ARTICLE IN PRESS
Journal of Insect Physiology 53 (2007) 285–294
www.elsevier.com/locate/jinsphys
Review
The respiratory proteins of insects
Thorsten Burmestera,, Thomas Hankelnb
a
Institute of Zoology, Biozentrum Grindel, University of Hamburg, Martin-Luther-King-Platz 3, D-20146 Hamburg, Germany
b
Institute of Molecular Genetics, Johannes Gutenberg University of Mainz, Becherweg, 32, D-55099 Mainz, Germany
Received 1 November 2006; received in revised form 10 December 2006; accepted 13 December 2006
Abstract
For a long time, respiratory proteins have been considered unnecessary in most insects because the tracheal system was thought to be
sufficient for oxygen supply. Only a few species that survive under hypoxic conditions were known exceptions. However, recently it has
become evident that (1) intracellular hemoglobins belong to the standard repertoire of insects and (2) that hemocyanin is present in many
‘‘lower’’ insects. Intracellular hemoglobins have been identified in Drosophila, Anopheles, Apis and many other insects. In all investigated
species, hemoglobin is mainly expressed in the fat body and the tracheal system. The major Drosophila hemoglobin binds oxygen with
high affinity. This hemoglobin type possibly functions as a buffer system for oxygen supply at low partial pressures and/or for the
protection from an excess of oxygen. Similar hemoglobins, present in much higher concentrations, store oxygen in specialized tracheal
organs of the botfly and some backswimmers. The extracellular hemoglobins in the hemolymph of chironomid midges are evolutionary
derivatives of the intracellular insect hemoglobins, which emerged in response to the hypoxic environment of the larvae. In addition,
several hemoglobin variants of unknown functions have been discovered in insect genomes. Hemocyanins transport oxygen in the
hemolymph of stoneflies, but also in the Entognatha and most hemimetabolan taxa. Apparently, hemocyanin has been lost in
Holometabola. At present, no physiological or morphological character is known that could explain the presence or loss of hemocyanins
in distinct taxa. Nevertheless, the occurrence of respiratory proteins in insects adds further complexity to our view on insect respiration.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Hemoglobin; Hemocyanin; Oxygen; Respiration; Trachea
1. Introduction
Animals that live under aerobic conditions consume
large amounts of O2, which is mainly used to sustain the
production of ATP in the respiratory chain of the
mitochondria. In Protozoa and small Metazoa, simple
diffusion is usually considered sufficient for the supply of
the inner layers of the body with O2. Larger animals,
however, require a variety of anatomical, physiological,
and molecular adaptations that enhance the O2 delivery to
the cells and eventually to the mitochondria. These
adaptations comprise respiratory organs, such as gills or
lungs, circulatory systems, as well as respiratory proteins
for transport or storage of O2 (Willmer et al., 2000).
Corresponding author. Tel.: +49 40 42838 3913;
fax: +49 40 42838 3937.
E-mail address: [email protected] (T. Burmester).
0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2006.12.006
The largely impermeable cuticle of insects (here, the
taxonomic terms ‘‘Insecta’’ and ‘‘Hexapoda’’ are used as
synonyms) and other arthropods constrains the uptake of
O2 by diffusion across the body’s surface. Terrestrial
insects and myriapods acquire O2 by the aid of trachea.
The tracheal system consists of highly branched air-filled
tubes that connect the inner tissues with the atmosphere
(Kestler, 1985; Brusca and Brusca, 2002). Aquatic insects
usually have other specialized respiratory organs such as
gills, tracheal gills or a plastron (Willmer et al., 2000).
Within the tracheal system, O2 is distributed through the
body mainly by passive diffusion (Krogh, 1920; Kestler,
1985), although some active breathing may occur (Komai,
1998; Westneat et al., 2003). O2 uptake by the cells takes
place mainly at the tips of the smallest branches, the
tracheoles, which have only a thin cuticle. In highly active
organs such as the insect flight muscle, the tracheoles may
even enter the cells and connect with the mitochondria.
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Due to high diffusion rates and capacity coefficients,
O2 is delivered about 200,000–300,000 times more efficient
in the tracheal air than in the aqueous environment
of the hemolymph or blood (Krogh, 1920; Kestler, 1985).
