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Developmental and Comparative Immunology 35 (2011) 273–284
Contents lists available at ScienceDirect
Developmental and Comparative Immunology
journal homepage: www.elsevier.com/locate/dci
The two suborders of chiropterans have the canonical heavy-chain
immunoglobulin (Ig) gene repertoire of eutherian mammals
John E. Butler a,∗ , Nancy Wertz a , Yaofeng Zhao b , Shuyi Zhang c , Yonghua Bao b ,
Sara Bratsch d , Thomas H. Kunz e , John O. Whitaker Jr. f , Tony Schountz g
a
Department of Microbiology, University of Iowa, Iowa City, IA, United States
State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China
School of Life Sciences, East China Normal University, Shanghai, China
d
Department of Biology, University of Wisconsin-River Falls, United States
e
Center for Ecology and Conservation Biology, Department of Biology, Boston University, Boston, MA, United States
f
Department of Biology, Indiana State University, Terra Haute, IN, United States
g
School of Biological Sciences, University of Northern Colorado, Greeley, CO, United States
b
c
a r t i c l e
i n f o
Article history:
Received 23 July 2010
Received in revised form 24 August 2010
Accepted 25 August 2010
Available online 9 September 2010
Keywords:
Bat
Immunoglobulin genes
Phylogeny
a b s t r a c t
Bats comprise 20% of all mammals, yet little is known about their immune system and virtually nothing
about their immunoglobulin genes. We show that four different bat species transcribe genes encoding
IgM, IgE, IgA and IgG subclasses, the latter which have diversified after speciation; the canonical pattern
for eutherian mammals. IgD transcripts were only recovered from insectivorous bats and were comprised
of CH1, CH3 and two hinge exons; the second hinge exon was fused to CH3. IgA in all species resembles
human IgA2 with the putative cysteine forming the bridge to the light chain found at position 77. Sequence
comparisons yielded no evidence for a diphyletic origin of the suborders. Bats show no close similarity to
another mammalian order; the strongest association was with carnivores. Data reveal that CH diversity
and VDJ and CDR3 organization are similar to other eutherian mammals, although the expressed VH3
family repertoire was unusually diverse.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Among living vertebrates, bats and birds are unique in their ability to fly, and this common feature sets them apart ecologically
and physiologically from other groups. Bats are in some ways the
nocturnal equivalent of birds, having evolved and radiated into a
diversity of forms to fill many of the same niches. Bats have successfully colonized almost every continental region on earth (except
Antarctica) as well as many oceanic islands and archipelagos (Kunz,
1982; Kunz and Lumsden, 2003). Bats impact the environment in
several ways. Insectivorous species suppress insect populations
(Cleveland et al., 2006; Kalka et al., 2008; Williams-Guillén et al.,
2008; Jones et al., 2009), and plant-visiting bats serve as pollinators and disperse seeds (Fujita and Tuttle, 1991; Shilton et al., 1999;
Muscarella and Fleming, 2007; Fleming et al., 2009). Insectivorous
species like the common little brown bat (Myotis lucifugus) may
consume their entire body mass each night in insects (Kurta et al.,
∗ Corresponding author at: Department of Microbiology, University of Iowa, 3550
BSB, 51 Newton Road, Iowa City, IA 52242, United States. Tel.: +1 319 335 7776;
fax: +1 319 335 9006.
E-mail address: [email protected] (J.E. Butler).
0145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2010.08.011
1989). Thus, they are the most important predators of nocturnal
insects and consume 5–10 times more insects than swallows and
flycatchers. So-called “megabats” or “flying foxes” of the suborder
Yinpteropodidae are important pollinators and seed dispersers, but
sometimes can be pests when they damage fruit crops.
Bats comprise the second largest order of mammals (next to
rodents) in total number of species and may exceed all other groups
in overall abundance (Kunz, 1982; Wimsatt, 1970). Of the >5400
recognized species of mammals, bats comprise 20% (Teeling et al.,
2002; Simmons, 2005). Bats are believed to have evolved early and
morphologically have changed very little over the past 52 million
years (Hill and Smith, 1984; Simmons et al., 2008). Bats are divided
into 18 families that comprise two major suborders: Yinpteropodidae and Yangochiroptera. The former suborder includes the
family Pteropodidae which comprises the so-called “megabats”
or “flying foxes” and the families Rhinolophidae, Hipposideridae,
Megadermatidae, Rhinopomidae and Crasonycyeteridae while the
Yangochiroptera includes all other families including the insectivorous “microbats” (Teeling et al., 2002). Except for the genus
Rousettes, all of the families Pteropodidae are non-echolators compared to all other species of both suborders. The appearance of
certain megabats (flying foxes) and neurological features of the eye
and prestrinate cortex lead Pettigrew to propose that the order was
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diphyletic, i.e. the Yinpteropodidae arose from a primate/lemurs
stock while all other bat families evolved from the Insectivora
(Pettigrew, 1986). This was an intriguing hypothesis because it
implied that mammalian flight evolved twice. However, advances
in molecular analyses, including what we report here, have largely
laid to rest the diphyletic hypothesis for the Chiroptera (Murphy
et al., 2007; Teeling et al., 2005; Adkins and Honeycutt, 1991;
Mindell et al., 1991). Bats were originally considered members of
the superorder Archonta which also included primates, tree shrews
and flying lemurs (Gregory, 1910; Simpson, 1945). Recent analyses places bats closer to ungulates, whales, horses and carnivores
(Teeling et al., 2002, 2005) [a clade referred to as Laurasiatheria].
