Introduction

Insects represent the majority of eukaryotic biodiversity in the terrestrial ecosystem (Grimardi and Engel, 2005). One of the factors that have driven the prominent diversification may be their ability to use nutritionally incomplete or difficult food resources, such as woody material that is hard to digest and contains low nitrogen, plant sap that contains little proteins and lipids, vertebrate blood that is deficient in B vitamins, etc. In many, if not all, of these cases, it has been shown that symbiotic microorganisms have important roles in compensating for such nutritional deficiencies. In termites, for example, intestinal protozoans and bacteria enable their hosts to digest cellulose, and some bacteria are involved in nitrogen fixation (Breznak and Brune, 1994; Ohkuma, 2003). In aphids, an endocellular bacterium Buchnera aphidicola synthesizes essential amino acids that are scarce in their plant sap diet (Douglas, 1998; Shigenobu et al., 2000). In tsetse flies, an endocellular bacterium Wigglesworthia glossinidia provides B vitamins that are deficient in their blood meal (Nogge, 1981; Akman et al., 2002).

The feeding habit on vertebrate blood has evolved in some 14 000 insect species representing 5 orders, namely Siphonaptera (fleas), Phthiraptera (lice), Diptera (mosquitoes and flies), Hemiptera (bedbugs and kissing bugs) and Lepidoptera (an exceptional case of nocturid moth Calyptra eustrigata) (Adams, 1999; Lehane, 2005). Some of the insects such as mosquitoes and fleas feed on vertebrate blood only at their adult stage mainly for gonad maturation and egg production, whereas their larvae live on different food sources. On the other hand, lice, tsetse flies, bedbugs and others depend on vertebrate blood as the sole food source throughout their life. Notably, the latter insect groups generally possess a highly developed endosymbiotic system, consisting of specialized cells called bacteriocytes for harboring specific microbes and symbiotic organs called bacteriomes constituted by bacteriocytes and other types of cells. These patterns suggest the pivotal necessity of microbial associates for such insects that are highly adapted to obligate blood-feeding lifestyles (Buchner, 1965; Bourtzis and Miller, 2003).

Members of the dipteran superfamily Hippoboscoidea are highly specialized ectoparasitic insects, encompassing four family-level taxa: tsetse flies feeding on mammalian blood (Glossinidae), louse flies associated with birds or mammals (Hippoboscidae) and bat flies obligatorily ectoparasitic on bats (Streblidae and Nycteribiidae) (McAlpine, 1989). Hippoboscoid species are generally associated with bacterial endosymbionts, whereas their microbial partners and their symbiotic organs are remarkably different between the families (Roubaud, 1919; Zacharias, 1928; Aschner, 1931; Buchner, 1965). Among them, the endosymbiotic system in tsetse flies, consisting of an obligate endosymbiont W. glossinidia in a midgut-associated bacteriome (Aksoy, 1995) and a facultative endosymbiont Sodalis glossinidius (Dale and Maudlin, 1999), is the best studied, reflecting their medical importance as trypanosome vectors (Aksoy, 2000; Aksoy and Rio, 2005). The complete genome sequences of Wigglesworthia and Sodalis have already been determined (Akman et al., 2002; Toh et al., 2006). By contrast, much less attention has been paid to the endosymbiotic systems of hippoboscids, streblids and nycteribiids (Dale et al., 2006; Trowbridge et al., 2006; Nováková et al., 2009), except for early histological descriptions (Roubaud, 1919; Zacharias, 1928; Aschner, 1931; Buchner, 1965).

