Transovarial transmission of Rickettsia spp. and organ-specific infection of the whitefly Bemisia tabaci

The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations. Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected by Rickettsia included the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association between Rickettsia and the B. tabaci digestive system might be important for digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.     

Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor "Candidatus Portiera aleyrodidarum," an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Horizontal transmission of the insect symbiont Rickettsia is plant-mediated

Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.     

An Invasive Whitefly Feeding on a Virus-Infected Plant Increased Its Egg Production and Realized Fecundity

Background

Plant-pathogenic begomoviruses have a complex association with their insect vectors. The interactions of begomoviruses and reproduction of their vectors are poorly understood. Bemisia tabaci is known to transmit many begomoviruses, and the spread of B. tabaci, especially the B and Q ‘biotypes’, has been accompanied by the epidemics of begomoviruses. One of these identified disease-causing agents was Tomato yellow leaf curl China virus (TYLCCNV).

Methodology/Principal Findings

In this study, we compared the egg production and realized fecundity of two ‘biotypes’ or putative species of the whitefly B. tabaci, including the alien invasive B and the indigenous ZHJ1 from Zhejiang, China, feeding on either healthy or TYLCCNV-infected tobacco plants. The ovary of the whitefly was composed of 12–22 telotrophic ovarioles. According to the morphology of the oocytes and level of yolk content, oocytes in ovarioles were divided into four developmental phases (I-IV). Significantly higher proportion of immature oocytes (phase II, III) and mature oocytes (phase IV) was observed in ovary of females that fed on TYLCCNV-infected tobacco compared to that on healthy plants. Moreover, there was significant increase of eggs laid of B whitefly that fed on TYLCCNV-infected tobacco plants during the early developmental stages. In contrast, the proportion of oocytes of different developmental phases and eggs laid had no significant differences between ZHJ1 whiteflies feeding on TYLCCNV-infected and non-infected host plants.

Conclusions/Significance

The invasive B whitefly benefits from feeding on a begomovirus-infected plant through increased egg production and realized fecundity.     

Patterns of host cell inheritance in the bacterial symbiosis of whiteflies

Whiteflies possess bacterial symbionts Candidatus Portiera aleyrodidium that are housed in specialized cells called bacteriocytes and are faithfully transmitted via the ovary to insect offspring. In one whitefly species studied previously, Bemisia tabaci MEAM1, transmission is mediated by somatic inheritance of bacteriocytes, with a single bacteriocyte transferred to each oocyte and persisting through embryogenesis to the next generation. Here, we investigate the mode of bacteriocyte transmission in two whitefly species, B. tabaci MED, the sister species of MEAM1, and the phylogenetically distant species Trialeurodes vaporariorum. Microsatellite analysis supported by microscopical studies demonstrates that B. tabaci MED bacteriocytes are genetically different from other somatic cells and persist through embryogenesis, as for MEAM1, but T. vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect, likely mediated by the degradation of maternal bacteriocytes in the embryo. These two alternative modes of transmission provide a first demonstration among insect symbioses that the cellular processes underlying vertical transmission of bacterial symbionts can diversify among related host species associated with a single lineage of symbiotic bacteria.     

The telotrophic ovary known from Neuropterida exists also in the myxophagan beetle <Emphasis Type="Italic">Hydroscapha natans</Emphasis>