All bat species can be vectors for human viral diseases
(Messenger et al., 2003; Calisher et al., 2006; Wibbelt et al., 2009).
There are >85 viruses known from bats (Calisher et al., 2006) and
several are associated with animal and human epidemics (Wong et
al., 2007). These include Hendra and Nipah viruses (horse, humans
and swine; Mackenzie and Field, 2004) SARS (rodents, cattle swine,
dogs, human; Dobson, 2005; Li et al., 2005; Mueller et al., 2007;
Shi and Hu, 2008) Ebola (human and other primates; Swanepoel
et al., 1996; Leroy et al., 2005) and West Nile viruses (horses and
humans; Lau et al., 2005). Recently there is concern over their role
in zoonotic diseases (Calisher et al., 2006; Wibbelt et al., 2009; Lau
et al., 2005; Halpin et al., 2008). This includes white nose syndrome
(WNS) which has caused unprecedented mortality in little brown
bat populations in the Northeastern US (Blehert et al., 2009; Frick
et al., 2010).
However, little is known about the immune system of bats,
especially their immunoglobulins (Igs) and the manner which they
generate their antibody repertoire.
The paucity of information on the bat immune system raised
the question of whether their immune system might show any
deviation from other mammals that would suggest an adaption
for flight. Birds have an immune system disparate from mammals.
They lack IgD and IgE and both chicken IgG (IgY) and IgA differ in
domain structure from their mammalian counterparts (Ratcliffe,
1991). Furthermore, birds lack lymph nodes, have determinant B
cell lymphopoiesis and use somatic gene conversion (SGC) to generate their antibody repertoire (Reynaud et al., 1989; McCormack
et al., 1991). Since the work of Teeling et al. (2002, 2005) leaves
little doubt that bats are indeed part of the mammalian lineage,
it is unlikely that their immune system would resemble that of
birds. However, there is considerable variation among mammals
regarding their antibody repertoire (Butler, 2006), thus investigating the Ig genes of a mammalian order not previously studied,
might uncover variations in the canonical mammalian Ig pattern
that could be valuable in future functional studies involving the
immune system.
In the present study, we demonstrate that the Igs of bats
possess the canonical pattern characteristic of other eutherian
mammals, including apparent diversification of IgG into subclasses
after speciation. Given the sequences for both Yinpteropodidae and
Yangochiroptera were available, including fruit-eating and insectivorous species, we found no evidence for a polyphyletic origin
for bats. However, we did find immunological evidence for the
divergence of the two suborders. Our analysis revealed a surprising
and unexplained diversity among expressed VH3 genes of the little
brown bat, M. lucifugus, which raises the question of whether bats
might use SCG similar to that of birds, extensive somatic hypermutation (SHM) or combinatorial diversity to develop their specific
antibody repertoire. The latter question has been addressed in a
separate study that shows extensive germline diversity but a surprising lack of SHM (Bratsch et al., in press).
2. Materials and methods
2.1. Source of materials
Spleen and other lymphoid tissues were removed from euthanized little brown bats (M. lucifugus) that had been collected
at a maternity roost in eastern Massachusetts. The same tissues
were also collected from the big brown bat (Eptiscus fuscus) from
western Indiana. Both species are members of the family Vespertilionidae, suborder Yangochiroptera. The same tissues were
collected from the Seba’s short-nosed fruit bat, Carollia perspicillata
(family Phyllostomidae suborder Yangochiroptera) as by-products
of viral research at Northern Colorado University. CDNA from the
short-nosed fruit bat Cynopterus sphinx (family Pteropodidae), was
prepared at the East China Normal University and at the China
Agricultural University. Apart from the latter, all samples were collected and frozen in liquid nitrogen and shipped on dry ice to the
University of Iowa.
2.2. Preparation of RNA and cDNA
RNA was extracted from pulverized frozen tissue or from TriZolstored material following the manufacturer’s protocol (Invitrogen,
Carlsbad, CA) and as previously described (Sun et al., 1998). RNA
concentration was determined spectrophotometrically using a
NanoDrop 1000. CDNA was prepared using Superscript II as previous described (Butler et al., 2006).
2.3. Recovery of bat Ig transcripts by PCR
Transcripts encoding bat IgM, IgD, IgG, IgA and IgE were recovered by PCR using degenerate primer sets. Table 1 indicates the
Table 1
Primers used for recovery of bat heavy-chain Ig transcripts.
Forwarda
Isotype
Product lengthb
Reverse
PCR1
PCR2
VH FR 1 5
VH FR 1 3
agctggtggagtctggagga
actctcctgtgcagcctctg
IgM CH4
gtggactttcctctcggt
PCR1
PCR2
VH FR 1 5
VH FR 1 3
agctggtggagtctggagga
actctcctgtgcagcctctg
IgG3 UTRc
tttatttcatgctggdcccggg
IgD
PCR1
PCR2
VH FR 1 5
VH FR 1 3
agctggtggagtctggagga
actctcctgtgcagcctctg
IgD CH3
tgcctgacctcgcatgtgtag
IgA
PCR1
PCR2
VH FR 1 5
VH FR 1 3
agctggtggagtctggagga
actctcctgtgcagcctctg
IgA CH3
tgacgtgggtgggtttacccg
PCR1
PCR2
agctggtggagtctggagga
actctcctgtgcagcctctg
IgE CH3
aaagaagttctggatcaggcaggtg
IgM
IgG
IgE
a
b
c
VH FR 1 5
VH FR 1 3
FR1 = framework 1 of VH3 genes. FR1 3 is a nested primer that targets a sequence downstream from FR1 5 .