These features make the tracheal system an extremely
efficiently transport apparatus, which has been thought
to comply with the O2 requirements of even the largest
insect known on earth. Therefore, until recently the
occurrence of respiratory proteins that would enhance O2
supply has been largely unknown and considered unnecessary (Mangum, 1985; Law and Wells, 1989; Locke,
1998; Willmer et al., 2000). Only a few insect species
that live in aqueous and hypoxic environments were
known to harbor hemoglobin (Weber and Vinogradov,
2001; see below). However, in recent years it has become
evident that O2 transport and storage proteins are
much more widespread among insects than previously
thought (Fig. 1; Burmester and Hankeln, 1999; Hankeln
et al., 2002, 2006; Hagner-Holler et al., 2004; Burmester
et al., 2006). Here we summarize the present state of
knowledge on the respiratory proteins of insects, and
analyze their occurrence in a physiological and evolutionary context.
2. Respiratory proteins
Respiratory proteins reversibly bind molecular O2 for
the purpose of transport or storage. They enhance the O2
transport capacitance of the body fluid, facilitate intracellular O2 diffusion or enable O2 storage for long or shortterm periods. In the animal kingdom, three types of metalcontaining respiratory proteins are known: (hemo-)globins
(Hb), hemerythrins and hemocyanins (Hc). Hemerythrins
are restricted to only a few animal phyla and have not been
detected in insects (Vinogradov, 1985). They will not be
considered here.
(Hemo-)globin (Hb) is a small globular protein, usually
consisting of about 140–150 amino acids that comprise
eight a-helical segments (named A–H). It displays a
characteristic 3-over-3 a-helical sandwich structure (Dickerson and Geis, 1983; Berg et al., 2002), which includes an
iron-containing heme group (Fe2+-protoporphyrin IX).
The Fe2+ is connected to the globin chain via the proximal
histidine in F8 (i.e., 8th amino acid of helix F). The gaseous
ligand, typically O2, but also CO or NO, can bind between
the Fe2+ ion and the distal amino acid at position E7. Hbs
are phylogenetically ancient molecules, whose complex
Fig. 1. Distribution of respiratory proteins in insect orders. The phylogenetic tree of the insects is based on Hennig (1969), Kukalovà-Peck (1991) and
Wheeler et al. (2001).
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evolution is demonstrated by their widespread occurrence
in bacteria, fungi, plants, invertebrate and vertebrate
animals (Weber and Vinogradov, 2001; Vinogradov
et al., 2005). Although the various Hbs may display only
little sequence resemblance, tertiary structures are usually
conserved. Invertebrate Hbs may assemble to multimers of
up to 144 Hb subunits or domains. Hbs are involved in
many different aspects of O2 supply. In almost all
vertebrates and many invertebrates, Hb is present in the
circulatory system. Hbs may either be contained in
specialized cells (erythrocytes) or are freely dissolved in
the blood. Hbs may also occur in distinct tissues. The
functions of these ‘‘tissue-globins’’ are not always obvious.
The best-studied example is myoglobin, a tissue-globin that
meets the high O2 demands of muscle tissues in both
vertebrates and invertebrates. Although there is no doubt
that it has respiratory functions, it is still not clear whether
myoglobin facilitates O2 diffusion from the cell surface to
mitochondria, or whether it acts as a short-term O2 store
(Wittenberg and Wittenberg, 2003). The physiological role
of other specialized tissue-globins, like neuroglobin in the
nervous system of vertebrates (Burmester et al., 2000) and
cytoglobin in various cell types (Burmester, 2002), is still a
matter of debate (Burmester and Hankeln, 2004; Hankeln
et al., 2005).
Hcs are copper-containing respiratory proteins that only
occur in the hemolymph of several arthropod and mollusk
species, but not outside these phyla (Markl and Decker,
1992; van Holde et al., 2001). Although arthropod and
mollusk Hcs have similar binuclear Cu-binding centers,
these proteins are most likely phylogenetically not related
and have emerged from different types of copper-proteins
(Burmester, 2001, 2002). Arthropod Hcs form either
hexamers or multi-hexamers of six similar or identical
subunits, with structures up to 8 6-mers (Markl and
Decker 1992; van Holde et al., 2001). A typical arthropod
Hc subunit comprises about 650 amino acids (75–80 kDa
polypeptide). Each subunit consists of three structural
domains and harbors two Cu+ ions, which can bind one
O2. Each Cu+ ion is coordinated by three histidines, which
reside in domain 2. In contrast to some Hbs, Hcs are never
intracellular proteins and always occur freely dissolved in
the hemolymph. In an evolutionary perspective, arthropod
Hcs derived from phenoloxidases, Cu+-containing enzymes of the melanin-pathway which are involved in
immune response, wound-healing and cuticle formation
(Burmester and Scheller, 1996; Burmester, 2001, 2002). Hcs
are also related to some non-respiratory proteins: crustacean pseudo-hemocyanins, insect hexamerins and dipteran
hexamerin receptors (Beintema et al., 1994; Burmester and
Scheller, 1996; Burmester, 2001, 2002). These proteins have
lost the ability to bind Cu+, and thus O2, and are mainly
used for amino acid and energy storage, but also have
other functions (Burmester, 1999a, b, 2002; Terwilliger
et al., 1999).