The ovary structure of the myxophagan beetle, Hycdoscapha natans, was investigated by means of light and electron microscopy for the first time. Each of the two ovaries consists of three ovarioles, the functional units of insect oogenesis. The ovary type is telotrophic meroistic but differs strongly from the telotrophic ovary found among all polyphagous beetles investigated so far. All characters found here are typical of telotrophic ovaries of Sialidae and Raphidioptera. Both taxa belong to the Neuropterida. As in all telotrophic ovaries, all nurse cells are combined in an anterior chamber, the tropharium. The tropharium houses two subsets of germ cells: numerous nurse cell nuclei are combined in a central syncytium without any cell membranes in between, surrounded by a monolayer of single-germ cells, the tapetum cells. Each tapetum cell is connected to the central syncytium via an intercellular bridge. Tapetum cells of the posterior zone, which sufficiently contact prefollicular cells, are able to grow into the vitellarium and develop as oocytes. During previtellogenic and early vitellogenic growth, oocytes remain connected with the central syncytium of the tropharium via their anterior elongations, the nutritive cords. The morphological data are discussed in the light of those derived from ovaries of other Coleoptera and from the proposed sister group, the Neuropterida. The data strongly support a sister group relationship between Coleoptera and Neuropterida. Furthermore, several switches between polytrophic and telotrophic ovaries must have occurred during the radiation of ancient insect taxa.     

昆虫共生细菌Rickettsia的研究进展

Rickettsia是传播和引起人类与其他脊椎动物疾病的胞内共生菌。引起脊椎动物疾病的这些Rickettsia, 其部分生活史是在节肢动物体内完成的;而另外许多Rickettsia, 其整个生活史都是在宿主节肢动物体内完成。为了叙述方便, 把前者称为脊椎动物Rickettsia, 后者称为节肢动物Rickettsia。过去的研究主要集中在医学上具有重大意义的脊椎动物Rickettsia, 而关于节肢动物Rickettsia的生物学特性等研究则相对较少。近年来, 研究者们加大了对昆虫Rickettsia的研究, 发现昆虫Rickettsia广泛分布于昆虫中, 且有两种存在形式。其可以通过垂直卵传的方式在世代间传递, 也可以通过寄生蜂和寄主植物达到在昆虫之间传播的目的。昆虫Rickettsia可通过诱导孤雌生殖、 诱导杀雄等方式影响宿主的生殖行为。其对不同宿主昆虫可产生对宿主有利或有害的作用;可增强宿主昆虫抵御高温和寄生蜂的能力, 与宿主昆虫对药剂的敏感性相关。最后, 昆虫Rickettsia具有一个简化的基因组, 且存在进一步减小的可能性。     

Incidence of male-killing Rickettsia spp. (alpha-proteobacteria) in the ten-spot ladybird beetle Adalia decempunctata L. (Coleoptera: Coccinellidae)

The diversity of endosymbiotic bacteria that kill male host offspring during embryogenesis and their frequencies in certain groups of host taxa suggest that the evolution of male killing and the subsequent spread of male-killing symbionts are primarily determined by host life history characteristics. We studied the 10-spot ladybird beetle, Adalia decempunctata L. (Coleoptera: Coccinellidae), in which male killing has not been recorded previously, to test this hypothesis, and we also assessed the evolution of the male killer identified by DNA sequence analysis. Our results show that A. decempunctata harbors male-killing Rickettsia (alpha-proteobacteria). Male-killing bacteria belonging to the genus Rickettsia have previously been reported only for the congeneric two-spot ladybird beetle, Adalia bipunctata L. Phylogenetic analysis of Rickettsia DNA sequences isolated from different populations of the two host species revealed a single origin of male killing in the genus Rickettsia. The data also indicated possible horizontal transfer of symbionts between host species. In addition, A. bipunctata is known to bear at least four different male-killing symbionts in its geographic range two of which coexist in the two locations from which A. decempunctata specimens were obtained for the present study. Since only a single male-killing taxon was found in A. decempunctata, we assume that the two closely related ladybird beetle species must differ in the number and/or geographic distribution of male killers. We discuss the importance of these findings to our understanding of the evolution and dynamics of symbiotic associations between male-killing bacteria and their insect hosts.     

