Intestinal Endocellular Symbiotic Bacterium of the Macaque Louse Pedicinus obtusus: Distinct Endosymbiont Origins in Anthropoid Primate Lice and the Old World Monkey Louse

A symbiotic bacterium of the macaque louse, Pedicinus obtusus, was characterized. The symbiont constituted a gammaproteobacterial lineage distinct from the symbionts of anthropoid primate lice, localized in the midgut epithelium and the ovaries and exhibiting AT-biased genes and accelerated molecular evolution. The designation “Candidatus Puchtella pedicinophila” was proposed for it.Sucking lice (Insecta: Phthiraptera: Anoplura), ectoparasitic insects that feed exclusively on the blood of their specific mammalian hosts, are generally associated with endosymbiotic bacteria (2, 13). Recent molecular studies have demonstrated that symbiotic bacteria of sucking lice are of multiple evolutionary origins (1, 8, 10, 16).From primates, three louse genera, Pediculus, Pthirus, and Pedicinus, have been recorded. Three Pediculus and two Pthirus species are known from anthropoid primates, harboring gammaproteobacterial “Candidatus Riesia spp.” in the stomach disc (1, 16). Meanwhile, 14 Pedicinus species, whose symbiotic bacteria have not been characterized, have been recorded from Old World monkeys (3).Samples of Pedicinus obtusus were collected in 2008 at the Jigokudani Monkey Park, Nagano, Japan, where a wild population of the Japanese macaque, Macaca fuscata, is exhibited to the public under careful control. When animals were caught and inspected, samples of P. obtusus were collected from the anesthetized animals under the supervision of a veterinary doctor (permission number 19-26-24, Nagano Prefecture, Japan). Samples of P. obtusus were collected in November 2008 by courtesy of Kiyoyasu Kowaki from a subspecies of the Japanese macaque, M. fuscata yakui, that is endemic in Yakushima Island, Kagoshima, Japan. Each of the insect samples was subjected to DNA extraction, and a 1.5-kb segment of the 16S rRNA gene (5) and a 1.6-kb segment of the groEL gene (7) were amplified by PCR, cloned, and sequenced. Molecular phylogenetic analyses were performed using the programs PAUP 4.0b10 (Sinauer Associates, Sunderland, MA), RAxML version 7.0.0 (17), and MrBayes 3.1.2 (15).The 1,490-bp 16S rRNA gene sequences formed a distinct lineage in the Gammaproteobacteria, exhibiting no phylogenetic affinity to the symbionts of other louse species, including those of human lice and the chimpanzee louse (see Fig. S1 in the supplemental material). The 1,601-bp groEL gene sequences also constituted a gammaproteobacterial lineage, exhibiting no phylogenetic affinity to the symbiont of human lice (Fig. ​(Fig.1)1) These results indicated that the endosymbiotic bacterium of the Old World monkey louse evolved independently of the endosymbiotic bacteria of the anthropoid primate lice. Considering that the date of divergence of Old World monkeys and anthropoid primates has been inferred as 23 to 31 million years ago, the endosymbiotic evolution in the primate lice must have occurred within this time scale (12).Open in a separate windowFIG. 1.groEL gene sequence-based molecular phylogenetic analysis of the symbiont of P. obtusus in the Gammaproteobacteria. A maximum-likelihood tree inferred from 1,040 unambiguously aligned nucleotide sites is shown. Bayesian and neighbor-joining analyses gave essentially the same results (data not shown). Statistical support values higher than 70% are indicated at the nodes in the order of maximum-likelihood/Bayesian/neighbor-joining values. Asterisks indicate statistical support values lower than 70%. Sequence accession numbers are shown in brackets. AT contents of the sequences are also shown. The sequences from the symbionts of human and monkey lice are highlighted in boldface. P-symbiont, primary symbiont; S-symbiont, secondary symbiont.The molecular evolutionary rates of the symbiont gene sequences were analyzed with a relative rate test using the program RRTree (14). The evolutionary rates of the 16S rRNA and groEL gene sequences in the lineage of the P. obtusus symbiont were significantly higher than those in the lineages of related free-living gammaproteobacteria (see Table S1 in the supplemental material).The AT contents of the 16S rRNA and groEL gene sequences of the P. obtusus symbiont were 53.5% and 64.8%, respectively. These values were equivalent to those of obligate endosymbionts of other insects (>50% for the 16S rRNA gene and >60% for the groEL gene) and were remarkably higher than those of allied free-living gammaproteobacteria (∼45% for the 16S rRNA gene and 45 to 50% for