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* School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd, UK; and
Department of Life Sciences, King’s College London, London, UK
1Correspondence: School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, UK. E-mail: bss40c{at}bangor.ac.uk
SPECIFIC AIMS
We investigated the diversity of endosymbionts and their relationship to host development in parthenogenetic book lice. The unexpected identity of the symbiont led us to examine the complexity of the evolution of symbiotic and pathogenic interactions. We questioned whether we could observe transitional stages of adaptation in the ontogeny of a single individual host between extracellular bacteria and organ-forming bacteria.
PRINCIPAL FINDINGS
1. Rickettsia are primary and sole endosymbionts
Only Rickettsia sequences were amplified from Liposcelis bostrychophila and Cerobasis guestfalica. These two parthenogenetic species and a sexual species as a control, were studied by confocal microscopy.
2. Mycetomic Rickettsia have multiple origins
The 16S genes of the two Rickettsia differ by >6% and are associated with two very different clades. The Liposcelis Rickettsia are closest to the spotted fever group whereas the Cerobasis Rickettsia join a diverse ancestral group around R. limoniae.
3. Mycetomic Rickettsia are essential for egg development in parthenogenetic species
Rickettsia-specific primers did not detect Rickettsia in 26 sexual species of booklice and barklice. L. bostrychophila were reared at 37°C to eliminate the rickettsiae. The egg production was reduced by >90%. The eggs laid did not contain Rickettsia and did not hatch. The curing did not affect the life span.
4 Rickettsia change their cellular association during development
Early oocytes harbored clusters of rickettsiae in the first anterior quarter of the ooplasm as they were received from the nurse cells. Heavy infection was seen in the newly invaginated germ cells.
First instar nymphs showed rickettsial loads in cells lining the ventral side of the thorax. A single heavily infected mycetocyte had migrated into the fat body on each side.
In second instar nymphs, the Rickettsia progressively spread into the posterior part of the gut epithelium. Some cells showed Rickettsia inside their nuclei. The primordial ovaries showed localized infections at the anterior end just adjacent to fat body and midgut.
5. Mycetomic Rickettsia
During the third instar, small globular mycetocytes aggregated to shape a pair of structures at the posterior end of the midgut, at the ventral site, between midgut and incipient ovaries. These aggregates lead to the mycetomes of the adults.
In the fourth instar, a pair of mycetomes with an elongated shape was now growing between the midgut and the developing ovaries. In tenerals, the mycetomes were built of 4 to 8 big and 8 to 12 small, nucleated rhomboid mycetocytes. Bacterial cells were repeatedly detected in the surrounding of the mycetome unassociated with cells, which might suggest free migration in the hemolymph.
The bacteria were concentrated in the two mycetomes: the four Malphigian tubules and the remaining ovarioles. The two mycetomes of adults were similar in size; their mycetocytes inside became compact, globular in shape, and crowded with slightly smaller Rickettsia bodies (Fig. 1
A–C). The Rickettsia had the smallest size in the late mycetome and in the early oocyte; size ranged between 700 and 1000 nm long and 350 and 450 nm wide.
The Malpighian tubules showed the heaviest rickettsial infection in the first third from the gut and only scattered infection in the remaining length. This first third developed into a thick and strongly hypertrophic vessel (Fig. 1D
). In one adult specimen of L. bostrychophila we observed six Malpighian tubules instead of four. The two extra tubules were only developed to a third of the length and contained masses of Rickettsia.
One teneral and one adult of L bostrychophila displayed an almost identical duplication of the mycetome on one side of their body leading to three mycetomes (Fig. 1E, F
).
CONCLUSIONS AND SIGNIFICANCE
Mycetomic Rickettsia
The Rickettsia and the mycetomes have been observed in disparate species of booklice that belong to opposing ends in the evolution of Psocoptera. Cerobasis guestfalica (Trogiidae) belongs to the Trogiomorpha, which represents a primitive suborder of the book- and barklice order Psocoptera; Liposcelis bostrychophila (Liposcelididae) has been associated with the advanced suborder Troctomorpha, but is now considered to represent a highly derived lineage in the biting and chewing lice order Phthiraptera. This suggests that the mycetome might be an organ belonging to the ancestor of Psocoptera and Phthiraptera. Mycetomes of varying structures have been well described for most members of the Phthiraptera but have been unknown for the Psocoptera.
