Differential diagnosis between Tunga penetrans (L., 1758) and T. trimamillata Pampiglione et al., 2002 (Insecta, Siphonaptera), the two species of the genus Tunga parasitic in man

Of the ten currently known species of sand fleas, only two, Tunga penetrans and Tunga trimamillata, are known to be parasites of man, besides other warm blooded animals, most of which are peridomestic. The hosts of the other eight are limited to a few genera of wild mammals. T. trimamillata was only recently identified and differentiated from T. penetrans by features of the gravid female phase. In the present paper the different morphological characters of both for non-gravid females and males of the two species are described. In the non-gravid-females the distinguishing characters are: a) differences in the length of the first two segments of the maxillary palps (the first is the longest in T. trimamillata, whereas the second is the longest in T. penetrans), this feature is associated with another character i.e. the presence of short, thick spines in addition to the bristles on the surface of the segments only in T. trimamillata; b) the presence, only in T. trimamillata, of a row of spines on the antero-medial surface of the libia of the 3rd pair of legs; c) the last abdominal spiracle protrudes in T. trimamillata but not in T. penetrans; d) the hood of the hilla in spermatheca is surrounded by a papilla only in T. penetrans. The following morphological characters differentiate males of T. trimamillata and T. penetrans: a) T. trimamillata has a row of spines on the antero-medial surface of the tibia of the 3rd pair of legs; b) the diameter of the abdominal spiracles of T. trimamillata is smaller than that of T. penetrans and the edges of the spiracles are more regular; c) the claspers and aedeagus of T. trimamillata are shorter than those of T. penetrans; d) the profile of the oedeagal apodeme differs between the two species.     

Molecular characterization of Tunga trimamillata and T. penetrans (Insecta, Siphonaptera, Tungidae): taxonomy and genetic variability

A new species of the genus Tungo, T. trimamillata has recently been described on the basis of several morphological traits. To explore the taxonomic status of this flea with respect to T. penetrans, we undertook a molecular analysis of cytochrome oxydase II and 16S rDNA mitochondrial genes and of the internal transcribed spacer 2 nuclear marker on samples of both species. Maximum Parsimony evaluations of the three data set indicate a differentiation compatible with a specific rank between the two fleas with very high levels of divergence. Both mitochondrial and nuclear data are in line with a recent bottleneck in the Malagasy population of T. penetrans, possibly due to the recent colonisation of Africa via human transportation. Further, significantly lower mitochondrial variability in the Ecuadorian populations of T. penetrans with respect to the T. trimamillata ones is also evidenced.     

Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites

In search of Wolbachia in human parasites, Wolbachia were identified in the sand flea Tunga penetrans. PCR and DNA sequencing of the bacterial 16S rDNA, the ftsZ cell division protein, the Wolbachia surface protein (wsp) and the Wolbachia aspartate aminotransferase genes revealed a high similarity to the respective sequences of endosymbionts of filarial nematodes. Using these sequences a phylogenetic tree was generated, that indicates a close relationship between Wolbachia from T. penetrans and from filarial parasites, but possibly as a member of a new supergroup. Ultrastructural studies showed that Wolbachia are abundant in the ovaries of neosomic fleas, whereas other, smaller and morphologically distinct, bacteria were observed in the lumen of the intestine. Wolbachia were labeled by immunohistology and immunogold electron microscopy using polyclonal antibodies against wsp of Drosophila, of the filarial parasite Dirofilaria immitis, or against hsp 60 from Yersinia enterocolitica. These results show that as in filariasis, humans with tungiasis are exposed to Wolbachia. Furthermore, antisera raised against proteins of Wolbachia from arthropods or from filarial parasites can be immunologically cross-reactive.     

Blood-feeding of Tunga penetrans males

The jigger Tunga penetrans (Linnaeus, 1758: type-species of the family Tungidae) is the smallest known species of flea (Siphonaptera), causing serious ectoparasitosis of humans and domestic animals. The adult female Tunga lodges in the epidermis of the mammalian host, grows by neosomy, becomes gravid and expels eggs. Relatively little is known about the free-living male Tunga adults. Among impoverished communities of Fortaleza in north-east Brazil, we observed T. penetrans males as well as females penetrating the skin of human hosts. After penetrating the epidermis for a few hours, evidently for capillary feeding from the dermis, males withdrew their mouthparts and crawled away, whereas the females remained completely embedded, hypertrophying to become gravid, eventually dying in situ after oviposition. Caged rats were placed on the sandy soil and examined periodically for Tunga infestation. On five rats we obtained 140 females embedded and we detected 75 males biting, with rat erythrocytes observed in the proventriculus and midgut of all five males dissected and examined microscopically. This confirms that T. penetrans males are hamatophagous ectoparasites of mammals.     

