Chloroplasts in the epidermis of Sarracenia (the American pitcher plant) and their possible role in carnivory - An immunocytochemical approach

A photosynthetic apparatus is present in the epidermis of the bottom zone of the pitcher of Sarracenia purpurea L. ssp. purpurea. This has been demonstrated using conventional light and electron microscopy, as well as fluorescent and immunohistochemical techniques. Red intrinsic fluorescence by these chloroplasts indicates photochemical activity. Antibodies against the coupling factor of chloroplast ATPase and against the subunits of ribulose-bis-phosphate-carboxylase were bound to the epidermal chloroplasts. This has been visualized using a ferritin-isothiocyanate labeled second antibody. These results unequivocally prove the existence of the two main proteins which are associated with the photophosphorylation (membrane protein) and carbon dioxide fixation (stromal protein). The possible implication of this system to interrelationships between the carnivorous plant and aquatic insects inhabiting its leaves is discussed.     

Interspecific and intraspecific variation in seed size and germination requirements of Sarracenia (Sarraceniaceae)

Seed size and germination requirements of eight (of nine) Sarracenia species, and 13 populations of S. purpurea were studied. All species except for S. purpurea are restricted to the southeastern United States, whereas S. purpurea ranges across Canada, southward along the eastern United States into Maryland and Virginia (S. purpurea ssp. purpurea), and from New Jersey southward into northern Florida and the coast of the Gulf of Mexico (S. purpurea ssp. venosa). I tested the hypotheses that dormancy-breaking requirements vary predictably among species across a latitudinal gradient. I also sought to determine whether seed size and germination requirements were useful characters for resolving systematic and phylogenetic questions within this genus. Seed size varied significantly among species, but variability in seed size within S. purpurea exceeded the variability in seed size observed across all eight species studied. Seeds of all species are morphophysiologically dormant upon dispersal. Length of required cool, moist pretreatment varied among species, and germination in higher latitude populations is enhanced with longer pretreatment. In contrast, variability in germination requirements of subspecies, varieties, and populations of the geographically wide-ranging S. purpurea was not related clearly to geographic location (latitude or elevation). Germination requirements do not map onto a proposed phylogeny of Sarracenia, but observed differences in germination requirements of S. purpurea ssp. venosa var. burkii relative to other populations of S. purpurea support the recent proposal to elevate this variety to species status.     

Morphological variation in Sarracenia purpurea (Sarraceniaceae): geographic, environmental, and taxonomic correlates

Geographic variation in morphology reflects phenotypic responses to environmental gradients and evolutionary history of populations and species and may indicate local or regional changes in environmental conditions. The pitcher plant (Sarracenia purpurea) illustrates these principles. At local scales, its morphology reflects nutrient availability. At points along its broad geographic range (from Florida to northern Canada) morphology has been used to distinguish subspecies and varieties, but there has been no detailed study of the continuum of morphological variation across this entire range. Patterns of morphological variation in S. purpurea were characterized as a function of climatic and environmental conditions at 39 sites spanning its range. Differences in pitcher size and shape were strongly correlated with temperature, annual precipitation, and availability of ammonium and calcium in peat pore water. Pitcher shape (lip width, mouth diameter, and pitcher width) in Florida panhandle populations differed significantly from pitcher shape of all other populations, even after accounting for environmental correlations. In contrast, the northern and southern subspecies of S. purpurea (the latter exclusive of the Florida panhandle populations) cannot be distinguished based on these morphological measurements alone. These results support a recent proposal that identifies the Florida populations as a distinct species, Sarracenia rosea.     

Occurrence of the rotifer,Habrotrocha cf. rosa Donner,in the purple pitcher plant,Sarracenia purpurea L., (Sarraceniaceae) along the eastern seaboard of North America

Sarracenia purpurea L. plants were studiedin seven bogsalong the eastern seaboard of the United States fromNew Jersey to Georgia. Water sampled from 225 leaveswas found to contain several invertebrates. A singlebdelliod rotifer, a form of Habrotrocha cf. rosaDonner very similar to that described by Bateman(1987) in S. purpurea in western Newfoundland,was identified in 70% of the leaves. Investigationsare in progress to examine the possibility that thisstrong association indicates H. cf. rosa isspecific to S. purpurea.     

Detection of methanogenic archaea in the pitchers of the Northern pitcher plant (Sarracenia purpurea )

Carnivorous plants of the genus Sarracenia rely on microorganisms in their pitchers to decompose drowned insects. The environment inside pitchers is considered to be aerobic; however, there might be zones, such as at the bottom of the pitcher, where anaerobic conditions develop. Samples of the sediment at the bottom of Sarracenia purpurea pitchers were analyzed for the presence of archaea, using PCR and sequencing of the 16S rRNA gene. Archaeal DNA was detected in 20% of sampled pitchers. All sequences were closely related to Methanobrevibacter . Therefore, pitchers may contain anoxic zones inhabited by methanogens.     

