Evolutionary Relationships of “Candidatus Riesia spp.,” Endosymbiotic Enterobacteriaceae Living within Hematophagous Primate Lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “Candidatus Arsenophonus insecticola.” We propose that two additional species be added to the genus “Candidatus Riesia.” The new species proposed within “Candidatus Riesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.     

Assessment of bacterial endosymbiont diversity in <Emphasis Type="Italic">Otiorhynchus</Emphasis>spp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

BACKGROUND: Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species. RESULTS: High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of "Candidatus Blochmannia" and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and "Candidatus Blochmannia", we identified a new phylogenetic clade of Rickettsia as well as "Candidatus Nardonella" endosymbionts in Otiorhynchus spp. which are closely related to "Candidatus Blochmannia" bacteria. CONCLUSIONS: Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and "Candidatus Nardonella" endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.     

Molecular and histological characterization of primary (betaproteobacteria) and secondary (gammaproteobacteria) endosymbionts of three mealybug species

Microscopic localization of endosymbiotic bacteria in three species of mealybug (Pseudococcus longispinus, the long-tailed mealybug; Pseudococcus calceolariae, the citrophilus mealybug; and Pseudococcus viburni, the obscure mealybug) showed these organisms were confined to bacteriocyte cells within a bacteriome centrally located within the hemocoel. Two species of bacteria were present, with the secondary endosymbiont, in all cases, living within the primary endosymbiont. DNA from the dissected bacteriomes of all three species of mealybug was extracted for analysis. Sequence data from selected 16S rRNA genes confirmed identification of the primary endosymbiont as "Candidatus Tremblaya princeps," a betaproteobacterium, and the secondary endosymbionts as gammaproteobacteria closely related to Sodalis glossinidius. A single 16S rRNA sequence of the primary endosymbiont was found in all individuals of each mealybug species. In contrast, the presence of multiple divergent strains of secondary endosymbionts in each individual mealybug suggests different evolutionary and transmission histories of the two endosymbionts. Mealybugs are known vectors of the plant pathogen Grapevine leafroll-associated virus 3. To examine the possible role of either endosymbiont in virus transmission, an extension of the model for interaction of proteins with bacterial chaperonins, i.e., GroEL protein homologs, based on mobile-loop amino acid sequences of their GroES homologs, was developed and used for analyses of viral coat protein interactions. The data from this model are consistent with a role for the primary endosymbiont in mealybug transmission of Grapevine leafroll-associated virus 3.     

Isolation, pure culture, and characterization of "Candidatus Arsenophonus arthropodicus," an intracellular secondary endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis

Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as "Candidatus Arsenophonus arthropodicus." Phylogenetic analyses indicate that "Candidatus Arsenophonus arthropodicus" is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of "Candidatus Arsenophonus arthropodicus" in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.     

Symbiotic bacteria associated with stomach discs of human lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation "Candidatus Riesia pediculicola" for the louse symbionts.     

Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement

Intracellular symbiosis is widespread in the insect world where it plays an important role in evolution and adaptation. The weevil family Dryophthoridae (Curculionoidea) is of particular interest in intracellular symbiosis evolution with regard to the great economical and ecological features of these invasive insects, and the potential for comparative studies across a wide range of host plants and environments. Here, we have analyzed the intracellular symbiotic bacteria of 19 Dryophthoridae species collected worldwide, representing a wide range of plant species and tissues. All except one (Sitophilus linearis) harbor symbiotic bacteria within specialized cells (the bacteriocytes) assembled as an organ, the bacteriome. Phylogenetic analysis of the 16S rDNA gene sequence of the Dryophthoridae endosymbionts revealed three endosymbiotic clades belonging to gamma3-Proteobacteria and characterized by different GC contents and evolutionary rate. The genus name Candidatus Nardonella was proposed for the ancestral clade infesting Dryophthoridae 100 MYA and represented by five of nine bacterial genera studied. For this clade showing low GC content (40.5% GC) and high evolutionary rate (0.128 substitutions/site per 100 Myr), a single infection and subsequent cospeciation of the host and the endosymbionts was observed. In the two other insect lineage endosymbionts, with relatively high GC content (53.4% and 53.8% GC), competition with ancestral pathogenic bacteria might have occurred, leading to endosymbiont replacement in present-day last insects.     