Products of expected length were obtained, cloned and sequenced.
All genes encoding C␥ genes share a common UTR sequence.
1500
1400
1450
1300
1500
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J.E. Butler et al. / Developmental and Comparative Immunology 35 (2011) 273–284
primers used and the domain sequences targeted by the primers
employed in first (PCR1) and second (PCR2) round PCR amplification and the expected length of the final PCR products. Since the
VH3 family is considered most ancestral (Schroeder et al., 1990)
we used the FR1-5 primer based on the VH3 genes of swine and
other species that have VH3 genes in their genome (Butler, 2006;
Butler et al., 2006). A hemi-nested PCR system was used in which
275
the second round used the internal forward FR1-3 primer (Table 1),
but the same reverse primers that annealed to sequences encoding
the CH3, CH4 or UTR regions of the various isotypes. PCR product
length was evaluated on agarose gels and those of expected length
were excised from the gels and cloned into pCR2TOPO according to
the manufacturer’s protocol (Invitrogen, Carlsbad, CA). The ligation
mixture was plated on L–B plates. Blue/white selection was used
Fig. 1. The deduced amino acid sequences of the heavy chains of IgM, IgD, IgE and IgA from four bats fit the canonical pattern for the corresponding human Ig isotypes. Amino
acid positions of interest that are referred to in the text, are in boldface and are numbered according to the IMGT system (Lefranc and LeFranc, 2001). Cysteines involved in the
intradomain S–S loop are in bold with arrows indicating the loop. The cysteines involved in the covalent bond to the light chain, are also in bold and indicated as L–H. Potential
N-linked glycosylation sites are in boldface. Position in each domain start with 1 as is done in the IMGT system. The functional hinge of IgA is part of the 5 portion of the CH
2 domain. Dots indicate that the residue is the same as for the reference human sequence. Human IgA2 was used for comparison since the extended hinge of human IgA1 is
unique among mammals. Gaps are inserted into the alignment to obtain the best possible alignment with the human sequence that was used as a reference. Bat sequences
have been deposited in GenBank and the accession numbers and those for the human sequences that were used for comparison are as follows: M. lucifugus = IgA, HM134924;
IgD, HM134925, HM134926; IgE, HM134927; IgM, HM134936, HM134937; E. fuscus = IgA, HM134938; IgD, HM134939, HM134940; IgE, HM134941; IgM, HM134943; C.
perspicillata = IgA, HM134944; IgE, HM134945; IgM, HM134948; C. sphinx = IgA, HM134948, HM134949; IgE, HM134950; IgM, HM134953.
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J.E. Butler et al. / Developmental and Comparative Immunology 35 (2011) 273–284
Fig. 1. (Continued ).
to recover colonies that were subsequently prepared for sequence
analysis. The samples were then processed using a Qiagen Quick
lyses kit (Valencia, CA) and digested with EcoR1 for 2 h to release
the inserted DNA after which the product was again analyzed on an
agarose electrophoretic gel to confirm that products of the expected
length were recovered.
2.4. Sequence analysis
Clones processed as described above were sent to the core
sequencing facility of the University of Iowa where they were analyzed using an Applied Biosystems Model 3730 (96 capillary) DNA
sequencer. Our data are presented using the IMGT system in which
the start codon for each domain is number 1 (Fig. 1). We have no
evidence for variants at each position that are described for human
variants on the IMGT website http://www.imgt.org.
2.5. Recovery of genomic IgD
Sequences from expressed IgD from M. lucifugus were used
to recover genomic IgD sequences (scafford 15451, 11.5 kb) by
applying BLAST to the partially sequenced genome of M. lucifugus (http://www.ensembl.org/Myotis lucifugus/Info/Index). A new
primer was made by aligning the sequences from the transcribed
IgD from M. lucifugus with the sequence recovered from BLAST to
identify heavily conserved areas of bat IgD. This new primer recovered IgD from E. fuscus spleen cDNA.
2.6. Analysis of VDJ transcripts
VDJ sequences, generated as by-products of efforts to identify
the various Ig heavy-chain genes, were analyzed with regard to the
recovery of identical VH genes (Table 2) and for the characteristics of their CDR3 regions (Table 3). Sixty-six IgG VDJ transcripts
and seven VDJ transcripts for IgM, IgD, IgE and IgA were recovered
from the spleen of M. lucifugus. In addition, 51 VDJ transcripts were
also recovered from E. fuscus, C. perspicillata and C. sphinx during
the procedure to recover their genes transcribing IgM, IgD, IgG, IgA
and IgE. These transcripts allowed for a partial characterization of
transcribed VDJs.
2.7. IgG subclass analysis
Putative IgG subclass/allotype sequences recovered from all four
bat species were compared in PileUp. The translated sequences
were then grouped and aligned based on the PileUp so that major
difference within and among species could be identified. Because
major differences were identified in the hinge region, intraspecific comparisons of hinge sequences for different bat IgGs and for
human, mouse, cattle and swine IgG subclasses were compared.
2.8. Phylogenetic comparisons of immunoglobulin heavy-chain
constant regions genes
Phylogenetic trees were constructed with PHYLIP 3.67 software
combined with the TreeView package (Felsenstein, 1989; Page,
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J.E. Butler et al. / Developmental and Comparative Immunology 35 (2011) 273–284
277
Table 2
Recovery of identical FR4 sequences was common while recovery of identical transcribed VH genes was not.