Bat flies of the family Nycteribiidae are obligate ectoparasites of bats with strikingly specialized morphological traits, including lack of wings, reduced head and eyes, dorsoventrally flattened thorax, dorsally inserted legs, spider-like appearance due to these morphologies and adenotrophic viviparity with a highly developed uterus and milk glands (Lehane, 2005; Dick and Patterson, 2006). On account of their extreme specialization for ectoparasitic and blood-feeding lifestyle, intimate endosymbiotic associations may be expected for nycteribiid bat flies. However, previous works on the subject are quite limited. Aschner (1931) histologically inspected three nycteribiid species, namely Nycteribia biarticulata, Nycteribia blassi and Nycteribia blainvillei, and observed the presence of endosymbiotic bacteria in bacteriocytes that formed loose clusters in the abdominal body cavity in both males and females, and also in the cavity of milk gland tubules in females. Aschner (1946) also inspected a distinct nycteribiid species Eucampsipoda aegyptia, wherein endosymbiotic bacteria were restricted to milk gland tubules. Recently, Nováková et al. (2009) provided the first molecular characterization of nycteribiid endosymbionts. Three nycteribiid species, Nycteribia kolenati, Penicillidia monoceros and Penicillidia sp., were subjected to cloning and sequencing of the bacterial 16S rRNA gene, which yielded two groups of gammaproteobacterial sequences: a group of sequences were placed within the clade of Arsenophonus spp. that are facultative endosymbionts associated with diverse insects; and another group of sequences formed a distinct clade, which exhibited phylogenetic affinity to the clade of Riesia spp., obligate endosymbionts of primate lice and also to the clade of Arsenophonus spp. (Nováková et al., 2009). However, the early histological data and the recent molecular data are currently not connected to each other, and should be integrated into a coherent picture. It is obscure which of the bacterial clades detected from the nycteribiids correspond to the endosymbionts harbored in bacteriocytes and those located in the milk glands, respectively. Co-evolutionary aspects between the endosymbionts and their nycteribiid hosts are largely unknown and yet to be established.

In this study, we investigated Japanese nycteribiid bat flies, which represented 4 genera, 8 species and 27 populations, for their bacterial endosymbionts. We identified a distinct clade of gammaproteobacterial endosymbionts from all the nycteribiid species as reported previously (Nováková et al., 2009), and also unveiled a number of previously unknown aspects in the nycteribiid–bacterium association, including the discovery of a pair of large bacteriomes in the abdomen of a Penicillidia species, detailed in vivo localization and ultrastructure of the endosymbiont, remarkable reductive evolutionary patterns in the endosymbiont genome and strict host–symbiont co-speciation.

Materials and methods

Insect materials

Nycteribiid bat flies were collected by bat ecologists in their field surveys and provided for this study (Supplementary Table S1; Supplementary Figure S1). Most of the samples were preserved in acetone upon collection for DNA analyses (Fukatsu, 1999). Some insects of Penicillidia jenynsii were anesthetized by chilling on ice, dissected and subjected to either in situ hybridization, electron microscopy or pulsed-field gel electrophoresis.

DNA extraction and morphological inspection

Acetone-preserved bat flies were individually subjected to DNA extraction and morphological inspection. Each fixed insect was dried in air, and its abdomen was detached using forceps under a dissection microscope. The insect parts were placed in a 1.5-ml plastic tube and digested in 200 μl of a proteinase K-containing lysis buffer supplied with the QIAamp tissue mini kit (Qiagen, Venlo, The Netherlands) at 55 °C overnight. The lysate was subjected to DNA extraction using the kit, while the resultant exoskeleton was thoroughly washed with distilled water, stored in 70% ethanol and observed under a dissection microscope.

Cloning, genotyping and sequencing

Bacterial 16S rRNA, groEL and gyrB genes were amplified by PCR from the DNA samples, and subjected to cloning, restriction fragment length polymorphism (RFLP) genotyping using restriction endonuclease HaeIII, and DNA sequencing as described previously (Kikuchi et al, 2009). Insect mitochondrial COI (cytochrome oxidase I) and 16S rRNA genes were also amplified by PCR and subjected to direct sequencing. The primers and PCR conditions are summarized in Supplementary Table S2.