Genetic analysis of lice supports direct contact between modern and archaic humans

Parasites can be used as unique markers to investigate host evolutionary history, independent of host data. Here we show that modern human head lice, Pediculus humanus, are composed of two ancient lineages, whose origin predates modern Homo sapiens by an order of magnitude (ca. 1.18 million years). One of the two louse lineages has a worldwide distribution and appears to have undergone a population bottleneck ca. 100,000 years ago along with its modern H. sapiens host. Phylogenetic and population genetic data suggest that the other lineage, found only in the New World, has remained isolated from the worldwide lineage for the last 1.18 million years. The ancient divergence between these two lice is contemporaneous with splits among early species of Homo, and cospeciation analyses suggest that the two louse lineages codiverged with a now extinct species of Homo and the lineage leading to modern H. sapiens. If these lice indeed codiverged with their hosts ca. 1.18 million years ago, then a recent host switch from an archaic species of Homo to modern H. sapiens is required to explain the occurrence of both lineages on modern H. sapiens. Such a host switch would require direct physical contact between modern and archaic forms of Homo.     

Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.     

Cospeciation of psyllids and their primary prokaryotic endosymbionts

Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity. Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies. In addition, 0.54-kb fragments of the psyllid nuclear gene wingless were also amplified from 26 species. Phylogenetic trees derived from 16S-23S rDNA and from the host wingless gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies. This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont. In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology. The 3' end of the 16S rDNA of the P endosymbionts differs from that of other members of the domain Bacteria in the lack of a sequence complementary to the mRNA ribosome binding site. The rate of sequence change in the 16S-23S rDNA of the psyllid P endosymbiont was considerably higher than that of other bacteria, including other fast-evolving insect endosymbionts. The lineage consisting of the P endosymbionts of psyllids was given the designation Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.).     

Nuclear Genetic Diversity in Human Lice (Pediculus humanus) Reveals Continental Differences and High Inbreeding among Worldwide Populations

Understanding the evolution of parasites is important to both basic and applied evolutionary biology. Knowledge of the genetic structure of parasite populations is critical for our ability to predict how an infection can spread through a host population and for the design of effective control methods. However, very little is known about the genetic structure of most human parasites, including the human louse (Pediculus humanus). This species is composed of two ecotypes: the head louse (Pediculus humanus capitis De Geer), and the clothing (body) louse (Pediculus humanus humanus Linnaeus). Hundreds of millions of head louse infestations affect children every year, and this number is on the rise, in part because of increased resistance to insecticides. Clothing lice affect mostly homeless and refugee-camp populations and although they are less prevalent than head lice, the medical consequences are more severe because they vector deadly bacterial pathogens. In this study we present the first assessment of the genetic structure of human louse populations by analyzing the nuclear genetic variation at 15 newly developed microsatellite loci in 93 human lice from 11 sites in four world regions. Both ecotypes showed heterozygote deficits relative to Hardy–Weinberg equilibrium and high inbreeding values, an expected pattern given their parasitic life history. Bayesian clustering analyses assigned lice to four distinct genetic clusters that were geographically structured. The low levels of gene flow among louse populations suggested that the evolution of insecticide resistance in lice would most likely be affected by local selection pressures, underscoring the importance of tailoring control strategies to population-specific genetic makeup and evolutionary history. Our panel of microsatellite markers provides powerful data to investigate not only ecological and evolutionary processes in lice, but also those in their human hosts because of the long-term coevolutionary association between lice and humans.     

Coevolution between a cockroach and its bacterial endosymbiont: a biogeographical perspective

Cryptocercus are subsocial, xylophagous cockroaches that live in temperate forests. Like other cockroaches, Cryptocercus harbour endosymbiotic bacteria in their fat bodies. Two species of Cryptocercus occur in the palaearctic, one each in eastern Russia and south-central China. In the USA, there are five species: one in the north-west and four in the south-east. Little is known about the relationship between the Eurasian and North American Cryptocercus or the causes of the disjunct distribution. Here, a molecular phylogeny for six out of the seven Cryptocercus species and their endosymbionts is inferred in an attempt to understand the evolution and biogeography of the genus. Our analysis showed that the North American Cryptocercus are monophyletic, suggesting that a single colonization event was followed by vicariance. There was complete concordance between the host and endosymbiont phylogenetic trees. Divergence estimates based on endosymbiont DNA sequences suggested that the palaearctic and nearctic Cryptocercus diverged 70-115 million years (Myr) ago and the eastern- and western-USA species diverged 53-88 Myr ago. These divergence estimates were correlated with biogeographical events, and a hypothesis is presented to explain the current distribution of Cryptocercus. Our findings suggest that Cryptocercus has had a long evolutionary history, dating back to the Jurassic.     