VH
Total
M. lucifugus
E. fuscus
73
20
CDR1
Identical
0
0
Identical
CDR2
Sequence
Total
JH-FR4
Sequence
Total
Sequence
6
5
SYWMH
SSWMNa
4
3
AISDNGVKT
AINPNGGKI
29
20
WGQGTMVTVSS
WGQGTLVTVSSb
4
3
3
2
2
2
2
2
2
40
SYSMD
DSWMN
TSWMN
DYHMN
NSWMN
SYGMS
SYPMN
SYWMD
SYWMS
Unique
2
2
2
2
2
2
54
ADSGGST
LINVDGSTT
AISTNGGNT
RINWNGGYI
YINSGSSYI
YISSGSSYI
Unique
7
5
12
WGPGTLVTVSS
WGPGTLVTVSQ
Unique
2
18
SHYMS
Unique
20
Unique
11
3
2
WGQGTMVTVSS
WGQGTQVTVSSc
WGQGTLVTVSSb
4
12
WGQGTLVTVSSb
14
NYWMN
Unique
14
Unique
2
2
WGQGTQVTVSSc
Unique
2
SSWMNa
15
Unique
13
13
Unique
2
C. perspicillata
16
0
C. sphinx
15
0
a
b
c
2
INTGGDSTY
Unique
2
WGQGTLVTVSSb
Unique
Same CDR1 (underlined) found in two different species.
The same JH was found in all four species of bats (also underlined).
JH found in two of the bat species examined.
1996). Multiple sequence alignments for tree construction were
generated using CLUSTAL. Prodist and Neighbor programs in the
PHYLIP package were also used for tree construction. A consensus
tree was taken from 1000 bootstrapped phylogenetic trees.
3. Results
3.1. Bats possess the canonical isotype repertoire of eutherian
mammals
Fig. 1 provides an alignment of the deduced amino acid
sequences of IgM, IgD, IgE and IgA from four bat species with that
of the corresponding isotypes from human. Given that IgG isotypes
from bats appear to have diverged prior to the appearance of the
major mammalian orders (Fig. 2), their sequences are separately
compared (see below). The major motifs for the five human isotypes
(and other eutherian mammals; data not shown) are present in all
four bat species investigated in the present study. For those shown
in Fig. 1, the intrachain disulfide loop in each domain is also present
in the corresponding domains of bat Igs and the sequences adjacent
to the cysteines involved in these bridges are generally conserved.
With some exceptions, the major potential N-glycosylation sites are
conserved for each isotype in bats. In the case of IgM and IgA, these
are identical. This also appears to be the situation for IgD although
the CH2 domain of bat IgD is absent (Figs. 1 and 5). IgE is most divergent by this criterion since the N-glycosylation site in CH1 (position
Table 3
CDR3 sequences motifs and CDR3 lengths in VDJ recovered with various heavy-chain transcripts.
Species
Isotype
Sequence of CDR3
M. lucifugus
IgM
IgA
IgE
IgD
IgG
R
R
R
R
R
DRLGSFDV W
YGAGADLAY W
DRSTTVTDMEY W
HGSYGDFDV W
DWGHGRFDV W
E. fuscus
IgM
IgA
IgE
IgD
IgG
R
R
R
R
R
VYCASGICYGFDY W
DGWGTYYLDY W
DVRGIYDY W
PPPLVAGGFDY W
EGDAAGGFDY W
C. sphinx
IgM
IgA
IgE
IgDb
IgG
R EYQGFIGY W
R GGGAYNGMDY W
R DLTDLGY W
C. perspicellata
IgM
IgA
IgE
IgDb
IgG
a
b
a
a
R EGTTNTSPFGY W
Frequency
CDR3 length (range)
Mean CDR3 length
4
5
3
2
66
8–10
6–12
6–12
9–10
4–12
8.5
9.2
10
9.5
8.6
3
5
3
4
9
13
6–11
7–8
11–13
4–13
10
8.8
7.7
11.5
9.4
1
4
1
8
6–14
7
8
9.5
7
10
8–12
9.9
R DLNWDGY W
R GMGRYDFYY W
R GVVGGRYFDN W
5
4
2
7–9
4–14
10–12
8
8.8
11
R SRYYSYIYSY W
7
4–12
9.6
The boundaries of CDR3 between the terminal arganine (R) of the FR3 of VH3 family genes and the invariant tryptophan of FR4 (W) are indicated.
IgD has not been recovered from the cDNA of C. sphinx and C. perspicillata.
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Fig. 2. Genes encoding the heavy-chain genes of IgM, IgD, IgE and IgA of bats comprise a separate group with highest similarity with carnivores. The phylogenetic tree was
constructed by using amino acid sequences of the first and last CH domains (platypus ␦ CH7 was used as the last domain for comparison) of these heavy-chain isotype.
The heavy-chain constant regions are indicated with the appropriate Greek letters. M = M. lucifugus, E = E. fuscus, Ca = C. perspicillata; Cy = C. sphinz. For the convenience of
readers, the regions dealing with these four isotypes are indicated in boxes. The values supporting each node are derived from 1000 bootstrapped phylogenetic trees. The
accession numbers for the sequences used are for C␣: cattle, AF109167; dog, L36871; dolphin, AY621035; echidna, AF416951; horse, AY247966; human, J00220; mouse,
J00475; opossum, AF012110; panda, AY818387; swine, U12594; platypus, AY055778; rabbit, X51647; rat, AABR03049801; sheep, AF024645. C␦: cattle, AF515672; dog,
DQ297185; horse, AY631942; human, BC021276; mouse, V00786 and V00788; panda, AY818394; swine AF411239; platypus, EU503149; rat, AAO19643; sheep, AF411238.