Unraveling the structure of the variola topoisomerase IB-DNA complex: a possible new twist on smallpox therapy

Smallpox is a serious and highly contagious disease that is caused by the variola virus. It is one of the most severe infectious human diseases known, with mortality rates as high as 30%. A successful worldwide vaccination program led to the eradication of smallpox in 1980. However, the high transmission rate of variola virus, coupled with the deadly nature of smallpox, makes this virus a potentially devastating weapon for bioterrorism. Currently, there is no specific treatment for smallpox. However, a recent article on the structure of a variola topoisomerase IB-DNA complex provides an intriguing starting point for the rational design of drugs with potential activity against smallpox.     

Protection of Rabbits and Immunodeficient Mice against Lethal Poxvirus Infections by Human Monoclonal Antibodies

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.     

Do carnivorous plants use volatiles for attracting prey insects?

1.  Scientists have been fascinated by carnivorous plants for centuries and they have thoroughly investigated how these plants can benefit from insect capture for example through increased growth, earlier flowering, and increased seed production. How prey is actually lured into the traps, however, is less well understood. Trapping prey may be achieved in a random way, for example by camouflaging the traps (hiding them in the surrounding vegetation), so that prey is trapped by accidental landing on the trap leaves or wind drift, or in the other extreme, trapping may involve mimicry of other attractive resources such as fruits or flowers by using specific visual or olfactory signals to attract a specific prey assemblage.
2.  We investigated for the first time volatiles of the trapping leaves of carnivorous plant species by dynamic headspace methods. We present data on the venus flytrap Dionaea muscipula , the sundew Drosera binata , and the North American pitcher plants Sarracenia flava , Sarracenia leucophylla , Sarracenia minor , and Sarracenia purpurea . A large number of compounds and relatively high emission rates were found in three of the North American pitcher plants ( S. flava , S. leucophylla , and S. minor ) with compounds typically found in flowers or fruits. This suggests together with other features (e.g. colour, nectar production) that these traps are possibly flower or fruit mimics. The leaves of S. purpurea , Dionaea muscipula , and Drosera binata emitted much weaker scents with lower numbers of components, consisting mainly of volatiles typically emitted from green leaves.
3.  We discuss whether or not the use of volatiles for attracting prey animals is linked with specific trapping mechanisms and whether carnivorous plants can be grouped into specialized 'olfactory syndromes'.     

Immunopathogenesis of poxvirus infections: forecasting the impending storm

Variola virus, the causative agent of smallpox, is a member of the poxvirus family and one of the most virulent human pathogens known. Although smallpox was eradicated almost 30 years ago, it is not understood why the mortality rates associated with the disease were high, why some patients recovered, and what constitutes an effective host response against infection. As variola virus infects only humans, our current understanding of poxvirus infections comes largely from historical clinical data from smallpox patients and from animal studies using closely related viruses such as ectromelia, myxoma and monkeypox. The outcome of an infection is determined by a complex interaction between the type of immune response mounted by the host and by evasion mechanisms that the virus has evolved to subvert it. Disease pathogenesis is also a function of both host and viral factors. Poxviruses are not only cytopathic, causing host tissue damage, but also encode an array of immunomodulatory molecules that affect the severity of disease. The ability of the host to control virus replication is therefore critical in limiting tissue damage. However, in addition to targeting virus, the immune response can inadvertently damage the host to such a degree that it causes illness and even death. There is growing evidence that many of the symptoms associated with serious poxvirus infections are a result of a 'cytokine storm' or sepsis and that this may be the underlying cause of pathology.     

Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus

The use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naive and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and immunocompromised animals, and its efficacy against smallpox is currently being addressed. Here we directly compare the efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus and is therefore an important candidate for the protection of humans against smallpox.     