The sex ratio distortion in the human head louse is conserved over time

Background

At the turn of the 19^th century the first observations of a female-biased sex ratio in broods and populations of the head louse, Pediculus humanus capitis, had been reported. A study by Buxton in 1940 on the sex ratio of lice on prisoners in Ceylon is still today the subject of reanalyses. This sex ratio distortion had been detected in ten different countries. In the last sixty years no new data have been collected, especially on scalp infestations under economically and socially more developed conditions.

Results

Here we report a female bias of head lice in a survey of 480 school children in Argentina. This bias is independent of the intensity of the pediculosis, which makes local mate competition highly unlikely as the source of the aberrant sex ratio; however, other possible adaptive mechanisms cannot be discounted. These lice as well as lice from pupils in Britain were carrying several strains of the endosymbiotic bacterium Wolbachia pipientis, one of the most wide spread intracellular sex ratio distorters. Similar Wolbachia strains are also present in the pig louse, Haematopinus suis, suggesting that this endosymbiont might have a marked influence on the biology of the whole order. The presence of a related obligate nutritional bacterium in lice prevents the investigation of a causal link between sex ratio and endosymbionts.

Conclusions

Regardless of its origin, this sex ratio distortion in head lice that has been reported world wide, is stable over time and is a remarkable deviation from the stability of frequency-dependent selection of Fisher's sex ratio. A female bias first reported in 1898 is still present over a hundred years and a thousand generations later.

     

Genome-Wide Functional Divergence after the Symbiosis of Proteobacteria with Insects Unraveled through a Novel Computational Approach

Symbiosis has been among the most important evolutionary steps to generate biological complexity. The establishment of symbiosis required an intimate metabolic link between biological systems with different complexity levels. The strict endo-cellular symbiotic bacteria of insects are beautiful examples of the metabolic coupling between organisms belonging to different kingdoms, a eukaryote and a prokaryote. The host (eukaryote) provides the endosymbiont (prokaryote) with a stable cellular environment while the endosymbiont supplements the host's diet with essential metabolites. For such communication to take place, endosymbionts' genomes have suffered dramatic modifications and reconfigurations of proteins' functions. Two of the main modifications, loss of genes redundant for endosymbiotic bacteria or the host and bacterial genome streamlining, have been extensively studied. However, no studies have accounted for possible functional shifts in the endosymbiotic proteomes. Here, we develop a simple method to screen genomes for evidence of functional divergence between two species clusters, and we apply it to identify functional shifts in the endosymbiotic proteomes. Despite the strong effects of genetic drift in the endosymbiotic systems, we unexpectedly identified genes to be under stronger selective constraints in endosymbionts of aphids and ants than in their free-living bacterial relatives. These genes are directly involved in supplementing the host's diet with essential metabolites. A test of functional divergence supports a strong relationship between the endosymbiosis and the functional shifts of proteins involved in the metabolic communication with the insect host. The correlation between functional divergence in the endosymbiotic bacterium and the ecological requirements of the host uncovers their intimate biochemical and metabolic communication and provides insights on the role of symbiosis in generating species diversity.     

Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria)

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approximately 98.5%, have been reported to occur as obligate endosymbionts of ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S-23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex life cycle. These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and obligately free-living bacteria ever revealed.     