Rickettsia 'in' and 'out': two different localization patterns of a bacterial symbiont in the same insect species

Intracellular symbionts of arthropods have diverse influences on their hosts, and their functions generally appear to be associated with their localization within the host. The effect of localization pattern on the role of a particular symbiont cannot normally be tested since the localization pattern within hosts is generally invariant. However, in Israel, the secondary symbiont Rickettsia is unusual in that it presents two distinct localization patterns throughout development and adulthood in its whitefly host, Bemisia tabaci (B biotype). In the "scattered" pattern, Rickettsia is localized throughout the whitefly hemocoel, excluding the bacteriocytes, where the obligate symbiont Portiera aleyrodidarum and some other secondary symbionts are housed. In the "confined" pattern, Rickettsia is restricted to the bacteriocytes. We examined the effects of these patterns on Rickettsia densities, association with other symbionts (Portiera and Hamiltonella defensa inside the bacteriocytes) and on the potential for horizontal transmission to the parasitoid wasp, Eretmocerus mundus, while the wasp larvae are developing within the whitefly nymph. Sequences of four Rickettsia genes were found to be identical for both localization patterns, suggesting that they are closely related strains. However, real-time PCR analysis showed very different dynamics for the two localization types. On the first day post-adult emergence, Rickettsia densities were 21 times higher in the "confined" pattern vs. "scattered" pattern whiteflies. During adulthood, Rickettsia increased in density in the "scattered" pattern whiteflies until it reached the "confined" pattern Rickettsia density on day 21. No correlation between Rickettsia densities and Hamiltonella or Portiera densities were found for either localization pattern. Using FISH technique, we found Rickettsia in the gut of the parasitoid wasps only when they developed on whiteflies with the "scattered" pattern. The results suggest that the localization pattern of a symbiont may influence its dynamics within the host.     

Structure of the ovaries and oogenesis in Cixius nervosus (Cixiidae), Javesella pellucida and Conomelus anceps (Delphacidae) (Insecta, Hemiptera, Fulgoromorpha)

Ovary organization in representatives of two families of Fulgoromorpha, Cixiidae (Cixius nervosus) and Delphacidae (Javesella pellucida and Conomelus anceps), was examined by light and transmission electron microscopy. Ovaries of studied fulgoromorphans consist of telotrophic ovarioles. From apex to base individual ovarioles have four well defined regions: a terminal filament, tropharium (trophic chamber), vitellarium and pedicel (ovariolar stalk). Tropharia are not differentiated into distinct zones and consist of syncytial lobes containing multiple trophocyte nuclei embedded in a common cytoplasm. Lobes are radially arranged around a branched, cell-free trophic core. Early previtellogenic (arrested) oocytes and prefollicular cells are located at the base of the tropharium. The vitellarium houses linearly arranged developing oocytes each of which is connected to the trophic core by a broad nutritive cord. Each oocyte is surrounded by a single layer of follicular cells that become binucleate at the beginning of vitellogenesis.     

Spatial and temporal transcellular current patterns during oogenesis.

We have used the two-dimensional vibrating probe to examine spatial and temporal patterns in the transcellular current flow around telotrophic ovarioles of the insect Rhodnius prolixus. We demonstrate a dynamic pattern of currents which correlates with various stages of vitellogenesis. Asymmetries exist in the radial current pattern around intact ovarioles, particularly around the terminal follicle, and may correlate with early developmental axes. The extra-cellular current pattern is largely reflected by a similar, though weaker pattern of currents over the germ cell membranes, indicating that both germ cell and somatic cell membranes are involved in current generation. Current enters previtellogenic oocytes and leaves oocytes entering vitellogenesis. We speculate that current reversal and loss of trophic cord contact may represent an electrophysiological feedback control mechanism during oogenesis.     

