Distribution and transmission of endosymbiotic microorganisms in the oocytes of the pig louse,Haematopinus suis (L.) (Insecta: Phthiraptera)

Summary All anoplurans live symbiotically with prokaryotic microorganisms hosted in specialized cells, termed mycetocytes. In nymphs and males mycetocytes are distributed between midgut epithelial cells. In females, besides the midgut, mycetocytes are found in the reproductive organs where they are located at the base of ovarioles in contact with lateral oviducts. The mycetocyte-associated symbionts are transmitted from one generation to the next transovarially. Here, the results of histological and ultrastructural studies on the distribution and transmission of symbiotic microorganisms within the ovaries of the anopluranHaematopinus suis are presented. Interestingly, during advanced oogenesis (i.e., choriogenesis) of this species all symbionts are localized extracellularly and form a tight mass located at the posterior pole of the oocyte just below the hydropyle. In insects studied so far, such localization of transovarially transmitted microorganisms has been reported only in the closely related speciesHaematopinus eurysternus.     

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.     

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.     

Intracellular translocation of symbiotic bacteroids during late oogenesis and early embryogenesis of Bradysia tritici (syn. Sciara ocellaris) (Diptera: Sciaridae)

Abstract. The distribution of symbiotic prokaryotes (bacteroids) in ovarian follicles and young embryos of Bradysia ( Sciara ) was studied using light and electron microscopy. In mid-vitellogenic follicles (prior to oosome formation) isolated from 8-h-old midges, most symbionts were scattered in the ooplasm. Later, during oogenesis and concomitant with the formation of the oosome, symbionts aggregated at the posterior pole of the follicle. During early embryogenesis, large symbiont clusters were seen between the oolemma and oosome and lateral to the oosome. When the presumptive pole-cell nuclei moved into the oosome, the bacteroids became scattered in and around the prospective germ plasm, and many of them became incorporated into the pole cells. At the anterior pole, a nearly symbiontfree area could be recognized at first (anterior cone), but later on symbionts aggregated there (usually best seen in 1-h embryos). Thereafter, smaller symbiont clusters spread into more lateral parts of the thickened anterior cortex. Finally, the aggregates became dispersed when cleavage nuclei moved into the anterior egg cortex. The distribution of symbionts may reflect cytoplasmic streaming or other transport phenomena during development which could participate in oosome formation or in the histological differentiation of the anterior egg cortex.     

Intracellular symbiotes of the pea aphid,Acyrthosiphon pisum

Histological and ultrastructural studies on the mycetome of the pea aphid, Acyrthosiphon pisum, disclosed two types of symbiotes. The more common primary symbiotes were oval in shape and were found in large mycetocytes making up the bulk of the mycetome. The secondary symbiotes were smaller, rod-shaped, and were restricted to an apparently syncytial sheath partially enclosing the primary mycetocytes. Extensive rough endoplasmic reticulum and Golgi occurred in the sheath but not in the primary mycetocytes. Lysosomal breakdown occurred in both primary and secondary symbiotes but the two processes differed markedly. In the primary mycetocytes, a small number of symbiotes were broken down individually to form small, compact residual bodies. In the sheath, breakdown of secondary symbiotes was more extensive: large numbers were broken down within cytolysomes.     

Two Intracellular Symbiotic Bacteria from the Mulberry Psyllid Anomoneura mori (Insecta, Homoptera)

We characterized the intracellular symbiotic bacteria of the mulberry psyllid Anomoneura mori by performing a molecular phylogenetic analysis combined with in situ hybridization. In its abdomen, the psyllid has a large, yellow, bilobed mycetome (or bacteriome) which consists of many round uninucleated mycetocytes (or bacteriocytes) enclosing syncytial tissue. The mycetocytes and syncytium harbor specific intracellular bacteria, the X-symbionts and Y-symbionts, respectively. Almost the entire length of the bacterial 16S ribosomal DNA (rDNA) was amplified and cloned from the whole DNA of A. mori, and two clones, the A-type and B-type clones, were identified by restriction fragment length polymorphism analysis. In situ hybridization with specific oligonucleotide probes demonstrated that the A-type and B-type 16S rDNAs were derived from the X-symbionts and Y-symbionts, respectively. Molecular phylogenetic analyses of the 16S rDNA sequences showed that these symbionts belong to distinct lineages in the γ subdivision of the Proteobacteria. No 16S rDNA sequences in the databases were closely related to the 16S rDNA sequences of the X- and Y-symbionts. However, the sequences that were relatively closely related to them were the sequences of endosymbionts of other insects. The nucleotide compositions of the 16S rDNAs of the X- and Y-symbionts were highly AT biased, and the sequence of the X-symbiont was the most AT-rich bacterial 16S rDNA sequence reported so far.     