the groEL gene) (Fig. ​(Fig.1;1; see Fig. S1 in the supplemental material).Obligate endosymbiotic bacteria that cospeciate with their host insects commonly exhibit peculiar genetic traits, including AT-biased nucleotide composition, an accelerated rate of molecular evolution, and significant genome reduction (18). The AT-biased nucleotide composition and the accelerated evolution observed with the P. obtusus symbiont (Fig. ​(Fig.1;1; see Fig. S1 and Table S1 in the supplemental material) are suggestive of a stable and intimate host-symbiont association over evolutionary time.Fluorescent in situ hybridization targeting 16S rRNA of the symbiont was performed using the Alexa Fluor 555-labeled oligonucleotide probes Al555-ML439 (Al555-5′-ATAATATCTTCTTTCCTACCGA-3′) and Al555-ML1256 (Al555-5′-GCTAATTCTTGCGAATTTGCTT-3′) as described previously (9). In first-, second-, and third-instar nymphs, the symbiont signals were detected in the posterior half of the stomach in the abdomen (Fig. ​(Fig.2A).2A). In the posterior stomach, the signals exhibited a periodical and striated pattern (Fig. ​(Fig.2B).2B). Magnified images located the symbiont signals endocellularly in the intestinal wall tissue (Fig. ​(Fig.2C).2C). In some of the third-instar nymphs, the symbiont signals were found not only in the posterior stomach but also in the ovaries (Fig. ​(Fig.2A).2A). In adult females, the symbiont signals were restricted to the lateral oviducts (Fig. ​(Fig.2A),2A), where many bacteriocytes formed a pair of symbiotic organs called ovarial ampullae (Fig. ​(Fig.2D).2D). The ovarial ampullae were located adjacent to developing oocytes in the ovarioles, where the symbiont was passed to the developing eggs (Fig. ​(Fig.2E2E).Open in a separate windowFIG. 2.Localization of the symbiont in nymphs and adults of P. obtusus. (A) General localization of the symbiont in nymphal and adult insects. The symbiont signals are seen in the posterior stomach (green arrows) and the ovarial ampullae (yellow arrows). (B) An image of the posterior stomach of a second-instar nymph. Periodical and striped regions are densely populated by the symbiont. (C) An image of the posterior stomach of a third-instar nymph. The symbiont signals are restricted to the gut epithelial cells and not detected in the stomach lumen. Bacteriocytes are intercalated with uninfected cells, forming a striated pattern. (D) An enlarged image of the ovarial ampulla, consisting of a number of uninucleated bacteriocytes. (E) An image of the ovary in an adult female, wherein developing oocytes are found in the ovarioles. Ovarial ampullae (yellow arrows) are located at the anterior tip of the lateral oviducts, where symbiont transmission to oocytes occurs (white arrow). Panel A shows epifluorescent images, while panels B to E are confocal optical sectioning images. Red and blue signals reflect the symbiont 16S rRNA and the host nuclear DNA, respectively. Abbreviations: lu, stomach lumen; oc, oocyte.These results suggested that in third-instar female nymphs of P. obtusus, the symbiont localization drastically changes, from the posterior stomach to the ovarial ampullae (Fig. ​(Fig.2A).2A). Presumably, the endocellular symbiont escapes the host cells and somehow moves to the female reproductive organ, establishing a new endocellular association and finding a way to the next host generation. Interestingly, such symbiont migrations from a symbiotic organ to the ovaries in third-instar female nymphs have been reported for the human body louse Pediculus humanus (4, 13) and the slender pigeon louse Columbicola columbae (6, 13). Here it is notable that the symbiotic bacteria of P. obtusus, P. humanus, and C. columbae are phylogenetically not related to each other (see Fig. S1 in the supplemental material). The mechanisms underpinning the symbiont migration are currently unknown. Eberle and McLean (4) suggested by a series of experiments that the ovary of the human body louse might emanate a humoral factor that attracts the symbiotic bacteria to induce the migration. Whether or not this hypothesis is true deserves future experimental studies of these louse species and their symbiotic bacteria.On the basis of these results, we propose the designation “Candidatus Puchtella pedicinophila” for the novel endosymbiont lineage. The generic name honors Otto Puchta, who identified the biological role of the human louse symbiont as the provision of B vitamins (11). The specific name indicates association with a Pedicinus monkey louse. Whether the other monkey lice harbor symbiotic bacteria allied to the symbiont of P. obtusus deserves future studies.