Molecular phylogenetic, molecular evolutionary and genomic analyses

Multiple alignments of the nucleotide sequences were generated using the program Clustal W (Thompson et al., 1994). Phylogenetic analyses were conducted by neighbor-joining, maximum parsimony and maximum likelihood methods, using the programs Clustal W (Thompson et al., 1994), PAUP 4.0b10 (Swofford, 2001) and Phyml (Guindon and Gascuel, 2003), respectively. Relative rate tests were performed using the program RRTree (Robinson-Rechavi and Huchon, 2000). Co-evolutionary analyses were performed under the jungles algorithm using the program TreeMap v2.02β, which evaluates the extent of topological congruence between phylogenies (Charleston and Page, 2002). Randomization tests were conducted as described previously (Hosokawa et al., 2006). Pulsed-field gel electrophoresis of the endosymbiont genome was performed with the bacteriome samples dissected from 50 adult females of P. jenynsii. The tissues were gently homogenized in 50 μl of phosphate-buffered saline containing 10 mM EDTA, and the homogenate was filtered through a 10-μm nylon mesh, and the filtered suspension was mixed with 1% low-melting point agarose. The agarose plug was cut into an appropriate shape and size, and treated with proteinase K at 50 °C overnight. After thorough washing, the plug was subjected to digestion with restriction endonuclease I-CeuI. Pulsed-field gel electrophoresis was conducted using CHEF Mapper XA (Bio-Rad, Hercules, CA, USA).

Histological procedures

Insects were dissected and fixed in Carnoy's solution (ethanol–chloroform–acetic acid, 6:3:1) overnight, and treated with 6% hydrogen peroxide in 80% ethanol for 2 days to reduce autofluorescence of tissues (Koga et al., 2009). The dissected tissues were thoroughly washed with 100% ethanol and then with phosphate-buffered saline, and subjected to in situ hybridization with fluorochrome-labeled oligonucleotide probes targeting 16S rRNA of the endosymbionts, Al555-BF440 (5′-TTAATKCCTTCCTCACAACTG-3′) and Al555-BF1256 (5′-TCCCATCGCTGGCTAGCCT-3′), as described previously (Kikuchi et al., 2009). For transmission electron microscopy, insects were dissected with fine forceps in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde, and isolated tissues were processed into ultra-thin sections and observed as described previously (Fukatsu et al., 2000).

Results

16S rRNA genes from nycteribiid bat flies P. jenynsii and Nycteribia allotopa

During an ecological monitoring of a natural bat population at Sayo-cho (Hyogo, Japan), two bat fly species, larger P. jenynsii and smaller N. allotopa (Figure 1; Supplementary Table S1), were collected from the Eastern bent-winged bats Miniopterus fuliginosus. Two individuals of P. jenynsii and two individuals of N. allotopa were subjected to DNA extraction and PCR amplification of bacterial 16S rRNA genes. PCR products were cloned, and 30 clones per insect were subjected to RFLP genotyping. All clones from N. allotopa exhibited an identical RFLP genotype, whereas clones from P. jenynsii consisted of a major frequent genotype and a minor infrequent genotype. For each of the RFLP genotypes, three clones per insect were sequenced. All sequences of the major genotype from P. jenynsii were 1464 bp in size and completely identical to each other, exhibiting the BLAST top hit to 16S rRNA gene sequence of Arsenophonus endosymbiont from scale insect Australiococcus greville (92.6% (1358/1467) sequence identity; accession number AY264673). When ∼1 kb region of the sequence corresponding to the sequenced region of the 16S rRNA gene reported by Nováková et al. (2009) was subjected to the BLAST search, the higher hit was identified as the 16S rRNA gene sequence of endosymbiont from nycteribiid bat fly P. monoceros (97.2% (1030/1060) sequence identity; accession number FJ265817). Meanwhile, all sequences from N. allotopa were 1473 bp in size and almost completely identical to each other, but a single nucleotide site between the two individuals, exhibiting the BLAST top hit to the 16S rRNA gene sequence of Arsenophonus endosymbiont from citrus psyllid Diaphorina citri (92.5% (1362/1473) sequence identity; accession number AB038366). When ∼1 kb region of the sequence corresponding to the sequenced region of the 16S rRNA gene reported by Nováková et al. (2009) was subjected to the BLAST search, the higher hit was identified as the 16S rRNA gene sequence of endosymbiont from nycteribiid bat fly N. kolenatii (97.2% (1039/1069) sequence identity; accession number FJ265804). All sequences of the minor genotype from P. jenynsii were 1426 bp in size and identical to each other, exhibiting the BLAST top hit to the 16S rRNA gene sequence of Wolbachia endosymbiont from louse fly Pseudolynchia carariensis (99.6% (1420/1425) sequence identity; accession number DQ115537).

