated with the cell membrane. In contrast to other animals, the fourth ancient globin lineage, represented by neuroglobin, appears to be absent in arthropods, and the putative arthropod orthologs of the fifth metazoan globin lineage, androglobin, lack a recognizable globin domain. Thus, the remarkable evolutionary stability of some globin variants is contrasted by occasional dynamic gene multiplication or even loss of otherwise strongly conserved globin lineages in arthropod phylogeny.

Divergent roles of the Drosophila melanogaster globins.

In contrast to long-held assumptions, the gene repertoire of most insects includes hemoglobins. Analyses of the genome of the fruitfly Drosophila melanogaster identified three distinct hemoglobin genes (glob1, glob2, and glob3). While glob1 is predominantly associated with the tracheal system and fat body, glob2 and glob3 are almost exclusively expressed in the testis. The physiological role of globins in Drosophila is uncertain. Here, we studied the functions of the three globins in a cell culture system. Drosophila Schneider 2 (S2) cells were stably transfected with each of the three globins and the empty vector as control. Under hypoxia (1% atmospheric O2), only glob1 overexpression enhanced the activity of mitochondrial oxidases and the ATP content. However, the positive effect of glob1 expression disappeared after 24h hypoxia, suggesting metabolic adaptations of the S2 cells. glob2 and glob3 had no positive effect on hypoxia-survival. After application of oxidative stress by H2O2, glob2 dramatically enhanced the viability of S2 cells. Evaluation of the intracellular localization of the globins using specific antibodies and green fluorescent protein-fusion constructs suggested that glob1 and glob2 most likely reside in the cytoplasm, while glob3 is associated with structures that may represent parts of the intracellular transport machinery. In silico analyses of public RNA-Seq data from different developmental stages provided that glob1 is co-expressed with genes of the aerobic energy metabolism, while glob2 and glob3 expression can be related to spermatogenesis and reproduction. Together, the results indicate divergent functions of the Drosophila globins: glob1 may play a role in the O2-dependent metabolism while glob2 may protect spermatogenesis from reactive oxygen species.

Sexual size dimorphism predicts rates of sequence evolution of SPerm Adhesion Molecule 1 (SPAM1, also PH-20) in monkeys, but not in hominoids (apes including humans).

Based on a dataset comprising coding DNA sequences of 23 anthropoid primates, we herein investigate if rates of sequence evolution of SPerm Adhesion Molecule1 (SPAM1, also PH-20), which participates in sperm-egg interaction, is lower in more sexually dimorphic species. For comparison, we analyze sequence evolution of apolipoproteinA-IV (APOA4) and apolipoprotein A-V (APOA5), which should evolve under less or even no sexual selection given their expression in blood, digestive tract, liver, and lungs. Regression analyses provides significant support for a negative dependence of SPAM1 derived branch-specific ratios of non-synonymous to synonymous substitution rates (dN/dS) on sexual size dimorphism (SSD) in a subsample comprising New World and Old World monkeys. We moreover observed a tendency for a positive correlation of substitution rates of SPAM1 with relative testes weight (RTW) and significantly lowered dN/dS estimates in uni-male and uni-male/multi-male breeding monkeys. Importantly, the pattern was not reproduced when analyzing partial APOA4 and APOA5 sequences. These findings illustrate that different levels of sperm competition, probably fueled by female cryptic choice, account for species-specific sequence evolution of SPAM1 in monkeys. Remarkably, present data do not support a correlation of species-specific sequence evolution of SPAM1 with sexual selection levels in hominoids (apes including humans). This can partly be ascribed to a relaxation of functional constraint of SPAM1 in some hominoid species. Additional factors confounding regression analyses specifically in hominoids might be higher levels of sperm competition than reflected by SSD and RTW in some species, a rather strong effect of female mate choice on paternity rates in others, and - in particular in humans - socio-cultural factors not measurable by SSD and RTW.