Wolbachia infections in the Cimicidae: museum specimens as an untapped resource for endosymbiont surveys

Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality.     

High incidence of the maternally inherited bacterium Cardinium in spiders

Inherited bacteria are now recognized as important players in arthropod evolution and ecology. Here, we test spiders, a group recently identified as possessing inherited bacteria commonly, for the presence of two reproductive parasites, Cardinium hertigii (Bacteroidetes group) and Wolbachia (α-proteobacteria), estimating incidence, prevalence, any sex bias in infection, and infection diversity, for a panel of field-collected specimens. We identify spiders as a hotspot for Cardinium . Present in 22% of the sampled species, incidence was significantly higher than that previously recorded in insects. Where present, Cardinium infection occurred at medium prevalence without evidence of sex bias in prevalence that would indicate sex-ratio distortion activity. Wolbachia was present in 37% of species, but revealed a gradation from being rare to very common. In one case, Wolbachia was found significantly more commonly in females than males, indicating it may act as a sex-ratio distorter in some species. Breeding work conducted on two species confirmed that Wolbachia and Cardinium were transmitted maternally, which represents the first proof of inheritance of these symbionts in spiders. Overall, this study demonstrates that the majority of spider species are infected with inherited bacteria, and their role in host biology clearly requires determination.     

Anatomy of Tunga trimamillata Pampiglione et al., 2002 (Insecta, Siphonaptera, Tungidae) and developmental phases of the gravid female

This paper deals with some internal anatomical features observed in histological sections and freshly dissected mounts of Tunga trimamillata, a Siphonaptera recently discovered in Andean regions of Ecuador from several mammals, including man. It was possible to study in males and also non-gravid and gravid females, the location and anatomy of several organs not previously described for this species: the testes, epididymis, ganglia, Malpighian tubules, eyes, rectal ampulla with one of its pads and structures which could be interpreted as midgut diverticula, whose presence has not been recorded in the Siphonaptera. The process of neosomy in the female during pregnancy is illustrated by photographs of the consecutive developmental phases, taken at the stereomicroscope. Furthermore, some details of the exoskeleton, spermatheca during different phases of pregnancy of the gravid female and the presence of a foreign body (parasite?) within the haemocoel have been displayed in specimens cleared with Hoyer's medium.     

Selfish element maintains sex in natural populations of a parasitoid wasp

Genomic conflicts between heritable elements with different modes of inheritance are important in the maintenance of sex and in the evolution of sex ratio. Generally, we expect sexual populations to exhibit a 1:1 sex ratio. However, because of their biology, parasitoid wasps often exhibit a female-biased sex ratio. Sex-ratio distorters can further alter this optimum, sometimes leading to the complete loss of sexual reproduction. In the parasitoid wasp Trichogramma kaykai ca. 4-26% of females in field populations are infected with a bacterial sex-ratio distorter, Wolbachia, allowing virgin mothers to produce daughters. In some micro-Hymenoptera these infections have led to the complete loss of sex, but in field populations of T. kaykai the proportion of individuals infected remains relatively stable. We tested several hypotheses to explain this low infection level, including inefficient and horizontal transmission of Wolbachia, suppressor genes negating the effect of Wolbachia and the presence of male-biasing sex-ratio distorters. Here, a male-biasing sex-ratio distorter, a parasitic B chromosome, causing females to produce only sons, keeps the frequency of Wolbachia low. The male-biasing factor of T. kaykai is the second known case of a B chromosome manipulating the reproduction of a parasitoid wasp.     

The Spiroplasma heritable bacterial endosymbiont of Drosophila

Since the discovery of the small, gram-positive bacterium, Spiroplasma, as a sex-ratio distorting agent in Drosophila over 50 years ago, substantial progress has been made in understanding the relationship of this bacteria with its insect host. Thus far, spiroplasmas have been found as heritable endosymbionts in sixteen different species of Drosophila. In some species these bacteria cause a male-killing phenotype, where the males die during embryogenesis. In other species, however, Spiroplasma does not cause male-killing, and its fitness effects are unclear. Though recent research has identified multiple factors that affect the prevalence and transmission of spiroplasmas in Drosophila populations, much work remains to fully characterize this symbiosis. Spiroplasma is the only identified heritable bacterial endosymbiont of Drosophila, other than Wolbachia, and can serve as a useful as model for elucidating the nature of insect/bacterial interactions.     