Figure 1
figure 1

Nycteribiid bat flies, Penicillidia jenynsii (top) and Nycteribia allotopa (bottom). Acetone-preserved specimens are shown.

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Phylogenetic placement of bat fly endosymbionts

Figure 2 shows the phylogenetic placement of the endosymbionts of P. jenynsii and N. allotopa in the Gammaproteobacteria on the basis of their 16S rRNA gene sequences. The endosymbiont sequences formed a robust clade, supported by 100% bootstrap values, together with 16S rRNA gene sequences that had previously been obtained from other nycteribiid bat flies (Nováková et al., 2009). It was notable that the phylogenetic relationship of the bacterial sequences reflected the systematics of bat fly hosts: the sequences from Penicillidia spp., namely P. jenynsii, P. monoceros and P. sp., formed a clade, whereas the sequences from Nycteribia spp., namely N. allotopa and N. kolenatii, constituted a sister clade to it. Adjacent to the endosymbiont clade of the nycteribiid bat flies, endosymbionts of primate lice, Riesia spp., formed a sister clade. Adjacent to them, endosymbionts of a hippoboscid fly Lipoptena sp. and streblid bat flies Trichobius spp. formed a clade. Allied to them was a clade of facultative Arsenophonus endosymbionts associated with diverse insects (Nováková et al., 2009). All these Arsenophonus-allied endosymbionts as a whole constituted a robust clade, supported by 100% bootstrap values, in the Gammaproteobacteria.

Figure 2
figure 2

Phylogenetic placement of the endosymbionts of nycteribiid bat flies Penicillidia jenynsii and Nycteribia allotopa in the Gammaproteobacteria on the basis of 16S rRNA gene sequences. A neighbor-joining tree inferred from a total of 973 aligned nucleotide sites is shown, whereas maximum parsimony and maximum likelihood analyses produced substantially the same results. Bootstrap values no less than 50% are indicated at the nodes in the order of neighbor-joining/maximum parsimony/maximum likelihood, whereas asterisks indicate support values less than 50%. Sequence accession numbers and AT contents of the nucleotide sequences are in brackets and parentheses, respectively. As for insect symbionts, the information of the host insect may also be indicated in parentheses. P-symbiont, primary symbiont of prevalent infection and obligatory nature; S-symbiont, secondary symbiont of sporadic infection and facultative nature.

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In vivo localization and fine structure of bat fly endosymbionts

To identify in vivo localization of bat fly endosymbionts, adult insects of N. allotopa and P. jenynsii were subjected to whole-mount in situ hybridization with fluorochrome-labeled probes specifically targeting the endosymbiont 16S rRNA.

In the abdomen of adult insects of N. allotopa, signals of the endosymbiont were detected in clusters of small bacteriocytes (Supplementary Figure S2). The localization patterns of bacteriocytes were similar to those previously observed in N. biarticulata and N. blasii (Aschner, 1931).

In the abdomen of adult males of P. jenynsii (Figure 3a), signals of the endosymbiont were detected in clusters of small bacteriocytes just beneath the cuticle, the most densely on both sides of the second, third, fourth and fifth abdominal segments and less densely under the ventral plates (Figures 3b and c). The localization patterns of bacteriocytes were a little different, but not so divergent from those observed in Nycteribia spp. (Aschner, 1931; see Supplementary Figure S2). The bacteriocytes were ∼20 μm in diameter, their nucleus was located on the periphery of the cells, and their cytoplasm was filled with numerous endosymbiont cells (Figure 3d). In the male reproductive organs, few signals of the endosymbiont were detected (Figure 3e).