The multi-subunit structure of Hc and many Hbs
enhances the O2 transport capacity (Willmer et al., 2000;
287
Berg et al., 2002). The combination of O2 with one subunit
may increase the O2-affinity of the other subunits (and vice
versa), thus enhancing O2 uploading at high PO2 and O2
unloading at low PO2 (positive cooperativity). In many
cases, O2-binding can also be modulated by changes of the
pH (Bohr effect). E.g., a low pH may increase O2 release in
the metabolic active tissues, which tend to be acidic. Both
cooperativity and Bohr effect enhance efficiency of
respiratory proteins in delivering O2.
3. The extracellular hemoglobins of the Chironomidae
As early as in the 19th century, scientists noted that the
bright red color of the aquatic larvae of chironomid midges
(Diptera: Nematocera) is due to Hb (Rollett, 1861;
Lankester, 1872). According to our present knowledge,
the Chironomidae are the only insects that have Hbs in
their hemolymph. The larvae of many chironomid species
live in the sediment of eutrophic and polluted waters,
sometimes reaching considerable depths. In this often
chronically hypoxic environment the ambient O2 partial
pressures range from 10 to 50 Torr (1.3–7 kPa) (Osmulski
and Leyko, 1986), and the larvae absorb O2 via their
cuticular surface. There is little doubt that chironomid Hb
has a respiratory function analogous to vertebrate Hb. It
enhances the O2 capacitance of the hemolymph, thus
enabling O2 transport inside the larvae. In addition, Hb
may serve as a short time oxygen store during rhythmic
periods of hypoxia, which result when the larvae stop
irrigating inside their dwelling tubes (Walshe, 1951).
Accordingly, poisoning of the Hb with CO reduces filterfeeding in Chironomus plumosus larvae (Walshe, 1951) and
larvae increase Hb synthesis in poorly aerated water (Fox,
1955).
Genomic analyses in diverse chironomid species have
revealed that the number of recognized Hb protein variants
is even outnumbered by the number of non-allelic Hb gene
copies (Kao et al., 1995; Trewitt et al., 1995; Hankeln et al.,
1997, 1998). We have counted more than 40 Hb genes in
the genome of Chironomus tentans (T. Hankeln, H. Friedl
and E. R. Schmidt, unpublished), making this the largest
Hb gene family reported so far in any organism. The Hb
gene duplicates are clustered at two separate chromosomal
loci (Hankeln et al., 1988). In few cases, chironomid Hb
genes have turned into non-functional pseudogenes (Gruhl
et al., 2000) or appear to have experienced accelerated
sequence evolution indicative of novel functional properties (Hankeln et al., 1998). While systematic data on
expression patterns/levels and ligand binding features of all
Hb variants are missing, the intriguing Hb multiplicity in
chironomids remains unexplained at the functional level.
Preliminary indications for a differential regulation of Hb
gene variants at the mRNA and protein levels exist
(Saffarini et al., 1991; Green et al., 1998). It remains to
be shown whether Hb gene copies are evolutionarily
preserved in chironomid genomes due to a ‘‘distribution
of tasks’’ (sub-functionalization; Force et al., 1999) or
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288
simply due to a more general, selected increase in Hb
protein dosage (Gruhl et al., 1997).
Hb synthesis in Chironomidae appears to be largely
restricted to the aquatic larval and pupal stages (Osmulski
and Leyko, 1986), although Hbs (for unknown reasons)
have also been found in adult ovaries and eggs (Trewitt
et al., 1986). Hbs are produced mainly by the fat body cells
(Bergtrom et al., 1976) and are exported into the
hemolymph by the means of N-terminal signal peptides.