Multigene analysis of phylogenetic relationships and divergence times of primate sucking lice (Phthiraptera: Anoplura)

Cospeciation between hosts and parasites offers a unique opportunity to use information from parasites to infer events in host evolutionary history. Although lice (Insecta: Phthiraptera) are known to cospeciate with their hosts and have frequently served as important markers to infer host evolutionary history, most molecular studies are based on only one or two markers. Resulting phylogenies may, therefore, represent gene histories (rather than species histories), and analyses of multiple molecular markers are needed to increase confidence in the results of phylogenetic analyses. Herein, we phylogenetically examine nine molecular markers in primate sucking lice (Phthiraptera: Anoplura) and we use these markers to estimate divergence times among louse lineages. Individual and combined analyses of these nine markers are, for the most part, congruent, supporting relationships hypothesized in previous studies. Only one marker, the nuclear protein-coding gene Histone 3, has a significantly different tree topology compared to the other markers. The disparate evolutionary history of this marker, however, has no significant effect on topology or nodal support in the combined phylogenetic analyses. Therefore, phylogenetic results from the combined data set likely represent a solid hypothesis of species relationships. Additionally, we find that simultaneous use of multiple markers and calibration points provides the most reliable estimates of louse divergence times, in agreement with previous studies estimating divergences among species. Estimates of phylogenies and divergence times also allow us to verify the results of [Reed, D.L., Light, J.E., Allen, J.M., Kirchman, J.J., 2007. Pair of lice lost or parasites regained: the evolutionary history of anthropoid primate lice. BMC Biol. 5, 7.]; there was probable contact between gorilla and archaic hominids roughly 3 Ma resulting in a host switch of Pthirus lice from gorillas to archaic hominids. Thus, these results provide further evidence that data from cospeciating organisms can yield important information about the evolutionary history of their hosts.     

Bacteria associated with gut lumen of Camponotus japonicus Mayr

Camponotus ants harbor the obligate intracellular endosymbiont Blochmannia in their midgut bacteriocytes, but little is known about intestinal bacteria living in the gut lumen. In this paper we reported the results of a survey of the intestinal microflora of Camponotus japonicus Mayr based on small-subunit rRNA genes (16S rRNAs) polymerase chain reaction (PCR)-restriction fragment-length polymorphism analysis of worker guts. From 107 clones, 11 different restriction fragment-length polymorphism profiles were identified, and sequences blasting analysis found these represent four types of bacteria. Most (91.6%) of the clones were "Candidatus Blochmannia", the obligate endosymbionts of Camponotus ants, and 6.5% of the clones were "Candidatus Serratia symbiotica", a secondary endosymbiont of aphids; the remaining 2% clones were Fructobacillus fructosus and uncultured Burkholderiales bacterium, respectively. These results show that the diversity of gut bacteria in C. japonicus was low. "Candidatus Serratia symbiotica" was identified from Camponotus ants for the first time, an interesting result because Blochmannia's closest bacterial relative is also in the genus Serratia. This discovery supports the scenario that consumption of aphid honeydew or tissue provides an initial step in the evolution of an advanced symbiosis, and suggests that Camponotus ant could acquire other secondary endosymbionts from Hemiptera host through their diet. In addition, Burkholderiales bacterium also was identified from the gut of C. japonicus for the first time, and whether it is a nitrogen-recycling endosymbiont in Camponotus ants needs to be investigated further.     

Evolution of symbiotic organs and endosymbionts in lygaeid stinkbugs

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone'' in the middle of each germarium and in the ‘symbiont ball'' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri'', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola'' is proposed for the endosymbiont clade.     

Infection dynamics of coexisting beta- and gammaproteobacteria in the nested endosymbiotic system of mealybugs

