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.

Characterization of the hemoglobin of the backswimmer Anisops deanei (Hemiptera).

While O(2)-binding hemoglobin-like proteins are present in many insects, prominent amounts of hemoglobin have only been found in a few species. Backswimmers of the genera Anisops and Buenoa (Notonectidae) have high concentrations of hemoglobin in the large tracheal cells of the abdomen. Oxygen from the hemoglobin is delivered to a gas bubble and controls the buoyant density, which enables the bugs to maintain their position without swimming and to remain stationary in the mid-water zone where they hunt for prey. We have obtained the cDNA sequences of three Anisops deanei hemoglobin chains by RT-PCR and RACE techniques. The deduced amino acid sequences show an unusual insertion of a single amino acid in the conserved helix E, but this does not affect protein stability or ligand binding kinetics. Recombinant A. deanei hemoglobin has an oxygen affinity of P(50) = 2.4 kPa (18 torr) and reveals the presence of a dimeric fraction or two different conformations. The absorption spectra demonstrate that the Anisops hemoglobin is a typical pentacoordinate globin. Phylogenetic analyses show that the backswimmer hemoglobins evolved within Heteroptera and most likely originated from an intracellular hemoglobin with divergent function.

Changes of globin expression in the Japanese medaka (Oryzias latipes) in response to acute and chronic hypoxia.

Fishes live in an aquatic environment with low or temporally changing O(2) availability. Variations in O(2) levels require many anatomical, behavioral, physiological, and biochemical adaptations that ensure the uptake of an adequate amount of O(2). Some fish species are comparatively well adapted to tolerate low O(2) partial pressure (hypoxia). The Japanese ricefish medaka (Oryzias latipes) is an important model organism for biomedical research that shows remarkable tolerance towards hypoxia. We have investigated the regulation and role of globins under hypoxia. We applied four different regimes of chronic hypoxia (24 and 48 h at PO(2) = 2 or 4 kPa) as well as acute hypoxia (2 h at PO(2) = 0.5 kPa) to adult male medaka. Changes of mRNA levels of seven globin genes (adult hemoglobin α and ß, myoglobin, neuroglobin, cytoglobin 1 and 2, globin X), three hypoxia-response genes (lactate dehydrogenase b, phosphoglycerate kinase, adrenomedullin 1) and two putative reference genes (cyclophilin, acidic ribosomal phosphoprotein P0) were monitored by means of quantitative real-time reverse-transcription PCR. We observed strong upregulation of myoglobin, which is also expressed in the medaka brain, as previously demonstrated for carp, goldfish and zebrafish. The hemoglobin chains were found upregulated, whereas earlier studies found down-regulation of hemoglobin in hypoxic zebrafish. By contrast, neuroglobin mRNA was not affected by hypoxia in medaka, but had been found upregulated in zebrafish. Globin X is induced in medaka brain, but down-regulated in zebrafish. Thus, the patterns of hypoxia response of globins are strikingly different in various fish species, which can be interpreted as indication for different roles of the various globins in hypoxia response and for alternative metabolic strategies of fish species in coping with O(2) deprivation.