Hematophagous insects ingest several times their body weight with each blood meal, obtaining nutrients needed for reproduction and survival in addition to high levels of free heme. However, they survive the high toxicity imposed by free heme due to the development of heme detoxification mechanisms such as heme degradation by the enzyme heme oxygenase (HOs). Despite being present in several disease vector insects, including tsetse flies that transmit sleeping sickness, the role and biochemical features of HOs in blood feeding insects is poorly studied. Here, we have cloned and expressed HO from the tsetse fly Glossina morsitans morsitans to determine whether the enzyme was active, unveil its biochemical features and develop specific antibodies to investigate HO distribution in this insect. HPLC analysis of midgut contents from G. m. morsitans was performed to track the degradation of heme and conversion to biliverdin during the first 6 days following a single blood meal, showing that whilst heme concentration roses to 3 mM at 48 hours after a single blood meal, the maximum concentration reached by biliverdin is 0.5 μM after 96 hours, suggesting that despite HO being active in the insect, it may play a minor role in heme degradation. Comparison of recombinant HOs suggests that the heme oxygenase from G. m.morsitans (GmmHO) degrades heme slower and presents distinct spectroscopic features to HOs from other hematophagous insects and humans. However, it is similar to that observed in D. melanogaster. Confocal analysis of immunostained tissues and western blotting using antibodies raised against recombinant enzymes have been used to track the tissue distribution of GmmHO, demonstrating that the enzyme is ubiquitously expressed in the insect, particularly in the nuclei from oenocytes, liver-like cells involved in cuticle synthesis. Such results were supported by analysis of mRNA levels, which showed higher expression levels in reproductive tissues (6.5 fold), digestive tract (2.0 fold) and fat bodies/oenocytes (1.7 fold), evincing a role in reproduction. Time series analysis of HO mRNA levels after a single blood meal revealed that GmmHO is an early blood meal induced gene in digestive tract (3.4 fold) and fat bodies/oenocytes (2.2 fold), confirming a role in the defence against heme toxicity in the digestive tract as well as unveiling possible roles in the catabolism of lipids. Thus, this work has confirmed the presence and activity of HO in a hematophagous insect and revealed both biochemical and functional aspects of the enzyme, shedding new light on the role of HO in hematophagous insects.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:733909 |
| Date | January 2017 |
| Creators | Pacelli Gomes de Lima, G. |
| Contributors | Paine, M. ; Lycett, G. ; Lian, L. |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/3011220/ |
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