Male-killing Wolbachia in a flour beetle

The bacteria in the genus Wolbachia are cytoplasmically inherited symbionts of arthropods. Infection often causes profound changes in host reproduction, enhancing bacterial transmission and spread in a population. The reproductive alterations known to result from Wolbachia infection include cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males, fecundity enhancement, male killing and, perhaps, lethality Here, we report male killing in a third insect, the black flour beetle Tribolium madens, based on highly female-biased sex ratios of progeny from females infected with Wolbachia. The bias is cytoplasmic in nature as shown by repeated backcrossing of infected females with males of a naturally uninfected strain. Infection also lowers the egg hatch rates significantly to approximately half of those observed for uninfected females. Treatment of the host with antibiotics eliminated infection, reverted the sex ratio to unbiased levels and increased the percentage hatch. Typically Wolbachia infection is transmitted from mother to progeny, regardless of the sex of the progeny; however, infected T. madens males are never found. Virgin females are sterile, suggesting that the sex-ratio distortion in T. madens results from embryonic male killing rather than parthenogenesis. Based on DNA sequence data, the male-killing strain of Wolbachia in T. madens was indistinguishable from the CI-inducing Wolbachia in Tribolium confusum, a closely related beetle. Our findings suggest that host symbiont interaction effects may play an important role in the induction of Wolbachia reproductive phenotypes.     

Efficient procedure for purification of obligate intracellular Wolbachia pipientis and representative amplification of its genome by multiple-displacement amplification

Bacteria belonging to the genus Wolbachia are obligatory microendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 x 10(7) cells). We obtained sufficient amounts (8 to 10 microg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.     

Strategies for introducing Wolbachia to reduce transmission of mosquito-borne diseases

Certain strains of the endosymbiont Wolbachia have the potential to lower the vectorial capacity of mosquito populations and assist in controlling a number of mosquito-borne diseases. An important consideration when introducing Wolbachia-carrying mosquitoes into natural populations is the minimisation of any transient increase in disease risk or biting nuisance. This may be achieved by predominantly releasing male mosquitoes. To explore this, we use a sex-structured model of Wolbachia-mosquito interactions. We first show that Wolbachia spread can be initiated with very few infected females provided the infection frequency in males exceeds a threshold. We then consider realistic introduction scenarios involving the release of batches of infected mosquitoes, incorporating seasonal fluctuations in population size. For a range of assumptions about mosquito population dynamics we find that male-biased releases allow the infection to spread after the introduction of low numbers of females, many fewer than with equal sex-ratio releases. We extend the model to estimate the transmission rate of a mosquito-borne pathogen over the course of Wolbachia establishment. For a range of release strategies we demonstrate that male-biased release of Wolbachia-infected mosquitoes can cause substantial transmission reductions without transiently increasing disease risk. The results show the importance of including mosquito population dynamics in studying Wolbachia spread and that male-biased releases can be an effective and safe way of rapidly establishing the symbiont in mosquito populations.     

The Sex-Ratio Trait in Drosophila Simulans: Genetic Analysis of Distortion and Suppression

The sex-ratio trait described in several Drosophila species is a type of naturally occurring X-linked meiotic drive that causes males bearing a sex-ratio X chromosome to produce progenies with a large excess of females. We have previously reported the occurrence of sex-ratio X chromosomes in Drosophila simulans. In this species, because of the co-occurrence of drive suppressors, the natural populations and the derived laboratory strains show an equal sex-ratio even when sex-ratio X chromosomes are present at a high frequency. The presence of sex-ratio X chromosomes is established via crosses with a standard strain that is devoid of drive suppressors. In this article, we show first that the sex-ratio trait in D. simulans results from the action of several X-linked loci. Second we describe drive suppressors on each major autosome as well as on the Y chromosome. The Y-linked factors suppress the drive partially whereas the autosomal suppression can be complete.     

Wolbachia genomes: insights into an intracellular lifestyle

The genome sequence of the Wolbachia endosymbiont that infects the nematode Brugia malayi has recently been determined together with three partial Wolbachia genomes from different Drosophila species. These data along with the previously published Wolbachia genome from Drosophila melanogaster provide new insights into how this endosymbiont has managed to become so successful.     