Pole cells of Drosophila paulistorum: embryologic differentiation with symbionts.

The pole cells of young D. paulistorum embryos are destined to form the germinal cells of both male and female imagoes. In addition, specialized portions of the midgut may be derived from pole cell progenitors. In this initial study of their embryogenesis by means of electron microscopy, various stages of pole cell development are shown in both non-hybrid (potentially fertile) and intersemispecific hybrid (potentially sterile as males) materials. Originally, approximately 5 or 6 cells emerge to form the early polar cap and subsequently divide asynchronously until the 35-50 cells of the late polar cap are derived. Unlike other Drosophila species, however, mycoplasma-like symbionts, apparently an hereditary infection, have been traced to locations within the cytoplasm of these pole cells. They are depicted as arriving there after transmission via the egg cytoplasm, implicating this as their probable route of entry into the future germinal tissues of adult flies. It is postulated that these microorganisms function as an infectious reproductive isolating mechanism fostering hybrid male sterility between D. paulistorum semispecies.     

Cytogenetic studies following high dosage paternal irradiation in the mealy bug,Planococcus citri

Summary In the mealy bug, Planococcus citri, following high dosage paternal irradiation (60,000–120,000 rep), the survivors are mostly female (about 30–40% of the unirradiated control value) whereas very few males survive (about 5% of control value). After lower doses of paternal irradiation (P. I.), however, few or no females survive while the normal number of males (never less than the control value) survive.The females developing after high dosage P. I. are gynogenetic and are triploid or diploid or 3N/2N or 2N/N mosaics (Chandra 1963).The cytology of X₁ embryos following 90,000 rep is described in this report, in comparison with data from embryos following lower doses (8,000 r) of P. I. and unirradiated controls, to illustrate the chromosomal mechanisms leading to the production of gynogenetic females and the probable reasons for lethality of X₁ males after heavy P. I.It has been shown that triploid females stem from a fusion nucleus of the first and second polar bodies. This triploid polar nucleus, which normally participates in the formation of a polyploid sector in the young embryo, undertakes a successful embryogeny in many embryos when the zygote nucleus is unable to do so because of the heavily damaged paternal complement of chromosomes. Since the chromosomes are characterized by holokinetic activity, the irradiated paternal set manages to divide with the maternal complement but did not always segregate as successfully. Restitution divisions of the zygotic nuclei result in haploid, hyperhaploid, diploid and polyploid nuclei. Most of the diploid gynogenetic females probably originate from diploid nuclei of zygotic origin although it is possible that a few diploid females and the 2N/N mosaic females develop from polar bodies.From a dissertation submitted to the University of California, in partial satisfaction of the requirements for the degree of Doctor of Philosophy.Supported in part by a National Science Foundation Grant (No. G-9772) to Professor Spencer W. Brown.N. I. H. Predoctoral Trainee in Genetics 1961–1962.     

Ultrastructure of the endosymbionts of the whitefly,Bemisia tabaci andTrialeurodes vaporariorum

Summary The ultrastructure of the mycetocytes and mycetome micro-organisms of the sweetpotato whitefly,Bemisia tabaci Genn. andTrialeurodes vaporariorum West are described. InB. tabaci, two morphologically distinct types of micro-organisms were observed in mycetocytes. The predominant type lacked a distinct cell wall, was pleomorphic in shape with a surrounding vacuole. The second type was a coccoid organism, with inner and outer cell membranes. The coccoid organism was often found in groups of varying number within vacuoles, and in many cases appeared to be undergoing degradation. InT. vaporariorum mycetocytes, pleomorphic and coccoid organisms were found, although the coccoid micro-organism inT. vaporariorum, had a thicker cell wall than the coccoid micro-organism inB. tabaci.Abbreviations C coccoid micro-organism - P pleomorphic micro-organism     

Maternal Inheritance of Enzymes in the Mealybug PSEUDOCOCCUS OBSCURUS (Homoptera)

In the mealybug Pseudococcus obscurus Essig (Pseudococcidae) two esterases, a tyrosinase and a mannosephosphate isomerase, exhibited an unusual type of maternal inheritance. Electromorphs (alleles) were transmitted by both parent but segregation was delayed by one generation and full sisters always had the same phenotype. Moreover, for esterase-1, in which three alleles were present, some of the females exhibited all three alleles. Several other polymorphic loci exhibited normal transmission and segregation. This mode of inheritance can be readily explained by assuming that most or all of the enzymes coded for by these loci are produced by the mycetocytes. The mycetocytes house intracellular bacteria-like symbionts and are usually formed by the fusion ofthe polar bodies and one or more cleavage nuclei. For a locus with two alleles exhibiting this type of inheritance, the expected frequencies of the three phenotypes are p3, 3pq an equation is presented for estimating the frequency of alleles from the frequencies of the phenotypes and it is shown that for three samples from wild populations there is a good agreement between the expected and observed frequencies of the phenotypes.     

Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera)

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcid Antonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned from A. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a beta-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences of Spiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections of A. crawii and in situ hybridization with specific oligonucleotide probes. The gamma- and beta-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the gamma- and beta-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the beta-Proteobacteria symbiont was detected in all five pseudococcids, the gamma-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only in A. crawii.     

Meiotic parthenogenesis and heterochromatization in a soft scale,pulvinaria hydrangeae (Coccoidea: Homoptera)

Summary Meiotic parthenogenesis of a type not previously described was found in Pulvinaria hydrangeae Steinweden. During diakinesis 8 bivalents were formed. At prometaphase the spindle was tripolar but anaphase I was bipolar and normal. After completion of division of the primary oocyte, the following sequence occurred: 1. polar body I divided, usually into 3 products; 2. the secondary oocyte divided to yield the egg pronucleus and polar body II; 3. the egg pronucleus divided into its two haploid products; and 4. the second polar body divided. The products of the egg pronucleus fused while dividing to restore the diploid chromosome number; this division may be equated to the first cleavage division. The products of the polar bodies did not take part in the formation of the embryo proper or the mycetocytes.Among the embryos produced by females of two out of the three populations studied some of the embryos showed a heterochromatic chromosome set, characteristic of males in this and related families. The reproductive system of the females as well as the eggs did not contain any sperm; thus the male embryos were apparently produced parthenogenetically.The euchromatic and heterochromatic chromosome sets were genetically identical, since they both originated from the egg pronucleus by mitosis. The heterochromatization of one set but not the other might be due in part to a previous difference in their position in the cytoplasm.The females were completely homozygous yet they produced male and female embryos. Thus it appears that sex determination in the group does not depend on the segregation of genetic factors in either males or females.In addition to male and female embryos, three types of degenerating embryos were observed. It is believed that these embryos were formed by polyploid somatic cells which invaded abnormal eggs and embryos and took over development.     

Pattern of bacteriocyte formation in Periplaneta americana (L.) (Blattaria: Blattidae)

In the embryos of Periplaneta americana (L.) (Blattaria: Blattidae), bacterial symbionts, together with vitellophages, form a mycetomic structure inside the deutoplasm; this regresses between the 15th and 16th day after deposition of the ootheca. In this article we describe the migration of bacteria across the wall of the midgut from the mycetome, and the topographic distribution of pre-bacteriocyte cells. We also report that the pre-bacteriocytes are present only on the lateral surface of the internal abdominal fat bodies. We discuss the possible embryological origin and evolution of these cells, and put forward the hypothesis that pre-bacteriocytes are derived from oenocytes activated to perform phagocytosis.     

蚜虫与体内布赫纳氏菌及其次生共生菌的相互关系

对蚜虫与其内共生细菌共生关系的研究进展进行综述。布赫纳氏菌Buchnera普遍存在于蚜虫体内特殊的菌胞中,与蚜虫形成专性共生关系,为宿主蚜虫提供多种必需氨基酸和B族维生素。蚜虫体内的菌胞数量是一个动态变量,受蚜虫体内、外环境因子的影响。在某些蚜虫体内菌胞中还发现有若干种类次生共生细菌,其功能尚不完全清楚,可能与蚜虫的生态学特征有关。对蚜虫与其体内共生菌相互关系的研究提出一些新的问题。     

Endosymbiosis of Aphids with Microorganisms: a Model Case of Dynamic Endosymbiotic Evolution

Abstract Almost all aphids harbor prokaryotic intracellular symbionts in the cytoplasm of mycetocytes, huge cells in the abdomen specialized for this purpose. The aphids and their intracellular symbionts are in close mutualistic association and unable to live without their partner. The intracellular symbionts of various aphids are of a single origin; they are descendants of a prokaryote that was acquired by the common ancestor of the present aphids. The date of establishment of the symbiotic association is estimated to be 160–280 million years ago using 16S rRNA molecular clock calibrated by aphid fossils. Molecular phylogeny indicates that the intracellular symbiont belongs to a group of gut bacteria, suggesting the possibility that it was derived from a gut microbe of aphids. While the in-tracellular symbionts are universal and highly conserved amongst aphids, other types of symbiotic microorganisms are also present. In various aphids, bacterial “secondary” intracellular symbionts are found in addition to the standard symbionts. They are thought to be acquired many times in various lineages independently. Some Cerataphidini aphids do not have intracellular symbiotic system but harbor yeast-like extracellular symbionts in the hemocoel. In a lineage of this group, symbiont replacement from intracellular prokaryote to extracellular yeast must have occurred. The diversity of the endosymbiotic system of aphids illuminates a dynamic aspect of endosymbiotic evolution.     