Hb decomposition takes place in the Malphigian tubules
(Jarial, 1988). The Chironomus Hb polypeptides comprise
about 140 amino acids plus N-terminal signal peptides of
about 20 amino acids. In all known Chironomus thummi
Hbs the distal position E7 is occupied by a standard
histidine residue; only in the Hb variant IIIa this residue is
a glutamine. By contrast, the Hb variants of Tokunagayusurika akamusi, a Japanese chironomid, either have
histidine, leucine or isoleucine at the E7 distal site (Fukuda
et al., 1993). The functional consequences of these
differences are unknown. Hb chains are monomers, form
homodimers or occur in a monomer–dimer equilibrium
(Goodman et al., 1983; Weber and Vinogradov, 2001).
Other Chironomus species have distinct Hb aggregation
levels, ranging from exclusively monomers up to tetramers
(Weber and Vinogradov, 2001). This demonstrates the
evolutionary flexibility of Hb organization in the Chironomidae. The chironomid Hbs bind O2 with high affinity
(half-saturation pressure [P50] ¼ 0.5–1.5 Torr) and in a
non-cooperative manner (Table 1) (Amiconi et al., 1972;
Weber et al., 1985). Measurements of pH-dependence
of O2-binding revealed that both monomeric and dimeric
C. thummi Hbs exhibit a mild Bohr effect (Weber et al.,
1985; Ruf et al., 1994). The crystal structure of Hb chain
III from C. thummi shows a protein with a typical globin
fold, but a reversed orientation of the heme (compared to
vertebrate Hb) and an unusual open conformation of the
ligand binding site (Huber et al., 1971).
4. Intracellular insect hemoglobins with specialized O2
storage function
In some insects, specialized tissues store O2 by the virtue
of intracellular Hb (Weber and Vinogradov, 2001). The
presence of these Hbs can be easily recognized by the red
color of particular tissues, e. g. in the larvae of the horse
botfly Gasterophilus intestinalis (Diptera) (Keilin and
Wang, 1946) and in backswimmers (Hemiptera) (Miller,
1964; Bergtrom, 1977; Wells et al., 1981). There is little
doubt that the intracellular Hb of G. intestinalis has a
myoglobin-like role. The larvae of G. intestinalis live for
7–8 months as parasites in the alimentary tract of horses.
The early larvae are bright red due to the presence of Hb in
the fat body, in the parietal musculature and in the
hypodermis (Keilin and Wang, 1946). In older larvae, Hb
becomes concentrated at the posterior body part in
specialized tracheal cells, which are thought to be
derivatives of the fat body. The Hb assists in the extraction
of O2 from air bubbles, which have been engulfed by the
horses and which are only temporarily available in the
semi-fluid environment. The Hb stores O2 that is subsequently released during anoxic periods. Biochemical
evidence and the crystal structure showed that the Hb of
G. intestinalis is a dimer, which is encoded by at least
two distinct genes (Dewilde et al., 1998; Pesce et al., 2005).
The protein measures 152 amino acids and harbors
histidines in proximal position F8 and in distal position
E7. G. intestinalis Hb binds O2 in a non-cooperative
manner (Table 1). Early measurements of O2 binding
kinetics suggested a moderately high affinity of P50 ¼
4.9 Torr at 39 1C (Keilin and Wang, 1946), while more
recent data obtained by flash photolysis showed a higher
affinity of P50 ¼ 0.15 Torr at 25 1C (Dewilde et al., 1998).
The reason for this discrepancy is unknown.
Backswimmers are diving predators that breathe under
water by the aid of an abdominal air bubble that had been
captured at the surface. Anisops and Buenoa (Notonectidae) and Macrocorixa geoffroyi (Coryxidae) have Hbs at
high concentrations (20 mM) in tracheal organs, which
are penetrated by tracheoles (Hungerford, 1922; Miller,
1964, 1966; Bergtrom, 1977; Wells et al., 1981; Matthews
and Seymour, 2006). Poisoning of the Hb with CO reduces
dive durations dramatically (Miller, 1964, 1966; Wells
et al., 1981). A recent study employing the Australian
backswimmer Anisops deanei has demonstrated that during
the initial phase of the dive O2 is taken mainly from the
bubble, which thus looses volume (Matthews and Seymour,
2006). When the PO2 in the bubble falls below about
30 Torr (4.3 kPa) at a later phase of the dive, Hb becomes
Table 1
Biochemical characteristics of selected insect respiratory proteins
Species
Protein
Localization
Structure
P50 (Torr)
n50
pH
T (1C)
Anisops assimilis
Gasterophilus intestinalis
Hb
Hb
Tracheal organ
Musculature, hypodermis, fat body, tracheal cells
Monomer, hexamer
Dimer
Drosophila melanogaster
Chironomus thummi
Perla marginata
Hb
Hb
Hc
Tracheal cells, fat body
Hemolymph
Hemolymph
Monomer
Monomers, dimers
Hexamer
30–40
4.9a
0.15b
0.12
0.32–1.3
8
5.2
1
1
1
1
2
6.9
7.3a
7.0b
7.0
7.0
7.8
25
39a
25b
25
25
25
a
Keilin and Wang (1946).