Morphology of the ovaries of Sphaerodema (Diplonychus) rusticum (Heteroptera,Belostomatidae)

The female reproductive system of Sphaerodema rusticum consists of a pair of ovaries, two lateral oviducts, a median common oviduct, and a median spermatheca. Accessory glands are absent. Each ovary has five free ovarioles branching from the oviduct. Each ovariole consists of a terminal filament, germarium, vitellarium, brown mass, and an exceptionally long pedicel. The terminal filament consists of a central core, interstitial cells, and an outer sheath. In the germarium, which consists of trophic and prefollicular regions, the trophic region or nurse cell chamber is divided into four histologically differentiated zones, distinguished as zones I–IV. Nutritive cords, originating from the posterior end of the trophic core in zone IV extend centrally and join the developing oocytes in the prefollicular chamber and the vitellarium. The compact prefollicular tissue at the base of the trophic core gives rise to prefollicular cells which, after encircling the young oocytes, become modified into follicular epithelial cells, the interfollicular plug, and epithelial plug. The young oocytes descend into the vitellarium and gradually develop into mature oocytes. A compound corpus luteum is observed simultaneously in all the ovarioles of both ovaries after ovulation. Below the epithelial plug there is an accumulation of material, the “brown mass,” which develops cyclically in correlation with the ovulation cycle. Each pedicel stores five mature chorionated eggs ready for oviposition. The epithelium of the anterior region of the pedicel secretes a PAS-positive material. General morphology and histology of the subdivisions of the ovarioles are described.     

Sea urchin spectrin in oogenesis and embryogenesis: a multifunctional integrator of membrane-cytoskeletal interactions

Using indirect immunofluorescence microscopy on semithin cryosections of maturing ovarian tissue, eggs, and developing embryos, we have mapped the cellular distribution and dynamic redistribution of spectrin in oogenesis and early embryogenesis. During oogenesis, spectrin is initially found in the cortex of oogonia and previtellogenic oocytes, and later accumulates in the cytoplasm of vitellogenic oocytes on the surfaces of cortical granules, pigment granules/acidic vesicles, and yolk platelets. Following egg activation, spectrin undergoes a rapid redistribution coincident with three major developmental events including: (1) restructuring of the cell surface, (2) translocation of pigment granules/acidic vesicles to the cortex during the first cell cycle, and (3) amplification of the embryo's surface during the rapid cleavage phase of early embryogenesis. The synthesis and storage of spectrin during oogenesis appears to prime the egg with a preestablished pool of membrane-cytoskeletal precursor for use during embryogenesis. Results from this study support the hypothesis that spectrin may function as a key integrator and modulator of multiple membrane-cytoskeletal functions during embryonic growth and cellular differentiation.     

Structure of ovarioles in adult queens and workers of the common wasp, Vespula germanica (Hymenoptera: Vespidae)

The ovaries of the common wasp, Vespula germanica are polytrophic-meroistic and consist of 2-3 (workers) or 7 (queens) ovarioles. The ovarioles are differentiated into three regions: a terminal filament, a germarium, and a vitellarium. The germaria of both castes consist of two zones: an anterior zone of germ-cell cluster formation and a posterior one of germ-cell cluster differentiation. The vitellaria comprise 4-6 (workers) or 7-10 (queens) ovarian follicles (egg chambers). Each chamber consists of an oocyte and about 60 isodiametric nurse cells (trophocytes). The egg chambers have been arbitrarily classified into four developmental categories: early and late previtellogenic, vitellogenic, and choriogenic. The process of oogenesis in workers proceeds only up to the onset of the late previtellogenesis. Neither vitellogenic nor choriogenic egg chambers were observed in this caste. During early and late previtellogenesis the envelope of the oocyte nucleus proliferates and becomes highly folded. This process leads to the formation of characteristic organelles, termed accessory nuclei (AN). Although AN arise in the oocytes of both queens and workers, their number in the latter caste is always considerably lower. At the onset of the late previtellogenesis AN start to migrate towards the periphery of the oocyte where they reside till the end of oogenesis. The physiological state of the worker ovaries is discussed in the light of the presented results.     