C: cattle U63640; dog, L36872; echidna, AY099258; horse, AJ305046; human, J00222; mouse, X01857; opossum, AF035194; panda, AY818389; swine, U96100; platypus,
AY055780; rat, K02901; sheep, M84356. C␮: cattle, U63637; camel, AB091831; cat, AB016712; dog, EF207721 and EF207724; dolphin, AAG40853; echidna, AF416952; horse,
L49414; human, X14940; mouse, V00818; opossum, AF012109; panda, AY818392; swine, U50149; platypus, AY168639; rabbit, J00666; rat, AABR03049259; sheep, X59994.
Accession numbers for bat species are the same as those given in Figs. 1 and 3.
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J.E. Butler et al. / Developmental and Comparative Immunology 35 (2011) 273–284
279
Fig. 3. The IgG subclasses of bats diversified after speciation as in other eutherian mammals. The phylogenetic tree was constructed based on the amino acid sequences of
the first CH domain of all IgG subclasses which is highly species-specific. The designation for bat species is the same as for Fig. 2, i.e. M, E, C␣ and C␺. These designations
are shown in boldface. The accession numbers for the sequences compared are: cattle (X16701, M36946, U63638), camel (AJ421266, AJ131945), dog (AF354264, AF354265,
AF354266, AF354267), horse (AJ302055, AJ302056, AJ312379, AJ302058, AJ312380, AJ312381), human (J00228, J00230, X03604, K01316), mouse (J00453, V00825, V00763,
J00479, X00915), opossum (AF035195), swine (U03778, U03779, U03782, EU372658, EU372657, EU372655), possum (AF157619), rabbit (L29172), rat (AABR03048905,
AABR03049560, AABR03048905, AABR03049912), sheep (X69797, X70983). GenBank numbers for bats C␥ sequences are: M. lucifugus (HM134929, HM134930, HM134931,
HM134932, HM134933, HM134934, HM134935), E. fuscus (HM134942), C .perspicillata (HM134946), C. sphinx (HM134951, HM134952).
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J.E. Butler et al. / Developmental and Comparative Immunology 35 (2011) 273–284
Fig. 4. The IgG subclasses of bats, like other eutherian mammals, differ most in their hinge regions and hinge motifs are not shared among species. The amino acid sequences
of the hinge regions of IgG from different bat species and for subclasses of human, mice, cattle and swine are presented. Sequence motifs shared by the hinge regions of
different subclasses within a species are underlined. The −1, −2, etc. associated with subclasses in cattle and humans indicate the use of alternative hinge exons. Superscripts
for swine C␥ genes denote allotypic variants. The accession numbers for the cg hinge sequences are the same as those listed in the legend of Fig. 3.
21) is not present in any bat species we examined. Moreover, the
potential N-glycosylation motif in the CH2 (position 43) is missing in all bats and only one of the three potential N-glycosylation
sites in the CH3 domain of human IgE (position 65) is found in bat
IgE. Because of this observation we examined the IgE sequences of
several other mammalian IgEs including the duckbill platypus and
the Virginia opossum. We found that the N-glycosylation motif at
position 21 in CH1 was common to human, mouse, cattle and swine
but was missing in all bats, in the opossum and the platypus. Cattle
have an additional site at position 22.
Of particular interest is the absence of a cysteine in the CH1
domain of bat IgA that is part of the covalent S–S bond to the light
chains in human IgA1. Thus we used human IgA2 as our reference
because it also lacks this cysteine. Furthermore human IgA2 lacks
the unique 13 amino acid hinge of human IgA1 that is not found
in any other mammal. This means that bat IgA resembles human
IgA2 and that of cattle and swine with a cysteine at position 77
in CH 1 that is part of the putative L–H bridge, whereas IgA from
the Virginia opossum and the duckbill platypus apparently use the
cysteine at position 14 in CH1 to form the L–H bridge like in human
IgA1.
3.2. IGHC gene sequences of bats form a separate group most
similar to carnivores
Phylogenetic analysis of IgM, IgD, IgE and IgA gene sequences
from bats indicates that especially IgM and IgD share their greatest similarity to that of carnivores (Fig. 2). While IgE from bats also
shares sequence homology with carnivores, it is also related to IgE
from the horse, swine and the human; rodents and ruminant artiodactyls are more distantly related. A somewhat similar pattern of
sequence similarity was found for bat IgA. Noteworthy is that the
sequence similarity of IgE, IgM and IgA, which corresponds to the
accepted taxonomic relationship for the bats included in this study
(Fig. 2). Given that IgM and IgD are the two most primordial Ig isotypes among vertebrates (Ohta and Flajnik, 2006) the comparison
of the C␮ and C␦ from bats to those in other mammals should be
most reliable. Using this criterion, a common ancestry of bats with
carnivores is suggested.
3.3. Subclass diversification occurred after speciation
The branch points for the various IgG subclasses appear to have
occurred after species diversification (Fig. 3). Our data also show
that the IgG from the Neotropical, Seba’s short-tailed fruit bat, C.
perspicillata and those from the Paleotropical, short-tailed fruit bat
C. sphinx form separate branches from the one leading to the insectivorous members of the Yangochiroptera, M. lucifugus and E. fuscus,
of the family Vespertilionidae. On the basis of transcripts recovered,
IgG in M. lucifugus has diversified to the greatest extent; seven different transcripts were recovered, whereas the transcript of only a
single IgG was recovered from C. perspicillata. While some variants
may be alleles, this cannot explain all the variants in M. lucifugus or
C. sphinx because all sequences were recovered from tissues of the
same individuals.