Figure 3
figure 3

In vivo localization of the endosymbiont in adult males of Penicillidia jenynsii. (a) An external ventral view of the abdomen of an adult male. Inset is an image of the whole body. (b–e) Visualization of the endosymbiont by 16S rRNA-targeted fluorescent in situ hybridization. Red signals indicate the endosymbiont, whereas blue signals show host insect nuclei. (Panel b) A ventral view of the abdomen, wherein dense clusters of bacteriocytes are located on both sides of the third, fourth and fifth abdominal segments (arrows), and less dense clusters are also seen under the ventral plates (arrowheads). (Panel c) A ventral view of the dissected abdomen, the ventral plates of which are cut and opened. (Panel d) An enlarged image of the bacteriocytes. (Panel e) Dissected reproductive organs of a male, wherein few signals of the endosymbiont are seen. ag, accessory gland; ed, ejaculatory duct; mt, Malphigian tubule; rc, rectum; tes, testis; vs, vesicula seminalis.

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In the abdomen of adult females of P. jenynsii (Figure 4a), strikingly, the endosymbiont exhibited localization patterns quite different from those in males. In the female abdomen, the fifth ventral plate was well developed and equipped with a pair of swellings on both sides (Figure 4a, arrows). In situ hybridization identified very strong signals inside the swellings, where a pair of large bacteriomes consisting of a number of bacteriocytes was located (Figure 4b). The bacteriocytes were, like those in males, ∼20 μm in diameter with a peripheral nucleus and full of numerous endosymbiont cells in the cytoplasm (Figure 4c). The abdominal cavity of adult females was mostly occupied by highly developed reproductive organs consisting of a uterus encasing a developing larva inside, a few immature oocytes, and highly branched milk glands for supplying food secretion to the developing larva (Figures 4d and e). Interestingly, signals of the endosymbiont were detected inside the milk gland tubules (Figures 4e and f), indicating the presence of endosymbiont cells in milk gland secretion.

Figure 4
figure 4

In vivo localization of the endosymbiont in adult females of Penicillidia jenynsii. (a) An external ventral view of the abdomen of an adult female. Arrows indicate swellings of the fifth ventral plate. Inset is an image of the whole body. (b–f) Visualization of the endosymbiont by 16S rRNA-targeted fluorescent in situ hybridization. Red signals indicate the endosymbiont, whereas blue signals show host insect nuclei. (Panel b) A ventral view of the abdomen, in which a pair of large bacteriomes are visualized inside the swellings of the fifth ventral plate (arrows). (Panel c) An enlarged image of bacteriocytes in the bacteriome. (Panel d) A female abdomen full of a uterus and entwined milk gland tubules. (Panel e) Dissected reproductive organs of a female, consisting of a large uterus, a few immature oocytes, as well as huge and highly branched milk glands. It must be noted that signals of the endosymbiont are seen inside the milk gland tubules. Arrows indicate the bacteriomes fragmented during dissection. Red color on the uterus is due to autofluorescence. (Panel f) An enlarged image of the milk gland tubules. o, oocyte; u, uterus.

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Electron microscopy revealed numerous tubular bacterial cells packed in the cytoplasm of bacteriocytes (Figures 5a and b), and confirmed the presence of endosymbiont cells in milk gland secretion (Figure 5c).

Figure 5
figure 5

Transmission electron microscopy of the symbiotic organs in adult females of Penicillidia jenynsii. (a) Bacteriocytes in a female bacteriome, the cytoplasm is filled with endosymbiont cells. (b) An enlarged image of the endosymbiont cells. (c) An image of the milk gland epithelium with well-developed rough endoplasmic reticula, which is indicative of its high secretory activity, and the milk gland secretion containing endosymbiont cells in the inner cavity of the organ. In panels b and c, asterisks indicate endosymbiont cells. N, nucleus; MGS, milk gland secretion; RER, rough endoplasmic reticulum.