Structure and development of the telotrophic ovariole in ensign scale insects (Hemiptera, Coccomorpha: Ortheziidae)

The paired ovaries of young larva of the 3rd instar of Orthezia urticae are filled with numerous germ cell clusters that can be regarded as ovariole anlagen. Germ cells (cystocytes) belonging to one cluster form a rosette, in the centre of which a polyfusome occurs. Staining with rhodamine-phalloidin has revealed that polyfusomes contain numerous microfilaments. The number of cystocytes per cluster is not stable and varies considerably. The ovaries of older larva become elongated with numerous young ovarioles protruding into the body cavity. The ovarioles are not subdivided into the tropharium and vitellarium. In this stage germ cells differentiate into oocytes and trophocytes (nurse cells). The ovaries of adult females are composed of about 20 (Newsteadia floccosa) or 30 (O. urticae) ovarioles. Their trophic chambers contain trophocytes and arrested oocytes. In the vitellarium, at the given moment, only one oocyte develops. It has been observed that after maturation of the first egg the arrested oocytes may develop.     

A cellular basis for Wolbachia recruitment to the host germline

Wolbachia are among the most widespread intracellular bacteria, carried by thousands of metazoan species. The success of Wolbachia is due to efficient vertical transmission by the host maternal germline. Some Wolbachia strains concentrate at the posterior of host oocytes, which promotes Wolbachia incorporation into posterior germ cells during embryogenesis. The molecular basis for this localization strategy is unknown. Here we report that the wMel Wolbachia strain relies upon a two-step mechanism for its posterior localization in oogenesis. The microtubule motor protein kinesin-1 transports wMel toward the oocyte posterior, then pole plasm mediates wMel anchorage to the posterior cortex. Trans-infection tests demonstrate that factors intrinsic to Wolbachia are responsible for directing posterior Wolbachia localization in oogenesis. These findings indicate that Wolbachia can direct the cellular machinery of host oocytes to promote germline-based bacterial transmission. This study also suggests parallels between Wolbachia localization mechanisms and those used by other intracellular pathogens.     

Involvement of EDTP, an egg-derived tyrosine phosphatase, in the early development of Drosophila melanogaster

Previously, we purified a novel protein tyrosine phosphatase from eggs of the flesh fly, Sarcophaga peregrina. This protein tyrosine phosphatase, named egg-derived tyrosine phosphatase (EDTP), is expressed during oogenesis and early embryogenesis but is rapidly degraded in middle embryogenesis by lysosomal cathepsin L. Here, we demonstrate the requirement of EDTP in the development of the fruit fly, Drosophila melanogaster. Deletion of the Drosophila EDTP gene using transposase-catalyzed imprecise excision resulted in homozygous lethals during embryogenesis. Additionally, germline clones generated using the FLP-FRT-ovo(D) system showed severe defects in ovarian development during oogenesis. These results indicate that the Drosophila EDTP gene is crucial in oogenesis and embryogenesis.     

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of <Emphasis Type="Italic">Rhodnius prolixus</Emphasis> Stahl

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18–24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.     

Bicaudal-C plays a vital role in oogenesis in Nilaparvata lugens (Hemiptera: Delphacidae)

Bicaudal-C (Bic-C) was originally identified in a Drosophila melanogaster mutagenesis screen and plays vital roles in embryogenesis. In this study, we characterized the Bic-C gene in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), an insect pest that undergoes incomplete metamorphosis. Our result showed that N. lugens Bic-C (NlBic-C) is a female-specific gene in this species. It is specifically expressed in developing oocytes and is not expressed in laid eggs. Ribonucleic acid interference (RNAi) of NlBic-C arrested the uptake of vitelline by oocytes, and resulted in undeveloped ovaries and the complete inhibition of oocyte growth in the ovarioles, suggesting that NlBic-C is required for oogenesis and oocyte maturation. NlBic-C is extremely highly sensitive to RNAi, suggesting that it may be a potential target in RNAi-based insect pest management.