3.4. IgG subclass genes in bats differ in the hinge region and
differences appear order-specific
Alignment of the various C␥ transcripts recovered from different bat species revealed that the putative bat IgG subclass
genes show their greatest difference in their hinge region. This follows the pattern observed in other species (Butler et al., 2009).
Thus, we focused our comparison on variations in the hinge of
the various C␥ genes from the four species of bats available for
the present study. Since the nomenclature of subclasses in all
species is based on order of discovery, same-name homology is
not implied. This should also be apparent from Fig. 3, which shows
that bat IgG subclasses diversified after speciation, as observed for
other mammalian orders (Butler, 2006) making sequence comparisons of subclasses between different species of little phylogenetic
relevance. Therefore, sequence similarities between the four bat
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281
in the duckbill platypus, the African clawed frog (Xenopus sp.) and
the catfish (Ohta and Flajnik, 2006; Zhao et al., 2006, 2009).
3.6. The transcribed VH3 repertoire is diverse
Fig. 5. The domain structure of IgD of bats is rodent-like but unique because of the
fusion of the second hinge exon with the CH3 domain. The TM exons were missing
in the retrieved genomic sequence. IgD was only recovered from two bat species
and only the genomic domain structure of M. lucifugus is indicated.
species in the present study are postulated to reflect either convergent or parallel evolution.
The hinge sequences of IgG from different mammalian taxa,
including bats, illustrate several features of evolutionary and functional importance. First, nearly all hinges are characterized by the
presence of a combination of prolines (often paired) in association
with cysteines. Second, the actual motif for this feature appears to
be species- (or order-) specific; these are illustrated by the underlined motifs in Fig. 4. In some cases different IgG subclasses share
nearly the same hinge. This is demonstrated in swine (Butler et al.,
2009; Fig. 4) but has been observed in M. lucifugus, C. perspicillata
and C. sphinx. The exception is in cattle.
Alternative and duplicated hinges also occur. This is best illustrated by IgG3 in humans and cattle; this is also a common feature
of the domain structure of IgD in some species. Since the genomic
structure of the bat C␥ genes has not yet been determined, this may
also be true for some bat IgG subclasses.
3.5. The domain structure of bat IgD is unique but is otherwise
most similar to rodents
A partial genomic ␦ sequence (scafford 15451, 11.5 kb) of
M. lucifugus was retrieved from its genome database using a
BLAST-based approach. Although the typical transmembrane (TM)
encoding exons are missing in the retrieved sequence, two constant region (C) domain encoding exons, could be identified with
one corresponding to CH1, and the other to the CH3 of other known
placental mammalian IgD heavy chains. The genomic structure of
IgD from a variety of vertebrates was compared with the partial
genomic structure of IgD from M. lucifugus (Fig. 5). These data show
that the domain structure of IgD from M. lucifugus is most similar
to that of rodents in having only one hinge exon and in lacking a
CH2 domain; H2 and CH3 are fused exons in M. lucifugus. Despite
this rodent-like feature, sequence similarities place bat IgD closer
to that of carnivores than rodents (Fig. 2). In no eutherian mammals is there evidence for the multi-domain structure of IgD seen
The primers used to recover the various C-region genes in bats
involved the use of forward primers for the ancestral VH3 variable
gene family. Thus, the 140 transcripts recovered all contain VDJs
encoded by VH3 genes. Because VH3 family genes in all species differ primarily in CDR1 and CDR2, our analysis focused on these gene
segments. Sixty-one percent of the transcripts had CDR1 sequences
that were only recovered once, 13% had CDR1s that were recovered
twice and 4% had CDR1s that were recovered six times (Table 2).
Being a longer sequence, a higher proportion of CDR2 sequences
were unique (75%). By contrast only 14% of JH-FR4 region sequences
were unique suggesting that there are a limited number of JH genes
in the genome but many different VH3 genes.
Of the 73 VDJ sequences recovered form M. lucifugus, none
contained identical VH genes when both CDR1 and CDR2 were considered, although some of these VH genes share CDR regions with
other VH genes. Sharing was most common among the shorter (15
nucleotides) CDR1 segments. In one case an identical CDR1 from
M. lucifugus was also found twice in C. sphinx. By contrast, identical FR4 regions (JH encoded) were quite frequently recovered.
The number and sequences of the various JH segments is unknown
since the genome has not been mapped. In any case, the frequency
of FR4 recovery suggests that there are fewer JH than VH genes
and that M. lucifugus probably has at least five JH gene segments
in its genome. The limited diversity of JH is also supported by the
observation that all four bat species in the present study share one
identical FR4. Since the species studied represent different families and suborders; (E. fuscus and M. lucifugus belong to the same
Vespertilionidae, suborder Yangochiroptera, C. perspicillata to the
family Phyllostomidae, suborder Yangochiroptera, and C. sphinx to
the family Pteropodidae, suborder Yinpterochiroptera) this JH gene
is evolutionarily conserved.
All VDJ sequences were from transcripts so that the diversity
among these VH3 genes may be the result of somatic hypermutation (SHM) of CDR1 and CDR2 regions. This issue has been recently
addressed in a separate study (Bratsch et al., in press).