Dewilde et al. (1998).
b
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the main source of O2. This strategy helps to stabilize the
volume of the air bubble and to maintain neutral
buoyancy. Sequences of backswimmer Hbs are yet
unknown, but biochemical studies in Anisops or Buenoa
showed 17 kDa Hb monomers. In their oxy-form, the Hbs
are mainly monomeric, while in the deoxy-form, the Hb
occurs as a mixture of monomers and hexamers (Bergtrom,
1977; Wells et al., 1981). Anisops Hb has a low O2 affinity
in the range P50 ¼ 30–40 Torr and a high cooperativity
(Wells et al., 1981; Matthews and Seymour, 2006). This P50
correlates with the critical PO2 in the bubble, when Hb
becomes the main source of O2 (Matthews and Seymour,
2006).
5. Intracellular insect hemoglobins at lower concentrations
While the Hbs of Gasterophilus and the backswimmers
can easily be linked to their specialist lifestyle, the recent
identification of intracellular Hbs in other insect species
was unexpected. Hbs have been discovered in the
Drosophilidae (Diptera, Brachycera) (Burmester and Hankeln,
1999; Hankeln et al., 2002; Burmester et al., 2006), the
mosquitoes Anopheles gambiae and Aedes aegypti (Diptera,
Nematocera) (Burmester et al., in press) and the honeybee
Apis mellifera (Hymenoptera) (Hankeln et al., 2006). In
addition, Hb sequences are present in the genomes or ESTs
(expressed sequence tags) of the hemipterans Acyrthosiphon
pisum and Aphis gossypii, the coleopterans Dascillus
cervinus and Tribolium castaneum, the silkworm Bombyx
mori (Lepidoptera) and the fly Glossina morsitans (Diptera)
(Hankeln et al., 2006). The conservation of amino acids in
key positions of the globin chain suggests that these Hbs
are actually functional in binding O2 (Table 2).
The Drosophilidae have three distinct Hb genes
(glob1–3), of which glob1 features the highest level of
expression (Burmester et al., 2006). A. gambiae and
289
A. aegypti each harbor two Hb genes that duplicated early
in the evolution of mosquitoes (Culicidae) (Table 1). Hb
expression in D. melanogaster, A. gambiae and A. mellifera
is mainly associated with the tracheal system. Hb mRNA
has been detected in the walls of tracheal tubes and in
terminal cells (Hankeln et al., 2002, 2006; Burmester et al.,
2006). In all three species other sites of Hb synthesis have
been identified in organs such as the fat body and pharynx
muscle in D. melanogaster, the visceral muscles in A. gambiae
and the Malphigian tubules in A. mellifera. The intracellular concentrations of these Hbs in these tissues are
unknown; a rough estimate by comparing Western blot
signal intensities and EST numbers suggests that the glob1
protein of D. melanogaster makes up about 0.1% of all
adult proteins (Burmester et al., 2006). Taking into account
the restricted cellular expression of this protein, this is
certainly a significant amount. Glob1 expression regulation
was studied under experimental hypoxia employing transfected S2 cell lines (Gorr et al., 2004) or living embryonic,
larval and adult Drosophila (unpublished data). The data
agree in that low O2 conditions appear to trigger a downregulation of glob1 mRNA, although there are differences
among developmental stages (unpublished data).
Biochemical and crystallographic studies demonstrated
that D. melanogaster glob1 is a monomer, which covers 153
amino acids (Hankeln et al., 2002; de Sanctis et al., 2005).