Impact of a parasitoid on the bacterial symbiosis of its aphid host

Embryo production in aphids is absolutely dependent on the function of symbiotic bacteria, mainly Buchnera, and the growth and development of koinobiont parasitoids in aphids requires the diversion of nutrients from aphid embryo production to the parasitoid. The implication that the bacterial symbiosis may be promoted in parasitized aphids to support the growing parasitoid was explored by analysis of the number and biomass of mycetocytes, and the aphid cells bearing Buchnera, in the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) parasitized by the wasp Aphidius ervi Haliday (Hymenoptera: Braconidae). Aphids hosting a young larval parasitoid bore more mycetocytes of greater total biomass, and embryos of lower biomass than unparasitized aphids. Furthermore, one of the three aphid clones tested, which limited teratocyte growth (giant cells of parasitoid origin having a trophic role), bore smaller mycetocytes and larger embryos, than one or both of the two aphid clones with greater susceptibility to the parasitoid. These data suggest that susceptibility of the aphid‐Buchnera symbiosis to parasitoid‐mediated manipulation may, directly or indirectly, contribute to aphid susceptibility to parasitoid exploitation.     

The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.     

MYCETOCYTE SYMBIOSIS IN INSECTS

1. Non-pathogenic microorganisms, known as mycetocyte symbionts, are located in specialized 'mycetocyte' cells of many insects that feed on nutritionally unbalanced or poor diets. The insects include cockroaches, Cimicidae and Lygaeidae (Heteroptera), the Homoptera, Anoplura, the Diptera Pupiparia, some formicine ants and many beetles. 2. Most mycetocyte symbionts are prokaryotes and a great diversity of forms has been described. None has been cultured in vitro and their taxonomic position is obscure. Yeasts have been reported in Cerambycidae and Anobiidae (Coleoptera) and a few planthoppers. They are culturable and those in anobiids have been assigned to the genus Torulopsis. 3. The mycetocyte cells may be associated with the gut, lie free in the abdominal haemocoel or be embedded in the fat body of the insect. The mycetocytes are large polyploid cells which rarely divide and the symbionts are restricted to their cytoplasm. 4. The mycetocyte symbionts are transmitted maternally from one insect generation to the next. In many beetles (Anobiidae, Cerambycidae, Chrysomelidae and cleonine Curculionidae), the microoganisms are smeared onto the eggs and consumed by the hatching larvae. In other insects, they are transferred from mycetocytes to oocytes in the ovary, a process known as transovarial transmission. The details of transmission in the different insect groups vary with the age of the mother (adult, larva or embryo) at which symbiont transfer to the ovary is initiated; whether isolated symbionts or intact mycetocytes are transferred; and the site of entry of symbionts to the egg (anterior, posterior or apolar). 5. Within an individual insect, the biomass of symbionts varies in a regular fashion with age, weight and sex of the insect. Suppression of symbiont growth rate and lysis of 'excess' microorganisms may contribute to the regulation of symbionts (including freshly-isolated preparations of unculturable forms) are used to investigate interactions between the partners. However, some methods to obtain aposymbiotic insects (e.g. antibiotics and lysozyme) deleteriously affect certain insects and aposymbionts may differ from the symbiont-containing stocks from which they were derived. 7. The mycetocyte symbionts have been proposed to synthesize various nutrients required by the insect. The symbionts of beetles and haematophagous insects may provide B vitamins and those in cockroaches and the Homoptera essential amino acids. The role of symbionts in the sterol nutrition of insects is equivocal. 8. Mycetocyte symbionts may have evolved from gut symbionts or guest microorganisms. The association is monophyletic in cockroaches but polyphyletic in many groups, including the sucking lice, beetles and scale insects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Embryonic pole cells and mycoplasmalike symbionts in Drosophila paulistorum

Mycoplasmalike intracellular symbionts have been located in the pole cells of Drosophila paulistorum embryos. These cells are destined to form the germ cells of both sexes. The symbionts had been previously localized in larval and adult developing and mature ovaries and testes. It is via the egg cytoplasm that these microorganisms are transmitted between generations to apparently cause an infectious and hereditary hybrid male sterility.