We investigated the infection dynamics of endosymbiotic bacteria in the developmental course of the mealybugs Planococcus kraunhiae and Pseudococcus comstocki. Molecular phylogenetic analyses identified a betaproteobacterium and a gammaproteobacterium from each of the mealybug species. The former bacterium was related to the beta-endosymbionts of other mealybugs, i.e., "Candidatus Tremblaya princeps," and formed a compact clade in the Betaproteobacteria. Meanwhile, the latter bacterium was related to the gamma-endosymbionts of other mealybugs but belonged to distinct clades in the Gammaproteobacteria. Whole-mount in situ hybridization confirmed the peculiar nested formation in the endosymbiotic system of the mealybugs: the beta-endosymbiont cells were present in the cytoplasm of the bacteriocytes, and the gamma-endosymbiont cells were located in the beta-endosymbiont cells. In nymphal and female development, a large oval bacteriome consisting of a number of bacteriocytes was present in the abdomen, wherein the endosymbionts were harbored. In male development, strikingly, the bacteriome progressively degenerated in prepupae and pupae and became almost unrecognizable in adult males. In the degeneration process, the gamma-endosymbionts disappeared more rapidly than the beta-endosymbionts did. Quantitative PCR analyses revealed that (i) the population dynamics of the endosymbionts in female development reflected the reproductive activity of the insects, (ii) the population dynamics of the endosymbionts were strikingly different between female development and male development, (iii) the endosymbiont populations drastically decreased in male development, and (iv) the gamma-endosymbiont populations decreased more rapidly than the beta-endosymbiont populations in male development. Possible mechanisms underlying the uncoupled regulation of the beta- and gamma-endosymbiont populations are discussed in relation to the establishment and evolution of this unique prokaryote-prokaryote endosymbiotic system.     

Phylogenetic characterization and molecular evolution of bacterial endosymbionts in psyllids (Hemiptera: Sternorrhyncha)

Most sternorrhynchan insects harbor endosymbiotic bacteria in specialized cells (bacteriocytes) near the gut which provide essential nutrients for hosts. In lineages investigated so far with molecular methods (aphids, mealybugs, whiteflies), endosymbionts apparently have arisen from independent infections of common host ancestors and co-speciated with their hosts. Some endosymbionts also exhibit putatively negative genetic effects from their symbiotic association. In this study, the identity of endosymbionts in one major sternorrhynchan lineage, psyllids (Psylloidea), was investigated to determine their position in eubacterial phylogeny and their relationship to other sternorrhynchan endosymbionts. Small-subunit ribosomal RNA genes (16S rDNA) from bacteria in three psyllid species (families Psyllidae and Triozidae) were sequenced and incorporated into an alignment including other insect endosymbionts and free-living bacteria. In phylogenetic analysis, all sequences were placed within the gamma subdivision of the Proteobacteria. Three sequences, one from each psyllid species, formed a highly supported monophyletic group whose branching order matched the host phylogeny, and also exhibited accelerated rates of evolution and mutational bias toward A and T nucleotides. These attributes, characteristic of primary (P) bacteriocyte-dwelling endosymbionts, suggested that these sequences were from the putative psyllid P endosymbiont. Two other sequences were placed within the gamma-3 subgroup of Proteobacteria and were hypothesized to be secondary endosymbionts. The analysis also suggested a sister relationship between P endosymbionts of psyllids and whiteflies. Thus, a continuous mutualistic association between bacteria and insects may have existed since the common ancestor of psyllids and whiteflies. Calculations using a universal substitution rate in bacteria corrected for endosymbiont rate acceleration support the idea that this common ancestor was also the ancestor of all Sternorrhyncha. Compared with other P endosymbiont lineages, the genetic consequences of intracellular life for some psyllid endosymbionts have been exaggerated, indicating possible differences in population structures of bacteria and/or hosts.     

What's in a name: the taxonomic status of human head and body lice

Human head lice (Anoplura: Pediculidae: Pediculus) are pandemic, parasitizing countless school children worldwide due to the evolution of insecticide resistance, and human body (clothing) lice are responsible for the deaths of millions as a result of vectoring several deadly bacterial pathogens. Despite the obvious impact these lice have had on their human hosts, it is unclear whether head and body lice represent two morphological forms of a single species or two distinct species. To assess the taxonomic status of head and body lice, we provide a synthesis of publicly available molecular data in GenBank, and we compare phylogenetic and population genetic methods using the most diverse geographic and molecular sampling presently available. Our analyses find reticulated networks, gene flow, and a lack of reciprocal monophyly, all of which indicate that head and body lice do not represent genetically distinct evolutionary units. Based on these findings, as well as inconsistencies of morphological, behavioral, and ecological variability between head and body lice, we contend that no known species concept would recognize these louse morphotypes as separate species. We recommend recognizing head and body lice as morphotypes of a single species, Pediculus humanus, until compelling new data and analyses (preferably analyses of fast evolving nuclear markers in a coalescent framework) indicate otherwise.