It has an O2 affinity of P50 ¼ 0.15 Torr (Hankeln et al.,
2002), which is similar to that of the Chironomus and
Gasterophilus Hbs (Table 1). However, D. melanogaster
glob1 displays a so-called hexacoordinate binding scheme
in its deoxy-state, in which the Fe2+ is bound to the distal
histidine residue at helix position E7. Interestingly, the
closely related G. intestinalis Hb shows the ‘‘classical’’
pentacoordination (Fe2+ is only bound to four N-atoms of
the heme ring plus the proximal histidine at F8) (Pesce et al.,
2005). The functional consequences of hexacoordination
Table 2
Functionally important amino acid residues found at key positions in insect Hbs
Species
A12
B10
CD1
E7
E10
E11
F8
Human Mb
Chironomus Hb
G. intestinalis Hb
Drosophila glob1
Drosophila glob2
Drosophila glob3
Apis mellifera glob1
Anopheles gambiae glob1
Anopheles gambiae glob2
Aedes aegypti glob1
Aedes aegypti glob2
Acyrthosiphon pisum glob1
Aphis gossypii glob1
Dascillus cervinus glob1
Tribolium castaneum glob1
Bombyx mori glob1
Glossina morsitans glob1
Trp
Trp/Phe/Tyr
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Leu
Leu
Leu
Leu
Phe
Phe
Met
Phe
Leu
Phe
Met
Val
Phe
Leu
Phe
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
His
His
His
His
His
His
His
His
Gln
His
His
His
His
Gln
His
His
His
Thr
Arg
Arg
Arg
Ala
Arg
Gly
Asn
His
Asn
Asn
Lys
Lys
Ser
Asn
Asn
Arg
Val
Ile/Val
Ile
Ile
Met
Phe
Val
Leu
Ile
Leu
Val
Val
Val
Val
Val
Ile
Ile
His
His
His
His
His
His
His
His
His
His
His
His
His
His
His
His
His
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are unknown, although this structure is also known from
other globins such as vertebrate neuroglobin and cytoglobin (Pesce et al., 2003; de Sanctis et al., 2004).
6. Evolutionary conservation and diversity of insect globins
Although being identified only in Eumetabola (Fig. 1), it
can be assumed that Hb gene(s) belongs to the standard
repertoire of insects. Insect Hbs are most similar to
crustacean Hbs. Phylogenetic studies suggest a complex
pattern of evolution (Fig. 2). Drosophila glob1-type Hbs
are orthologous to the G. intestinalis Hb. The Drosophila
glob2 and glob3 genes are of ancient evolutionary origin, as
confirmed by their ancestral exon–intron pattern (Burmester et al., 2006). Interestingly, mosquito and the chironomid Hb genes are not orthologous, but have divergent
evolutionary origins before the radiation of Diptera
(Burmester et al., in press). Chironomid Hbs are associated
with G. intestinalis Hb, G. morsitans Hb and Drosophila
glob1 proteins, whereas the A. gambiae and A. aegypti Hbs
have a more basal position within the insect Hb tree. Thus
the extracellular Hbs of the Chironomidae evolved from an
intracellular Hb after the separation of the Chironomidae
(Nematocera) and the brachyceran flies (Burmester and
Hankeln, 1999; Burmester et al., 2006). An orthology of
Chironomus and vertebrate Hbs, and thus an ancient origin
of the Chironomus Hbs, as originally proposed, e.g. by
Goodman et al. (1983), is not evident. The phylogenetic
history of insect Hb genes can also be traced by the aid of
intron positions (Hankeln et al., 1997; Burmester et al., in
Fig. 2. Phylogeny of insect hemoglobins. An alignment of insect Hb amino
acid sequences was analyzed by MrBayes 3.1.1. A simplified tree is
displayed; branches with support values o0.9 have been collapsed.
press). Introns in helix-positions B12.2 (i.e. intron inserted
between the second and third bp of codon 12 of globin
helix B) and G7.0 are most likely ancestral in the
globin gene lineage (Hardison, 1996). The Hb genes of
T. castaneum, B. mori, the mosquito glob1 genes and
Drosophila glob2 and glob3 genes indeed have introns in
B12.2 and G7.0 (Fig. 2). This may be interpreted as an
indication of a basal position of these genes within the
insect globin tree. Mosquito glob2 genes have lost the
intron in G7.0, while A. aegypti glob1 has acquired an
additional intron in E18.0. This complex pattern shows the
dynamics of intron gain and loss in the insect globin gene
lineage. G. intestinalis Hb and the Drosophila glob1 have
introns in D7.0 and G7.0, indicating a loss of the B12.2
intron and an acquisition of an intron in D7.0 during the
evolution of the Brachycera (Burmester and Hankeln,
1999; Burmester et al., 2006). Furthermore, this pattern
confirms the evolutionary orthology of G. intestinalis Hb
and Drosophila glob1 genes. Most known present-day
chironomid Hb genes do not harbor any introns, but
during evolution some have acquired introns in central
positions E9.1, E15.0 and E12.2 (Hankeln et al., 1997; and
our unpublished data). In some cases, introns may have
laterally spread to neighbor gene duplicates by gene
conversion-like processes.