On the evolution of cytoplasmic incompatibility in haplodiploid species

The most enigmatic sexual manipulation by Wolbachia endosymbionts is cytoplasmic incompatibility (CI): infected males are reproductively incompatible with uninfected females. In this paper, we extend the theory on population dynamics and evolution of CI, with emphasis on haplodiploid species. First, we focus on the problem of the threshold to invasion of the Wolbachia infection in a population. Simulations of the dynamics of infection in small populations show that it does not suffice to assume invasion by drift alone (or demographic "accident"). We propose several promising alternatives that may facilitate invasion of Wolbachia in uninfected populations: sex-ratio effects, meta population structure, and other fitness-compensating effects. Including sex-ratio effects of Wolbachia allows invasion whenever infected females produce more infected daughters than uninfected females produce uninfected daughters. Several studies on haplodiploid species suggest the presence of such sex-ratio effects. The simple metapopulation model we analyzed predicts that, given that infecteds are better "invaders," uninfecteds must be better "colonizers" to maintain coexistence of infected and uninfected patches. This condition seems more feasible for species that suffer local extinction due to predation (or parasitization) than for species that suffer local extinction due to overexploiting their resource(s). Finally, we analyze the evolution of CI in haplodiploids once a population has been infected. Evolution does not depend on the type of CI (female mortality or male production), but hinges solely on decreasing the fitness cost and/or increasing the transmission efficiency. Our models offer new perspectives for increasing our understanding of the population and evolutionary dynamics of CI.     

Host-specific Wolbachia strains in widespread populations of Phlebotomus perniciosus and P. papatasi (Diptera: Psychodidae), and prospects for driving genes into these vectors of Leishmania

A single strain of Wolbachia (alpha-proteobacteria, Rickettsiales) was found in widespread geographical populations of each of two Phlebotomus species, within which there was no indication of 'infectious speciation'. The two strains were identified by sequencing a fragment of wsp (a major surface protein gene), amplified by polymerase chain reaction from DNA extracted from the body parts of individual sandflies. Infection rates were high in the males and females of both sandflies, but they were lower for the B-group wPrn strain of Wolbachia in Phlebotomus perniciosus Newstead (60.3% overall) than for the A-group wPap strain in P. papatasi (Scopoli) (81.7%). Infections were frequent in the thorax, where Leishmania develops infective forms, as well as in the abdomen, where Wolbachia must infect the reproductive tissues to ensure its vertical transmission. These findings were related to knowledge of the population biology of Wolbachia in other insects, leading to the conclusion that this endosymbiont could be useful for driving transgenes through wild populations of both sandflies. This will require characterizing the cytoplasmic incompatibility phenotypes of Wolbachia-sandfly combinations, as well as estimating for them the incidence of paternal transmission and the fidelity of maternal transmission. Paternal transmission is one explanation for finding a single Wolbachia strain associated with all mitochondrial haplotypes and lineages of each sandfly species. However, this distribution pattern could also result from multiple horizontal transmissions or the failure of wsp to provide strain markers.     

Evidence for low-titre infections in insect symbiosis: Wolbachia in the bark beetle Pityogenes chalcographus (Coleoptera, Scolytinae)

Wolbachia are obligatory endosymbiotic α-proteobacteria found in many insect species. They are maternally transmitted and often exhibit reproductive phenotypes like cytoplasmic incompatibility. Pityogenes chalcographus is a bark beetle causing severe damage in spruce stands. Its European populations are divided into several mitochondrial clades separated by partial crossing barriers. In this study, we tested a large sample set covering the natural range of the beetle in Europe for the presence of Wolbachia and associations between infection pattern and mitotypes using a highly sensitive nested PCR technique. 35.5% of the individuals were infected with the endosymbiont and two distinct strains were identified. Both strains occur in low titre not accessible by conventional detection methods. The infections are present all over Europe, unlikely to cause the partial crossing barriers in this host and uncoupled from mitochondrial clades. This pattern is indicative for populations evolving towards endosymbiont loss and for repeated intraspecific horizontal transfer of Wolbachia . Alternatively, the low-titre infections found in P. chalcographus are yet another example for Wolbachia that can persist in host species at low densities and frequencies.     

Wolbachia与昆虫寄主关系研究进展

Wolbachia pipientis是一种广泛存在于节肢动物和线虫生殖组织中的细胞内共生菌,通过母系生殖细胞在寄主种群内垂直传播。据分析,Wolbachia在昆虫中的感染率大约为66%,是昆虫中分布最广泛的胞内共生菌。Wolbachia能够以多种方式调控寄主的生殖行为,包括诱导细胞质不亲和、诱导孤雌生殖、雌性化、杀雄作用等。近10年来,Wolbachia的研究在多个领域都取得了长足进展。本文介绍了Wolbachia的多样性与分布、对寄主生殖行为的影响、基因组结构,以及其与寄主在基因组水平上的相互作用等领域的最新研究成果,并展望了Wolbachia研究的发展趋势。