Antifungal skin bacteria,embryonic survival,and communal nesting in four-toed salamanders, <Emphasis Type="Italic">Hemidactylium scutatum</Emphasis>

We examined a novel hypothesis for the maintenance of communal nesting in the salamander, Hemidactylium scutatum, namely that communal nests are more likely than solitary nests to be associated with cutaneous antifungal bacteria, which can inhibit fungal infections of embryos. A communal nest contains eggs of two or more females of the same species. The nesting behavior of H. scutatum females and survival of embryos were determined by frequent nest surveys at three ponds. For communal nests, embryonic survival tended to be higher and catastrophic nest failure was lower. Pure bacterial cultures of resident species were obtained from the salamanders’ skins by swabbing and tested against a fungal pathogen of embryos (Mariannaea sp.) in laboratory assays. We found that 27% of females had skin bacteria inhibitory to Mariannaea sp. Communal nests were more likely to have at least one female with antifungal bacteria than were solitary nests. Using a culture-independent assay (denaturing gradient gel electrophoresis of 16S rRNA gene fragments), we found that bacterial species on females and embryos were more similar to each other than they were to bacterial species found in soil within the nest, suggesting that females transmitted skin bacteria to embryos. The presence of anti-Mariannaea skin bacteria identified from the laboratory assays did not prevent fungal presence in field nests. However, once a nest was visibly infected with fungi, presence of anti-Mariannaea bacteria was positively correlated with survival of embryos. Microbe transmission is usually thought to be a cost of group living, but communal nesting in H. scutatum may facilitate the transmission of antifungal bacteria to embryos.     

Determinants of the prevalence of the cloacal cestode <Emphasis Type="Italic">Cloacotaenia megalops</Emphasis> in teal wintering in the French Camargue

Eurasian teal Anas crecca (n = 46,581) were inspected during ringing operations for the presence of the hymenolepidid cloacal cestode Cloacotaenia megalops between 1954 and 1971 while wintering in the Camargue, Southern France. These birds become infected when ingesting seed shrimps (Ostracoda) that act as intermediate hosts, largely while on migration across Western Europe. The prevalence ranged from 4% to 14% per year and increased significantly over time. This long-term trend was further supported by studying 366 teal shot in 2006–2008, for which prevalence of C. megalops was 27.6%. We found no evidence to suggest that this increase in prevalence has been caused by an increase in temperature, but eutrophication and an increase in duck densities are potential contributing factors. Adult teal were more likely to be infected than first-year birds and females more so than males, probably due to differences in diet and/or habitat use. Within a given age−sex class, heavier birds were more infected than lighter ones, suggesting low pathogenicity and a causal effect of ingestion rate. Within a year, the highest prevalence was observed in mid-winter.     

Phylogeny of a novel “<Emphasis Type="Italic">Helicobacter heilmannii</Emphasis>” organism from a Japanese patient with chronic gastritis based on DNA sequence analysis of 16S rRNA and urease genes

“Helicobacter heilmannii” is an uncultivable spiral-shaped bacterium inhabiting the human gastric mucosa. It is larger and more tightly-coiled than H. pylori. We encountered a patient with chronic gastritis infected a “H. heilmannii”-like organism (HHLO), designated as SH6. Gastric mucosa derived from the patient was orally ingested by specific pathogen free mice. Colonization of the mice by SH6 was confirmed by electron microscopy of gastric tissue specimens. In an attempt to characterize SH6, 16S rRNA and urease genes were sequenced. The 16S rRNA gene sequence was most similar (99.4%; 1,437/1,445 bp) to HHLO C4E from a cheetah. However, the urease gene sequence displayed low similarity (81.7%; 1,240/1,516 bp) with HHLO C4E. Taxonomic analysis disclosed that SH6 represents a novel strain and should constitute a novel taxon in the phylogenetic trees, being discriminated from any other taxon, with the ability of infecting human gastric mucosa.     