7. Intracellular hemoglobins as ‘standard’ respiratory
proteins in insects
The apparently universal presence of intracellular Hbs
among insects is striking and requires a physiological
interpretation. At the first glance, it may seem improbable
that Hbs expressed at low concentrations have a myoglobin-style O2 supply function, as it has been demonstrated
for botfly and backswimmer Hbs. This is mainly due to the
apparent lack of need of a respiratory protein in insect taxa
that live under largely normoxic conditions. Nevertheless,
at least in Drosophila, a respiratory function of glob1 is
conceivable (Hankeln et al., 2002; Burmester et al., 2006):
(1) although the cellular concentration of glob1 is
unknown, Western blot signals and EST numbers suggest
that local amounts are likely sufficient to contribute
significantly to O2 supply. (2) Embryos and larvae of
Drosophila compete efficiently with microorganisms for a
limited supply of O2 within fermenting fruits. Therefore, an
Hb in the tracheal system that enhances the extraction of
O2 from the environment would almost certainly be
beneficial. The same might be true for adult flies which
possibly meet short-term hypoxia when closing their
spiracles during flight in order to prevent excessive
desiccation (Lehmann, 2001). (3) Drosophila glob1 and
G. intestinalis Hb display similar tissue expression patterns
and O2-binding kinetics. In an evolutionary perspective,
G. intestinalis Hb and Drosophila glob1 are orthologues
(Fig. 2). Thus, it is most parsimonious in an evolutionary
sense to propose that glob1 fulfills a respiratory role just
like the botfly Hb.
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However, alternative physiological functions of intracellular insect Hbs may also be considered. Some globins
carry out enzymatic functions, such as the detoxification of
nitric oxide (NO; Flögel et al., 2001) and possibly other
noxious reactive nitrogen and oxygen species, or they may
serve as O2 sensing molecules (Freitas et al., 2005). A role
of glob1 as an O2 sensor is unlikely, because the observed
ligand affinity is too high for a sensor that works under
cellular O2 concentrations. By contrast, a hypothetical
function of glob1 in detoxification of reactive oxygen
species (ROS) would be in line with the observed tracheal
expression pattern and gene regulation data (unpublished
results). It must be considered that the cells adjacent to the
tracheal tubes may experience unusually high O2 partial
pressures, which are close to the atmospheric level (Krogh,
1920), whereas inner tissues normally work at PO2 in the
range of few kPa. High PO2, however, leads to the
generation of ROS and cell damage (Halliwell and
Gutteridge, 1999). In fact, it has been proposed that under
certain conditions the tracheal system has to adapt to avoid
the influx of an excess of O2 (Burmester, 2005; Hetz and
Bradley, 2005). The Hbs in the insect tracheal cells may
therefore scavenge ROS directly by transforming them into
harmless compounds.
One can also combine the oxygen-supply and protection
hypotheses. In this scenario, Hb in the tracheal walls may
act as an O2 buffer system that on the one hand supports a
constant flow of O2 from the trachea to the inner cells, but
also protects the cells from an excess of O2 that would lead
to the generation of ROS. In this context it is noteworthy
that intracellular Hbs at high levels are located in the
tracheal system of insects that experience intermittent
hypoxia, which results in dramatically changing PO2 in the
tracheal system. A similar cyclic pattern of oxygen
availability also exists in insects that periodically close
their spiracles. When the spiracles are closed, the PO2 in
the terminal cells slowly decreases, and O2 is released from
the Hb. With open spiracles, Hb binds to O2 and impedes
its flow from the atmosphere to the inner organs, thus
protecting the cells from an excess of O2.