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Molecular phylogenetic analyses of nycteribiid bat flies

In an attempt to understand the evolution of the host–symbiont association in the Nycteribiidae, we collected 41 individuals of nycteribiid bat flies representing 4 genera, 8 species and 27 populations in Japan, and several endosymbiont genes and host insect genes were cloned and sequenced from them (Supplementary Table S1; Supplementary Figure S1). Here, we focused on the bacteriocyte-associated endosymbiont, and thus sequenced clones of the major RFLP genotype for each of the insect samples. On the basis of these sequence data, we conducted a series of molecular phylogenetic analyses.

Phylogenetic analyses of endosymbiont genes

Phylogenetic relationships of endosymbionts inferred from the nucleotide sequences of the 16S rRNA gene (Supplementary Figure S3), gyrB gene (Supplementary Figure S4) and groEL gene (Supplementary Figure S5) consistently agreed with the systematics of the host insects. Supplementary Figure S6 shows the phylogenetic tree based on the combined sequence data of the three genes. The endosymbiont sequences from the same host species were completely or nearly identical to each other, and therefore sequences representing each of the host species formed a distinct monophyletic group. Moreover, the endosymbiont sequences from each of the same host genera, namely Nycteribia, Basilia and Penicillidia, constituted a well-supported monophyletic group.

Phylogenetic analyses of host insect genes

Phylogenetic relationships of the host insects inferred from the nucleotide sequences of the mitochondrial COI gene (Supplementary Figure S7) and the 16S rRNA gene (Supplementary Figure S8) reflected the host insect systematics. Supplementary Figure S9 shows the phylogenetic tree based on the combined sequence data of the two genes. Sequences of the same insect species were closely related to each other, and therefore sequences representing each of the insect species formed a distinct monophyletic group. Furthermore, the sequences from each of the same host genera, namely Nycteribia, Basilia and Penicillidia, constituted a well-supported monophyletic group.

Phylogenetic analysis of host–symbiont co-evolution in nycteribiid bat flies

Figure 6 contrasts the phylogeny of bat fly species and of their endosymbionts. Strikingly, the phylogenetic relationship of the host insects was almost in complete concordance with the phylogenetic relationship of their endosymbionts. Although the placement of Phthiridium hindlei was slightly different between the phylogenies, the bootstrap supports for it were extremely poor in the endosymbiont phylogeny. Given that the placement of Phthiridium hindlei was treated as multi-furcated, the endosymbiont phylogeny was perfectly congruent with the host phylogeny. On account of a total of 135 135 possible rooted tree topologies for 8 taxa, the chance that the symbiont tree will exactly match the host tree is expected to be <0.001%. The congruence was so complete that the jungles algorithm identified only one reconstruction of the co-evolutionary history with six co-divergence events (Figure 6). A randomization test also confirmed that such a high level of phylogenetic congruence is quite unlikely to occur by chance (P=0.002).

Figure 6
figure 6

Comparison between the phylogeny of the nycteribiid bat fly species and the phylogeny of their endosymbionts. (a) A neighbor-joining phylogeny of the host insect species on the basis of the data set of Supplementary Figure S9. (b) A neighbor-joining phylogeny of their endosymbionts on the basis of the data set of Supplementary Figure S6. Dots on the nodes indicate co-divergence events inferred by the jungles algorithm (Charleston and Page, 2002). Bootstrap values are indicated as in Figure 2.

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Molecular evolutionary and genomic aspects of the bat fly endosymbionts

Nucleotide compositions of the 16S rRNA gene of the bat fly endosymbionts were adenine/thymine (AT) rich, ranging from 50.3% to 51.2%, in comparison with those of allied facultative Arsenophonus endosymbionts ranging from 45.8% to 47.0% (Figure 2). Evolutionary rates of the 16S rRNA, gyrB and groEL genes of the bat fly endosymbionts were significantly higher than allied facultative Arsenophonus endosymbionts (Table 1). Pulsed-field gel electrophoresis of the bacteriomes of P. jenynsii estimated the genome size of the endosymbiont to be 0.76 Mb (Supplementary Figure S10).