The exclusive feeding of lice on blood and/or epidermis has been used to explain the presence of mycetomes. Our findings in the basal Cerobasis lineage in the booklice suggest that the evolution of mycetomic provisions might have predated the acquisition of blood feeding. The advanced Liposcelis lineage might have lost the parasitic lifestyle of lice. The continuing presence of mycetomes and the dependence on the mycetomic Rickettsia for reproduction might underpin the antiquity of symbiotic interactions regardless of food source.
Host adaptations
We did not detect any Rickettsia in the salivary glands as might have been expected for Rickettsia that are vectored or horizontally transmitted.
Treatment with high temperature confirmed that the Rickettsia in parthenogenetic L. bostrychophila is obligate for development of oocytes and egg laying.
With the infection of Malpighian tubules there is a clear host specialization in the first third of the tubules, with hypertrophy of the vessels and the formation of giant cells occupied by masses of Rickettsia. Transformation of the tubules occurs at the stage of the evagination of the ectodermal proctodeum, which seems to exclude any interpretation of the Malpighian structures as pathological host reactions and may suggest that the Malpighian tubules are one major location at which the endosymbionts fulfill their physiological duty. However, the curing of the parthenogenetic booklouse had no effect on life span. The only observed phenomenological difference of the loss of Rickettsia is complete cessation of egg development.
Intranuclear Rickettsia
With the exception of ciliates, intra- or endonuclear bacteria are quite unusual. Rickettsia belonging to the spotted fever group and Orientia tsutsugamushi are known to invade the nucleus of both their invertebrate vector and their vertebrate host but this is rare. Booklice rickettsiae are much more often detected inside the nucleus. Only the nuclei of the mycetomes disappear over time but not those of the mycetocytes.
Diversity of Rickettsia
In phylogenetic analysis, the Liposcelis Rickettsia cluster with a newly discovered Rickettsia associated with parthenogenesis in the parasitoid wasp Neochrysocharis formosa and are most closely related to R. felis. Although R. felis is grouped with the spotted fever rickettsiae, its biology and genome separate it from the spotted fever group proper. R. felis carries an unexpectedly high number of ankyrin motif-containing genes, which is more characteristic for endosymbiotic Wolbachia than for Rickettsia. The spotted fever group separates from the bellii group that assembles aphid, leafhopper, mite, and tick Rickettsia. The Cerobasis Rickettsia form part of the most divers grouping at the base of the genus. It assembles the crane fly Rickettsia, R. limoniae, with a Rickettsia associated with the amphizoic amoeba Nuclearia pattersoni of the gills of roaches and secondary and facultative Rickettsia found in leeches. The Liposcelis bacteria are the first Rickettsia that function as primary endosymbionts and have become obligatory for oogenesis.
Transitional stages in evolution
The interaction between Rickettsia and booklice is characterized by a multitude of transitional stages (Fig. 2
). The oocyte is infected through the germline and nurse cells (Fig. 1G
). Reproductive parasites are mainly transmitted through the germline and primary endosymbionts are transmitted via secondary tissues like nurse cells. Rickettsia are found in differentiated tissues, single cell mycetocytes, and an organ-forming mycetome. Fluctuating asymmetry is quite common in insects, but organ duplications are rare in the ontology of insects. In "old" mycetomic associations, the mycetomic endosymbiont does not infect other tissues. In most cases with centralized Malpighian infections, the nonadapted part of tubules is free from infections; in booklice it is not. The Rickettsia are both intra- and extracellular and are found in nuclei, which suggests we are witnessing the evolutionary transition from parasite to obligate nutritional or developmental symbiont.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5870fje
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