Phylogeny and biogeography of the carnivorous plant family Sarraceniaceae

The carnivorous plant family Sarraceniaceae comprises three genera of wetland-inhabiting pitcher plants: Darlingtonia in the northwestern United States, Sarracenia in eastern North America, and Heliamphora in northern South America. Hypotheses concerning the biogeographic history leading to this unusual disjunct distribution are controversial, in part because genus- and species-level phylogenies have not been clearly resolved. Here, we present a robust, species-rich phylogeny of Sarraceniaceae based on seven mitochondrial, nuclear, and plastid loci, which we use to illuminate this family's phylogenetic and biogeographic history. The family and genera are monophyletic: Darlingtonia is sister to a clade consisting of Heliamphora+Sarracenia. Within Sarracenia, two clades were strongly supported: one consisting of S. purpurea, its subspecies, and S. rosea; the other consisting of nine species endemic to the southeastern United States. Divergence time estimates revealed that stem group Sarraceniaceae likely originated in South America 44-53 million years ago (Mya) (highest posterior density [HPD] estimate = 47 Mya). By 25-44 (HPD = 35) Mya, crown-group Sarraceniaceae appears to have been widespread across North and South America, and Darlingtonia (western North America) had diverged from Heliamphora+Sarracenia (eastern North America+South America). This disjunction and apparent range contraction is consistent with late Eocene cooling and aridification, which may have severed the continuity of Sarraceniaceae across much of North America. Sarracenia and Heliamphora subsequently diverged in the late Oligocene, 14-32 (HPD = 23) Mya, perhaps when direct overland continuity between North and South America became reduced. Initial diversification of South American Heliamphora began at least 8 Mya, but diversification of Sarracenia was more recent (2-7, HPD = 4 Mya); the bulk of southeastern United States Sarracenia originated co-incident with Pleistocene glaciation, <3 Mya. Overall, these results suggest climatic change at different temporal and spatial scales in part shaped the distribution and diversity of this carnivorous plant clade.     

Progression of pathogenic events in cynomolgus macaques infected with variola virus

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.     

Fungal endophyte diversity in Sarracenia

Fungal endophytes were isolated from 4 species of the carnivorous pitcher plant genus Sarracenia: S. minor, S. oreophila, S. purpurea, and S. psittacina. Twelve taxa of fungi, 8 within the Ascomycota and 4 within the Basidiomycota, were identified based on PCR amplification and sequencing of the internal transcribed spacer sequences of nuclear ribosomal DNA (ITS rDNA) with taxonomic identity assigned using the NCBI nucleotide megablast search tool. Endophytes are known to produce a large number of metabolites, some of which may contribute to the protection and survival of the host. We speculate that endophyte-infected Sarracenia may benefit from their fungal associates by their influence on nutrient availability from within pitchers and, possibly, by directly influencing the biota within pitchers.     

LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.     

Characterization of microsatellite loci for the pitcher plant midge, Metriocnemus knabi Coq. (Diptera: Chironomidae)

As a component of the inquiline community of the purple pitcher plant (Sarracenia purpurea), the pitcher plant midge Metriocneus knabi has been the subject of various ecological studies. However, very little is known about its characteristics beyond the larval stage, in particular the dispersal ability of adults. This study presents new molecular tools developed for testing of evolutionary and ecological questions in natural populations of this species. We describe a set of 12 microsatellite loci specific to M. knabi that are sufficiently polymorphic to provide insight into population genetic structure and dispersal patterns.     

Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Genomic expression libraries for the identification of cross-reactive orthopoxvirus antigens

Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines.     

Endocytotic uptake of nutrients in carnivorous plants

Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.     

Cowpox Virus Outbreak in Banded Mongooses (Mungos mungo) and Jaguarundis (Herpailurus yagouaroundi) with a Time-Delayed Infection to Humans

Background

Often described as an extremely rare zoonosis, cowpox virus (CPXV) infections are on the increase in Germany. CPXV is rodent-borne with a broad host range and contains the largest and most complete genome of all poxviruses, including parts with high homology to variola virus (smallpox). So far, most CPXV cases have occurred individually in unvaccinated animals and humans and were caused by genetically distinguishable virus strains.

Methodology/Principal Findings

Generalized CPXV infections in banded mongooses (Mungos mungo) and jaguarundis (Herpailurus yagouaroundi) at a Zoological Garden were observed with a prevalence of the affected animal group of 100% and a mortality of 30%. A subsequent serological investigation of other exotic animal species provided evidence of subclinical cases before the onset of the outbreak. Moreover, a time-delayed human cowpox virus infection caused by the identical virus strain occurred in a different geographical area indicating that handling/feeding food rats might be the common source of infection.

Conclusions/Significance

Reports on the increased zoonotic transmission of orthopoxviruses have renewed interest in understanding interactions between these viruses and their hosts. The list of animals known to be susceptible to CPXV is still growing. Thus, the likely existence of unknown CPXV hosts and their distribution may present a risk for other exotic animals but also for the general public, as was shown in this outbreak. Animal breeders and suppliers of food rats represent potential multipliers and distributors of CPXV, in the context of increasingly pan-European trading. Taking the cessation of vaccination against smallpox into account, this situation contributes to the increased incidence of CPXV infections in man, particularly in younger age groups, with more complicated courses of clinical infections.