3.7. The CDR3 regions of VDJ transcripts resembles that of most
mammals
The CDR3 portion of transcribed VDJs mostly results from contributions by DH segments, differences in the 5 end of JH gene
segments, nucleotide additions and SHM. Table 3 summarizes data
from the most frequently encountered CDR3 regions transcribed
with different isotypes of the four bat species. Our data indicate
that the mean length of CDR3 was 9.1 ± 1.1 amino acids; the same
length found in mice, humans and swine. We found no significant
differences in CDR3 length among the isotype or the various species.
Identification of putative DH segments in CDR3 was not possible
because: (a) the same motif was never recovered twice among the
140 transcripts and (b) there is currently no information available
on the genomic DH repertoire of bats.
4. Discussion
Biologists have long recognized the importance that 20% of the
world’s mammals play in the ecosystem. They suppress aerial insect
populations, pollinate flowers and disperse seeds (Cleveland et al.,
2006; Kalka et al., 2008; Williams-Guillén et al., 2008; Shilton et
al., 1999; Fleming et al., 2009). Despite their ecological importance,
interest in their immune system has been almost entirely limited to
the Old World family Pteropodidae (suborder Yinpterochiroptera).
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Chakraborty and Chakravarty (1984) studied the immune system of
the Indian flying fox (Pteropus giganteus) using a hemolytic plaque
assay and (Omatsu et al. (2003) purified IgG from the Egyptian flying fox (Rousettus aegyptiacus, family Pteropodidae) and used it in
serological comparison with other mammals. In studies on histoplasmosis, McMurry et al. (1982) purified serum Igs from the New
World fruit bat, Artibeus lituratus and monitored changes during
infection. However, to our knowledge, no attention has been given
to the Igs of insectivorous bats in the suborder Yangochiroptera, and
no effort has been made to clone and characterize the heavy-chain
constant region Ig genes of any bat species.
The studies herein were undertaken for three reasons. First, by
addressing the growing concern for emerging diseases carried by
bats, we reasoned that an understanding of their immune system
might provide valuable insight that could indirectly aid in mitigating these diseases. Bats carry >85 viruses and are vectors for a
variety of human pandemic disease like SARS, Hendra and Ebola
(Calisher et al., 2006). WNS, a fungal disease of hibernating bats in
the eastern US (Blehert et al., 2009; Gargas et al., 2009), has caused
one of the most precipitous declines ever reported for North American wildlife, that is threatening regional extinction (Frick et al.,
2010). Second, we wished to determine whether bats might show
differences in their Ig repertoire or its formation from that seen
in other mammals. Finally, we reasoned that by comparing the Ig
genes of different bats species with those of other mammals, we
could improve our understanding of mammalian phylogeny in general and chiropteran phylogeny in particular. Various hypotheses
have been proposed linking bats with various taxa including primates, rodents, insectivores, carnivores and ungulates (Teeling et
al., 2002, 2005). A relatively recent hypothesis even proposed that
the two suborders (i.e. so-called mega- and microbats), evolved
from two separate lineages (Pettigrew, 1986)
Our findings are unambiguous in showing that Ig genes of bats
fit the canonical pattern of eutherian mammals, neither birds nor
proto- or protherian mammals. All four bat species examined in
the present study transcribed genes for IgM, IgG, IgA and IgE and
both insectivorous species of the suborder Yangochiroptera transcribed IgD (Figs. 1 and 2) with a domain structure characteristic
of eutherian (e.g. placental) mammals but not of Protheria (e.g.
monotremes) or lower vertebrates (e.g. amphibians and fish; Fig. 5).
We were unable to recover a transcript for IgD from either C. perspicillata or C. sphinx. IgD in mammals shows the least interspecific
homology among all Ig isotypes and has therefore been elusive to
recover (Butler, 2006; Butler et al., 1996). It is likely that when
genomic projects for bats other than M. lucifugus appear, IgD may
also be found in the genome of other bat species, with an altered
sequence and possibly as pseudogenes. Since IgD has been lost during evolution in some cases such as in birds, marsupials and even
the eutherian rabbit (Mage et al., 2006), its loss in one suborder of
bats may not be surprising.
The differences among mammals in the existence of the cysteine at position 15–18 (Fig. 1) in the CH1 domain that forms a
bridge to the light chain in human IgA, suggests that either bats,
human IgA2, cattle and swine: (a) have light chains that are noncovalently bonded to the C␣ chain or (b) the cysteine at position 77
(Fig. 1) is used in the L–H bridge. Because there is no biochemical
evidence of non-covalent L chains in swine and cattle, the latter
hypothesis is most likely. Our results suggest that in the diversification of mammalian IgA, the pathways diverged in C␣1 resulting
in the human being the only mammal outside of the exceptional
rabbit family, which has more than one subclass and retains both
motifs for covalent linkage to light chains.
Motifs for N-glycosylation are less likely to be conserved than
cysteine resides but may nevertheless distinguish evolutionary
pathways. In this respect, the pattern of N-glycosylation sites in
all bats studied to date resembles that of the Virginia opossum
and duckbill platypus, but not other mammals for which data are
available.
The diversification of IgG into subclasses is primarily a feature
of eutherian mammals; the Virginia opossum has only a single IgG
and the duckbill platypus has two very similar IgGs (Zhao et al.,
2009; Wang et al., 2009). The IgG-like Igs in other vertebrates have
only distant sequence similarities to those in eutherian mammals
(Fig. 3). The IgG-like IgY of the chicken and lizard also shows no
subclass diversification (Wei et al., 2009), although in the African
clawed frog (Xenopus sp.) a short Ig molecule, IgF, appears to have
been derived from IgY and further diversified in genus Xenopus
(Zhao et al., 2006). IgG has been regarded as the “flagship Ig” of
mammals and is not found in other vertebrates (Butler et al., 2009).