Discovery of a Novel Alveolate Pathogen Affecting Southern Leopard Frogs in Georgia: Description of the Disease and Host Effects

In April of 2006, we observed southern leopard frog (Rana sphenocephala) tadpoles in a pond in northeast Georgia that were dying from an unknown pathogen. Examination of affected specimens, as well as PCR characterization, revealed that all were infected with a novel alveolate pathogen closely related to freshwater and marine eukaryotic organisms and, to a lesser degree, to members of the genus Perkinsus. This pathogen has been documented in numerous mortality events in anuran tadpoles in the United States, although it has not yet been named nor clearly described. We subsequently conducted a systematic survey of this and four other ponds in the same area to document the extent of the pathogen and to describe the nature of infections in leopard frog tadpoles. Of 87 live tadpoles examined, 25% were infected with the alveolate pathogen, based on visual inspection of tadpole liver tissue. Affected tadpoles frequently had enlarged abdomens, swam erratically, and could be captured by hand. All organs of infected tadpoles were infiltrated but typically to a lesser extent than the liver and kidneys, which often had hundreds of thousands of the spherical, 6-μm organisms. Infected tadpoles tended to weigh more than noninfected ones, likely due to the massive organ swelling that coincided with infections. Infected tadpoles did not differ in developmental stage from noninfected tadpoles. Infection prevalence varied widely among ponds, and in one pond, we witnessed a rapid die-off of R. spenocephala tadpoles during our surveys, although we did find infected metamorphic frogs. The rapid mortality we observed as well as the vast number of organisms seen in specimens suggests that this pathogen has tremendous transmission potential, and therefore deserves further monitoring and study.     

Parasitemia in PCR‐detected Plasmodium and Haemoproteus infections in birds

Most comparative studies of avian blood parasites based on visual inspection of smears have reported Haemoproteus infections to be more prevalent than Plasmodium infections in both tropical and temperate locations. Recently, molecular techniques have increased our ability to detect infections often missed on blood smears. Here we quantify the bias in prevalence resulting from unrecognized infections by examining blood smears of infected passerine birds from the West Indies (312 individuals) and the Ozark Mountains of southern Missouri (134 individuals) for which we could identify parasites based on cytochrome b sequences. In the West Indian sample, 63 of 179 Haemoproteus infections (35%) and 121 of 133 Plasmodium infections (91%) were not detected among ca. 2,800 red blood cells examined per smear. In the Missouri sample, 19 of 77 Haemoproteus infections (25%) and 31 of 57 Plasmodium infections (54%) were not detected among ca. 10,000 red blood cells examined. Clearly, visual inspection of blood smears at this level of effort fails to recognize many malaria parasite infections ascertained by PCR screening, and this bias for Plasmodium parasites exceeds that for Haemoproteus parasites. The lower prevalence of Plasmodium compared to Haemoproteus reported in comparative studies based on blood smears likely reflects differences in detection rather than infection rates. Estimates obtained from visual inspection of blood smears would appear to be more indicative of parasite virulence and how well host individuals control infections than of the prevalence of infections in host populations.     

Effects of <Emphasis Type="Italic">Acanthocephalus</Emphasis> sp. (Acanthocephala: Echinorhynchidae) on the body size and reproduction of isopods (<Emphasis Type="Italic">Asellus hilgendorfi</Emphasis>)

 Isopods Asellus hilgendorfi were collected from a small lake in northern Japan and examined to determine whether their body size and reproduction were affected by infection with larval acanthocephalans (Acanthocephalus sp.). Seasonal changes in the breeding ratio of isopods and the prevalence of larval acanthocephalan infection showed a reverse trend. Acanthocephalan larvae occurred mainly in males and immature females and were rarely found in mature females. In late immature females, the body size, as indicated by the width of the pleotelson, of infected isopods was significantly larger than that of uninfected ones. These results suggest that acanthocephalans can prevent female isopods from attaining sexual maturity and increasing their body size. Received: January 9, 2002 / Accepted: December 16, 2002 Acknowledgments We thank Professor Shōichi Saito, Faculty of Education, Hirosaki University, for his encouragement of the present study. Thanks are also due to the Iwasaki Village Office and the Fukaura Forestry Office for giving us permission for the survey. Correspondence to:A. Ohtaka     

Investigations into the natural infection rate of <Emphasis Type="Italic">Haemaphysalis qinghaiensis</Emphasis> with Piroplasma using a nested PCR