Table 1 Relative rate test for comparing the molecular evolutionary rates of 16S rRNA, gyrB and groEL gene sequences between the lineage of nycteribiid endosymbionts and the lineage of facultative Arsenophonus endosymbionts
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Discussion

Host–symbiont co-evolution in nycteribiid bat flies

Among diverse insect endosymbionts of obligate nature, such as Buchnera of aphids (Moran et al., 1993), Wigglesworthia of tsetse flies (Chen et al., 1999), Carsonella of psyllids (Thao et al., 2000), Portiera of whiteflies (Thao and Baumann, 2004), Sulcia of many homopterans (Moran et al., 2005), Baumannia of sharpshooters (Takiya et al., 2006), Blochmannia of carpenter ants (Sauer et al., 2000), Nardonella of weevils (Conord et al., 2008) and others, the endosymbiont phylogeny generally mirrors the host phylogeny, indicating stable and intimate host–symbiont association over evolutionary time. Our results unequivocally indicate that the endosymbiont of nycteribiid bat flies has experienced a similar co-evolutionary history (Figure 6). Vertical symbiont transmission through host generations should facilitate host–symbiont co-speciation. The presence of the endosymbiont cells in the milk glands (Figures 4e and f; Figure 5c) strongly suggests a uterine transmission route of the nycteribiid endosymbiont, wherein the endosymbiont is vertically passed from mother to offspring through milk gland secretion in the uterus, as has been reported from tsetse flies and other hippoboscoids (Buchner, 1965; Attardo et al., 2008). The family Nycteribiidae consists of 3 subfamilies Nycteribiinae (7 genera, 210 species), Cyclopodinae (4 genera, 62 species) and Archinycteribiinae (1 genus, 3 species) (Dick and Patterson, 2006). All nycteribiid bat flies examined in this study (four genera, eight species) belong to the Nycteribiinae, suggesting that the origin of the host–symbiont association could be dated back to the common ancestor of the subfamily Nycteribiinae.

Reductive genome evolution of the nycteribiid endosymbiont

The AT-biased nucleotide composition (Figure 2), the accelerated molecular evolution (Table 1) and the reduced genome size (Supplementary Figure S10) consistently indicate that the nycteribiid endosymbiont has experienced a remarkable level of reductive genome evolution. The estimated genome size, 0.76 Mb, is similar to genome sizes of obligate insect endosymbionts like Buchnera of aphids (∼0.6 Mb) (Shigenobu et al., 2000) and Wigglesworthia of tsetse flies (∼0.7 Mb) (Akman et al., 2002), and remarkably smaller than the genome of allied facultative endosymbiont Arsenophonus nasoniae (∼3.2 Mb) (Wilkes et al., 2010), as well as the genomes of free-living gammaproteobacteria like Escherichia coli (∼4.6 Mb) (Blattner et al., 1997) and Vibrio cholerae (∼4.0 Mb) (Heidelberg et al., 2000). Such evolutionary patterns are probably attributable to the stable and nutrition-rich endocellular habitat for the endosymbiont and also attenuated purifying selection due to small population size and strong bottleneck associated with the lifestyle of the vertically transmitted endosymbiont (Wernegreen, 2002; Moran et al., 2008).

Evolutionary origin of the nycteribiid endosymbiont

Molecular phylogenetic analyses revealed that the nycteribiid endosymbionts form a robust clade in the Gammaproteobacteria, and the clade is certainly distinct but allied to the clade of Arsenophonus spp., which embrace facultative endosymbionts of diverse insects (Figure 2). These patterns suggest that the evolutionary origin of the obligate nycteribiid endosymbiont might be an Arsenophonus-like facultative endosymbiont. In this context, it seems meaningful that facultative Arsenophonus endosymbionts have been detected from several Nycteribia species in addition to the obligate endosymbionts (Nováková et al., 2009). The obligate bacteriome-associated endosymbionts of primate lice, Riesia spp., are also allied to the clade of Arsenophonus spp. (Sasaki-Fukatsu et al., 2006; Allen et al., 2007), which may represent a similar evolutionary trajectory from facultative to obligate endosymbiotic association. In the bedbug Cimex lectularius, its obligate bacteriome-associated endosymbiont was shown to have evolved from a Wolbachia lineage (Hosokawa et al., 2010), the members of which are commonly found in diverse insects as facultative and/or parasitic endosymbiotic associates (Bourtzis and Miller, 2003; Werren et al., 2008).