Thus, its presence in bats and in diversified form (subclasses) further supports the conclusion that bats possess the canonical isotype
repertoire of other eutherian mammals.
Also consistent with the pattern seen in eutherian mammals is
that post-speciation diversification of IgG into subclasses mostly
affects the sequence of the hinge exon (Butler et al., 2009). We
observed the same to be true in the Chiroptera. The hinge sequences
of the IgG variants from bats, together with that of other eutherian
mammals, are shown in Fig. 4. These data show that specific hinge
sequence motifs are often shared among the hinges of different IgGs
in one species but not shared between species. Seven variants were
recovered from a single specimen of M. lucifugus, so that assuming this individual was heterozygous, there must be 3–4 subclass
eugenes while the remainder could be alleles of these subclasses.
At this time only a single IgG has been recovered from Seba’s shorttailed fruit bat (family Phyllostomidae, suborder Yangochiroptera;
Fig. 3).
IgGs with extended hinges such as human and swine IgG3 are
best-suited for complement activation (Butler et al., 2009; Lefranc
and LeFranc, 2001). In humans all but IgG2 are actively transported
across the placenta (Lefranc and LeFranc, 2001) and IgG1 of cattle is selectively transported across the acinar epithelial cells of
the mammary gland (Hammer et al., 1969; Brandon et al., 1971).
Subclass IgGs also differ in their affinity for Fc␥R1, Fc␥RII and
Fc␥RIII that are distributed among phagocytic cells and lymphocytes, resulting in a further division of labor among subclasses
(Ravetch and Kinet, 1991).
Differential subclass expression can depend on whether a
pro- or anti-inflammatory cytokine microenvironment is present
(Mossman and Coffman, 1989). Based on the known division
of function among IgG subclasses in well-studied species, the
expressed subclass diversity we report here for bats predicts a similar diversification of function for bat IgGs like that seen among
human IgG subclasses and in other eutherian mammals.
The organization of the heavy-chain variable region appears
similar to that of other eutherian mammals. However, the low frequency of recovering a particular VH gene during a second attempt
in >70 chances in M. lucifugus (Table 2) suggests an exceptionally
diverse germline VH3 repertoire or a high rate of somatic hypermutation. The same applies to the recovery of putative DH segments
(Table 3). A separate study indicates that diversity is germline
encoded and not the result of extensive SHM (Bratsch et al., in
press).
As in laboratory mice, the rabbit family and humans, at least five
different FR4 regions were recovered suggesting as many as five JH
regions. Furthermore one of these is highly conserved since it was
found in all bat species examined in this study and was recovered
20 and 13 times respectively, in species from two different suborders (Table 2). CDR3 length was in the same range as in humans,
laboratory mice and swine with no evidence for the unusually long
CDR3 regions that are seen in cattle (Berens et al., 1997) or camels
(Nguyen et al., 2000). Data summarized in Tables 2 and 3 therefore
suggests a VH, DH and JH repertoire at least similar to humans and
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J.E. Butler et al. / Developmental and Comparative Immunology 35 (2011) 273–284
laboratory mice. These results are in stark contrast to swine (Sun
et al., 1998; Butler et al., 2006).
Regarding the issue of phylogenetic origins, our data offer no evidence to support the diphyletic origin of bats (Pettigrew, 1986) and
thus are consistent with the conclusions reached by others (Mindell
et al., 1991; Nikaida et al., 2000; Teeling et al., 2005). Regarding
their relationship to other mammalian orders, the sequence similarity of the genes encoding IgM and IgD may be most valuable
because these are regarded as phylogenetically the two oldest Ig
genes. Using this criterion, bats are closely related to carnivores
(Fig. 2). While sequence similarity of carnivores and bat IgD is
strong, the domain structure is more rodent-like due to the lack
of a CH2 domain (Fig. 5). Analogous with IgA, the hinge exon of bat
IgD is fused to the CH3 domain. In the case of IgD, this has not been
reported in other mammals. When genes for IgA and IgE are considered, a relationship with carnivores is still present but there is
also a relationship with hoofed mammals (e.g. ungulates), humans
and the dolphin (Fig. 2). Our data linking Chiroptera, Carnivora and
Perissodactyla (horses) with the Laurasiatheria is consistent with
studies using retroposon insertion (Nishihara et al., 2006) and noncoding intron sequences (Matthee et al., 2007). The relationship to
the Perissodactyla was also reported by Murphy et al. (2007). Thus,
from our analysis there is no evidence to support the hypothesis
that bats evolved from flying primates or rodents. Since IgG diversified following the appearance of bats (Fig. 3) sequence similarity
among eutherian mammals provides no insight into phylogeny but
suggest that IgG diversification is convergent.
Results from our study unambiguously support the hypothesis
that the Ig genes of both the Yinpteropodidae and the Yangochiroptera have the same canonical pattern of organization
as in other eutherian mammals, and thus are not in support of
a diphyletic origin for the Chiroptera. Their surprisingly diverse
variable heavy-chain diversity appears to be germline encoded
(Bratsch et al., in press). All other factors predict that the humoral
immune system of bats will function in much the same manner
as in well-studied species such as laboratory rodents. This means
that the humoral immune responses of bats to their pathogens,
the viruses they transmit and vaccines that may be developed, are
likely to follow the same pattern as in well-studied, non-volant
eutherian mammals.
Acknowledgement
Research supported in part by NIH Contract AI25489.
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