Here we describe the natural infection rate in China of Haemaphysalis qinghaiensis with four Piroplasma species, namely Theileria uilenbergi, T. luwenshuni, T. sinensis and Babesia motasi. Specifically, a nested PCR was designed based on 18S ribosomal RNA genes and its specificity and sensitivity were established. The result showed that 62 flat adult field H. qinghaiensis ticks (27 females and 35 males) out of 136 (55 females and 81 males) were infected by one or more parasites. All 62 (45.6%) were infected with T. uilenbergi; nine (five males and four females; 6.6%) were infected with T. luwenshuni; two (1.5%) females were infected with T. sinensis; and one (0.7%) male was infected with B. motasi. Twelve (19.4%) were infected with more than one pathogen. There was no significant difference in infection rate between males and females. The high figure 45.6% Theileria infection rate indicates the serious prevalence of theileriosis; while the presence of T. sinensis and B. motasi implies the potential existence of the corresponding diseases in the area studied.     

Detection of Batrachochytrium dendrobatidis in Mexican Bolitoglossine Salamanders Using an Optimal Sampling Protocol

The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further studies would be required to draw a causal link.     

Evidence of <Emphasis Type="Italic">Babesia microti</Emphasis> infection in multi-infected <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

To detect Babesia-infected Ixodes persulcatus Shulze in a suburb of St. Petersburg, Russia, 738 adult ticks were studied using Babesia specific primers and PCR techniques. The entire sample (more than 1,200 individuals) was screened for the presence of Borrelia spp., Ehrlichia spp. and tick-borne encephalitis virus (TBEV). All 7 ticks infected with Babesia microti, were also infected with other pathogens (all 7 among 417 infected ticks, zero amongst the remaining 321 naive ones (χ² = 5.25, p < 0.05). Babesia microti occurred twice with Borrelia afzelii, 3 times with Borrelia garinii, once with both, and once with both B. garinii and TBEV. The prevalence of infection with Borrelia spp. was 34.0%, with Ehrlichia spp. 6.2%, with TBEV 1.5%, and with Ba microti 0.9%. Babesia microti infection was not found in combination with Ehrlichia sp. or Borrelia burgdorferi sensu stricto. The latter pathogen (prevalence 2.6%), just like Ba. microti, was not encountered as a monoinfection. The data suggest that Ba. microti infection can only survive in I. persulcatus in combination with Borrelia spp. (7 of 7 infections). The disease in humans is more severe and longer-lasting when more than one pathogen is involved. Our observations show that the well known St. Petersburg focus of tick-borne encephalitis and Lyme disease is also a focus of ehrlichiosis and babesiosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Associations Between Coinfection Prevalence of <Emphasis Type="Italic">Borrelia lusitaniae</Emphasis>, <Emphasis Type="Italic">Anaplasma</Emphasis> sp., and <Emphasis Type="Italic">Rickettsia</Emphasis> sp. in Hard Ticks Feeding on Reptile Hosts

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host–vector–pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host–vector–pathogen system—green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia–Anaplasma coinfection rates.     

A novel filamentous planctomycete of the <Emphasis Type="Italic">Isosphaera-Singulisphaera</Emphasis> group isolated from a <Emphasis Type="Italic">Sphagnum</Emphasis> peat bog

Planctomycetes are common inhabitants of northern wetlands. A significant proportion of these bacteria revealed in peat with the Planctomycetes-specific probes PLA46 and PLA886 is represented by filamentous forms which have not been cultured under laboratory conditions. In the present work, one of such organisms was isolated from a Sphagnum peat bog in a monoculture. The organism had large spherical cells assembled in long filaments. It could grow only in associations with bacterial satellites; attempts to isolate it in pure culture were unsuccessful. The organism was identified by PCR amplification, cloning, and subsequent analysis of its 16S rRNA gene fragment. Comparative sequence analysis revealed its affiliation with the Isosphaera-Singulisphaera group within the order Planctomycetales. The nucleotide sequence of the 16S rRNA gene of the new organism exhibited 94–96% similarity to those of the unicellular peat-inhabiting planctomycete Singulisphaera acidiphila and uncharacterized filamentous planctomycete “Nostocoida limicola III” from activated sludge. The new planctomycete utilized heteropolysaccharides of microbial origin as growth substrates and could grow at the low pH and temperatures typical of the northern wetlands.     