Commonality and variability of the endosymbiotic system among nycteribiid bat flies

A previous study (Aschner, 1931) identified loose clusters of bacteriocytes in the abdominal body cavity of bat flies of the genus Nycteribia, which was confirmed by our histological observation of N. allotopa (Supplementary Figure S2). In P. jenynsii, by contrast, we discovered a previously unknown type of symbiotic organ: in adult females, many bacteriocytes constitute a pair of distinct bacteriomes inside the swellings of the fifth abdominal ventral plate (Figure 4). These observations suggest that different symbiotic organs have evolved in the subfamily Nycteribinae, whereas the endosymbionts harbored therein have been conserved. Meanwhile, endosymbiont localization in the milk glands has been consistently observed among all nycteribiid bat flies ever examined histologically: Nycteribia spp., P. jenynsii and E. aegyptia (Aschner, 1931, 1946; Figure 4), which should be relevant to the importance of uterine vertical transmission of the endosymbiont for the host bat flies.

Relationship to endosymbionts of other hippoboicoid families

The families Glossinidae (tsetse flies), Hippoboscidae (louse flies), Streblidae (sterblid bat flies) and Nycteribiidae (nycteribiid bat flies) form a well-defined superfamily Hippoboscoidea (McAlpine, 1989; Nirmala et al., 2001; Petersen et al., 2007), all members of which are obligate blood feeders and commonly associated with bacterial endosymbionts (Roubaud, 1919; Zacharias, 1928; Aschner, 1931; Buchner, 1965), although the endosymbionts are microbiologically distinct between the host families (see Figure 2). Tsetse flies are associated with W. glossinidia, which is phylogenetically not related to Arsenophonus spp. in the Gammaproteobacteria (Aksoy, 1995). Meanwhile, diverse Arsenophonus-related endosymbionts have been detected from louse flies and stereblid bat flies (Dale et al., 2006; Trowbridge et al., 2006; Nováková et al., 2009), which are certainly allied to but distinct from the clade of nycteribiid endosymbionts (Figure 2; Nováková et al., 2009). These patterns favor the hypothesis that the hippoboscoid endosymbionts are of multiple evolutionary origins and have experienced repeated acquisitions and/or replacements from different bacterial sources to meet their physiological requirement for their obligate blood-feeding lifestyle.

Biological function of the endosymbiont for nycteribiid bat flies

On account of the long-lasting host–symbiont association in the Nycteribiidae (Figure 6), it seems plausible that the endosymbiont has important biological roles for its nycteribiid host, although its exact function is yet to be established. As for other endosymbionts of obligate blood-feeding insects such as Wigglesworthia of tsetse fly, Riesia of human body louse and Wolbachia of bedbug, their biological function has been demonstrated as provisioning of B vitamins that are deficient in vertebrate blood (Puchta, 1955; Nogge, 1981; Hosokawa et al., 2010). It is conceivable that the nycteribiid endosymbiont has a similar role for the host insect. Endosymbiont genome analyses revealed that biosynthetic genes for B vitamins are encoded in the main chromosome of Wigglesworthia (Akman et al., 2002) while present in a plasmid of Riesia (Kirkness et al., 2010). Sequencing of the genome of the nycteribiid endosymbiont will provide a crucial insight into its biological function, in particular involvement in provisioning of B vitamins.

Proposal of candidate name

On account of those distinct and coherent microbiological, phylogenetic and evolutionary traits described above, we propose the designation ‘Candidatus Aschnera chinzeii’ for the endosymbiotic bacterial clade associated with bat flies of the family Nycteribiidae. The generic name honors Manfred Aschner, who first described the endosymbionts of nycteribiid bat flies (Aschner, 1931, 1946). The specific name is after a Japanese biologist Yasuo Chinzei, who significantly contributed to the biochemistry and molecular biology of blood-sucking insects (Yuda et al., 1996; Kadota et al., 2004).