8. Insects with blue blood
Since many years, the presence of Hc in the hemolymph
of most spiders (Chelicerata) and the malacostracan
Crustacea is common knowledge (for review, see: Markl
and Decker, 1992; van Holde et al., 2001). It has been
demonstrated that Hcs also occur in Onychophora and
some Myriapoda (Jaenicke et al., 1999; Kusche and
Burmester, 2001; Kusche et al., 2002, 2003). Hcs had been
unknown in insects, although hexamerins, storage proteins
that had derived from Hc, are widespread in this taxon
(Telfer and Kunkel, 1991; Burmester et al., 1998; Burmester, 1999a). Hexamerins and Hcs can easily be distinguished by the absence of the conserved Cu-binding
histidines in hexamerins. In 1998, however, Sànchez and
colleagues identified an Hc-like protein in the embryonic
291
hemolymph of the grasshopper Schistocerca americana.
This ‘‘embryonic hemolymph protein’’ (EHP) measures
674 amino acids (including an N-terminal signal peptide of
20 residues) and shows conservation of the three structural
domains and the six Cu-binding histidines. Although this
finding was the first indication that true Hcs may occur in
insects, a role of EHP in O2 transport was uncertain
because neither O2-binding nor its expression in later
developmental stages could be demonstrated (Sánchez
et al., 1998).
Recently, however, the presence of a true Hc was
demonstrated in the stonefly Perla marginata (Plecoptera)
(Hagner-Holler et al., 2004). This Hc occurs in the
hemolymph at moderately high concentrations in nymphs
and adults, and there is no doubt that it acts as respiratory
protein. P. marginata Hc binds O2 reversibly with a P50 of
8 Torr and shows moderate cooperativity. Thus the O2
affinity of P. marginata Hc is lower than that of
Chironomus Hb, which is likely related to the higher PO2
in the stonefly’s environment. Stonefly Hc is a hexamer
with a mass of about 460 kDa, which consists of two
distinct subunits that assemble in unknown stoichiometry.
Both subunits contain N-terminal signal sequences for
transmembrane transport. The native Hc subunits comprise 659 and 655 amino acids with molecular masses of
77.2 and 76.3 kDa, respectively. In both subunits the six
Cu-binding site histidines in the second domain, as well as
phenylalanines in the first and second domain that stabilize
the binding of O2, are conserved. Isolated subunits have
high O2 affinities of P50 ¼ 0.063 and 0.023 Torr. Interestingly, the two Hc subunits appear to have divergent
origins. While Hc1 resembles the EHP of S. americana
(thus providing further indirect evidence for a respiratory
function of this protein) and forms a common clade
with the insect hexamerins, Hc2 has a more ancient
evolutionary origin and diverged before Crustacea and
insects split (Fig. 3).
The leading phylogenetic hypothesis suggests a close
relationship between insects and crustaceans, or even the
inclusion of the insects within a Pancrustacean taxon
(Hwang et al., 2001; Giribet et al., 2001). This view is also
supported by hemocyanin phylogeny (Fig. 3). Fundamental changes in the mechanisms of gas exchange must have
accompanied the diversification of insects from crustaceans
and their invasion of terrestrial and aerial environments
(Kukalovà-Peck, 1991). Nevertheless, Hcs are still present
in many lower insects. This finding shows that neither
terrestrialization, nor the evolution of a tracheal system for
efficient O2 supply was accompanied by the loss of a
hemolymph-based respiratory protein. A similar observation has been made in Onychophora and Myriapoda,
which possess both trachea and Hc (Jaenicke et al., 1999;
Kusche et al., 2002, 2003).
We have also investigated other insect species for the
presence of Hc (C. Pick and T.B., unpublished data).
Although the picture is far from being complete, the data
show that Hcs are common in many different ‘‘lower’’
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Forschungsgemeinschaft (Bu 956/5 and Bu956/9 to T.B.,
and Bu 956/6 to T.B. and T.H.).
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Fig. 3. Phylogeny of insect hemocyanin subunits. A Bayesian phylogenetic
tree was deduced from the analyses of an amino acid alignment of insect
hexamerins and hemocyanins. All nodes are supported with 40.95. Insect
hemocyanin subunits are shaded in gray.
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of the backswimmers, Gasterophilus and the chironomids),
they had to recruit Hb from the intracellular globin gene
that appears to be available in the genome of all insects.
Acknowledgments
We thank C. Pick (Hamburg, Germany), H. Decker, E.
Gleixner, S. Hagner-Holler, S. Klawitter, M. Krämer
(Mainz, Germany), J. Marden (University Park, PA,
USA), M. Marden (Paris, France), S. Dewilde, L. Moens
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