Survey of Ixodes pacificus Ticks in California Reveals a Diversity of Microorganisms and a Novel and Widespread Anaplasmataceae Species

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called ‘Candidatus Cryptoplasma californiense’. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Inoculum Sources for the Spread of Leaf Scald Disease of Sugarcane Caused by Xanthomonas albilineans in Guadeloupe

Leaf scald of sugarcane, caused by Xanthomonas albilineans, is thought to be spread mainly in infected cuttings and transmitted on infested cutting implements. Several observations made in Guadeloupe indicated that other means of spreading also occur. The dispersal of the pathogen outside sugarcane was investigated with plants inoculated by an antibiotic-resistant marked strain of X. albilineans and with plants naturally infested with wild strains of the pathogen. The bacteria were isolated in water droplets (rain or dew) on the surface of sugarcane leaves at dawn. It was also detected on the surface of dry leaves during the day by leaf imprinting onto a selective culture medium. The bacteria were much more frequently isolated from the surface of symptomatic leaves than from symptomless ones. Aerial dispersal of X. albilineans was investigated by placing Petri dishes containing selective culture medium between sugarcane plants but without direct contact with the leaves. The pathogen was isolated in four out of 270 dishes which were randomly set 3–14 h in a diseased field. These results indicated that the pathogen exuded from the leaves and then was spread by aerial means (rain, insects, …) or by leaf contact. The bacteria were also found in roots and rhizospheric soil of infested sugarcane stools suggesting that X. albilineans could be transmitted by root to root contact or by the soil. Finally, isolations of the pathogen in sugarcane inflorescences were positive. So, fuzz transmission may also occur.     

Cutaneous Bacteria of the Redback Salamander Prevent Morbidity Associated with a Lethal Disease

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an infectious disease that causes population declines of many amphibians. Cutaneous bacteria isolated from redback salamanders, Plethodon cinereus, and mountain yellow-legged frogs, Rana muscosa, inhibit the growth of Bd in vitro. In this study, the bacterial community present on the skin of P. cinereus individuals was investigated to determine if it provides protection to salamanders from the lethal and sub-lethal effects of chytridiomycosis. When the cutaneous bacterial community was reduced prior to Bd exposure, salamanders experienced a significantly greater decrease in body mass, which is a symptom of the disease, when compared to infected individuals with a normal bacterial community. In addition, a greater proportion of infected individuals with a reduced bacterial community experienced limb-lifting, a behavior seen only in infected individuals. Overall, these results demonstrate that the cutaneous bacterial community of P. cinereus provides protection to the salamander from Bd and that alteration of this community can change disease resistance. Therefore, symbiotic microbes associated with this species appear to be an important component of its innate skin defenses.     

Inoculum Sources for the Spread of Leaf Scald Disease of Sugarcane Caused by Xanthomonas albilineans in Guadeloupe

Leaf scald of sugarcane, caused by Xanthomonas albilineans, is thought to be spread mainly in infected cuttings and transmitted on infested cutting implements. Several observations made in Guadeloupe indicated that other means of spreading also occur. The dispersal of the pathogen outside sugarcane was investigated with plants inoculated by an antibiotic-resistant marked strain of X. albilineans and with plants naturally infested with wild strains of the pathogen. The bacteria were isolated in water droplets (rain or dew) on the surface of sugarcane leaves at dawn. It was also detected on the surface of dry leaves during the day by leaf imprinting onto a selective culture medium. The bacteria were much more frequently isolated from the surface of symptomatic leaves than from symptomless ones. Aerial dispersal of X. albilineans was investigated by placing Petri dishes containing selective culture medium between sugarcane plants but without direct contact with the leaves. The pathogen was isolated in four out of 270 dishes which were randomly set 3-14 h in a diseased field. These results indicated that the pathogen exuded from the leaves and then was spread by aerial means (rain, insects,…) or by leaf contact. The bacteria were also found in roots and rhizospheric soil of infested sugarcane stools suggesting that X. albilineans could be transmitted by root to root contact or by the soil. Finally, isolations of the pathogen in sugarcane inflorescences were positive. So, fuzz transmission may also occur.     

Identification,distribution and occurrence of the ascomycete <Emphasis Type="Italic">Metschnikowia typographi</Emphasis> in the great spruce bark beetle, <Emphasis Type="Italic">Dendroctonus micans</Emphasis>

This is the first report on the ascomycete Metschnikowia typographi from the adults and larvae of the great spruce bark beetle Dendroctonus micans in Turkey. In total, 910 of 1928 adults and 44 of 149 larvae investigated during the two years were infected by the pathogen. In a fresh smear the asci of the pathogen measure 18.5 ± 2.05 μm (14.7–22.3) in length and 2.1 ± 0.4 μm in width (n = 35). The ascospores are about 2 μm shorter than asci, having an average length of 16.4 ± 1.5 μm (14.2–18.0). The total infection rate of D. micans was 47.2 %. The prevalence of M. typographi infections differed between localities and years. Different infection rates of male and female beetles of D. micans were not recognized.     

Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus)

Beneficial cutaneous bacteria on amphibians can protect against the lethal disease chytridiomycosis, which has devastated many amphibian species and is caused by the fungus Batrachochytrium dendrobatidis. We describe the diversity of bacteria on red-backed salamanders (Plethodon cinereus) in the wild and the stability of these communities through time in captivity using culture-independent Illumina 16S rRNA gene sequencing. After field sampling, salamanders were housed with soil from the field or sterile media. The captive conditions led to different trajectories of bacterial communities. Eight OTUs present on >90% of salamanders in the field, through time, and in both treatments were defined as the core community, suggesting that some bacteria are closely associated with the host and are independent of an environmental reservoir. One of these taxa, a Pseudomonas sp., was previously cultured from amphibians and found to be antifungal. As all host-associated bacteria were found in the soil reservoir, environmental microbes strongly influence host–microbial diversity and likely regulate the core community. Using PICRUSt, an exploratory bioinformatics tool to predict gene functions, we found that core skin bacteria provided similar gene functions to the entire community. We suggest that future experiments focus on testing whether core bacteria on salamander skin contribute to the observed resistance to chytridiomycosis in this species even under hygenic captive conditions. For disease-susceptible hosts, providing an environmental reservoir with defensive bacteria in captive-rearing programs may improve outcomes by increasing bacterial diversity on threatened amphibians or increasing the likelihood that defensive bacteria are available for colonization.     

Does multiple paternity influence offspring disease resistance?

It has been suggested that polyandry allows females to increase offspring genetic diversity and reduce the prevalence and susceptibility of their offspring to infectious diseases. We tested this hypothesis in wild‐derived house mice (Mus musculus) by experimentally infecting the offspring from 15 single‐ and 15 multiple‐sired litters with two different strains of a mouse pathogen (Salmonella Typhimurium) and compared their ability to control infection. We found a high variation in individual infection resistance (measured with pathogen loads) and significant differences among families, suggesting genetic effects on Salmonella resistance, but we found no difference in prevalence or infection resistance between single‐ vs. multiple‐sired litters. We found a significant sex difference in infection resistance, but surprisingly, males were more resistant to infection than females. Also, infection resistance was correlated with weight loss during infection, although only for females, indicating that susceptibility to infection had more harmful health consequences for females than for males. To our knowledge, our findings provide the first evidence for sex‐dependent resistance to Salmonella infection in house mice. Our results do not support the hypothesis that multiple‐sired litters are more likely to survive infection than single‐sired litters; however, as we explain, additional studies are required before ruling out this hypothesis.     

High‐prevalence male‐killing Wolbachia in the butterfly Acraea encedana

Inherited bacteria which kill males during early development are widely distributed throughout the insects, but have been little studied outside of a single family of beetles, the Coccinellidae. We have investigated a male‐killing bacterium discovered in the butterfly Acraea encedana. This bacterium belongs to the genus Wolbachia and is identical in wsp gene sequence to a male‐killer in the closely related butterfly A. encedon, suggesting that it has either recently moved between host species or was inherited from a common ancestor of the butterflies. The prevalence of Wolbachia is remarkably high, 95% of females are infected and only 6% of wild caught butterflies are male. Measurements of the vertical transmission efficiency were used to calculate that this high prevalence is the result of infected females producing at least 1.79 times the number of surviving daughters as uninfected females (lower confidence limit is 1.25).