Variation in vector competency depends on chrysanthemum yellows phytoplasma distribution within Euscelidius variegatus

Phytoplasmas are plant‐pathogenic Mollicutes transmitted by leafhoppers, planthoppers, and psyllids in a persistent propagative manner. Chrysanthemum yellows phytoplasma (CY) is a member of ‘Candidatus Phytoplasma asteris’, 16Sr‐IB, and is transmitted by at least three leafhopper species, Macrosteles quadripunctulatus Kirschbaum, Euscelidius variegatus Kirschbaum, and Euscelis incisus Kirschbaum (all Homoptera: Cicadellidae: Deltocephalinae). Although M. quadripunctulatus transmits CY with very high efficiency (near 100%), 25% of E. variegatus repeatedly fail to transmit CY. The aims of this work were to correlate vector ability with different pathogen distribution in the insect body and to investigate the role of midgut and salivary glands as barriers to CY transmission. Euscelidius variegatus individuals acquired CY by feeding on infected plants or by abdominal microinjection of a phytoplasma‐enriched suspension. Insects were individually tested for transmission on daisy seedlings [Chrysanthemum carinatum Schousboe (Asteraceae)], and thereafter analysed by real‐time polymerase chain reaction (PCR) for CY concentration on whole insects or separately on heads and the rest of the body. Hoppers were classified as early and late transmitters or non‐transmitters, according to the time inoculated plants required for expression of CY symptoms. Similar transmission efficiencies were achieved following feeding or abdominal microinjection, suggesting that salivary glands may be a major barrier to transmission. Following acquisition from infected plants, all transmitters tested positive by PCR, and 60% of non‐transmitters also tested positive although with a significantly lower CY concentration. This indicates that a minimum number of phytoplasma cells may be required for successful transmission. The midgut may have prevented phytoplasma entry into the haemocoel of PCR‐negative non‐transmitters. Results suggest that both midgut and salivary glands may act as barriers. To assess the effect on CY transmission of a specific parasitic bacterium of E. variegatus, tentatively named BEV (Bacterium Euscelidius variegatus), we established a BEV‐infected population by abdominal microinjection of BEV bacteria. The presence of BEV did not significantly alter the efficiency of CY transmission.     

Origin and Examination of a Leafhopper Facultative Endosymbiont

Eukaryotes engage in intimate interactions with microbes that range in age and type of association. Although many conspicuous examples of ancient insect associates are studied (e.g., Buchnera aphidicola), fewer examples of younger associations are known. Here, we further characterize a recently evolved bacterial endosymbiont of the leafhopper Euscelidius variegatus (Hemiptera, Cicadellidae), called BEV. We found that BEV, continuously maintained in E. variegatus hosts at UC Berkeley since 1984, is vertically transmitted with high fidelity. Unlike many vertically transmitted, ancient endosymbioses, the BEV–E. variegatus association is not obligate for either partner, and BEV can be cultivated axenically. Sufficient BEV colonies were grown and harvested to estimate its genome size and provide a partial survey of the genome sequence. The BEV chromosome is about 3.8 Mbp, and there is evidence for an extrachromosomal element roughly 53 kb in size (e.g., prophage or plasmid). We sequenced 438 kb of unique short-insert clones, representing about 12% of the BEV genome. Nearly half of the gene fragments were similar to mobile DNA, including 15 distinct types of insertion sequences (IS). Analyses revealed that BEV not only shares virulence genes with plant pathogens, but also is closely related to the plant pathogenic genera Dickeya, Pectobacterium, and Brenneria. However, the slightly reduced genome size, abundance of mobile DNA, fastidious growth in culture, and efficient vertical transmission suggest that symbiosis with E. variegatus has had a significant impact on genome evolution in BEV.     

Phylogenetic affiliation of BEV,a bacterial parasite of the leafhopperEuscelidius variegatus,on the basis of 16S rDNA sequences

The phylogenetic relationship of a nonflagellated, Gram-negative, rod-shaped intracellular bacterial parasite (BEV) of the leafhopperEuscelidius variegatus to other bacteria within the classProteobacteria was determined by sequence analysis of 16S rDNAs. The presence of specific signature nucleotides showed this bacterium to be a member of the -3 subdivision of theProteobacteria. Phylogenetic analysis based on maximum parsimony placed BEV within a clade in theEnterobacteriaceae, which includes a number of bacteria that are facultative symbiotes of insects and have a common ancestor withProteus vulgaris. Within this clade, BEV is most closely related to a bacterium identified as the secondary endosymbiote of another homopteran, the pea aphid,Acyrthosiphon pisum.     

Transmission via plants of an insect pathogenic bacterium that does not multiply or move in plants

A bacterial parasite (designated as BEV) of the leafhopper Euscelidius variegatus, which is passed transovarially to offspring, was transmitted from insect to insect via feeding of the insects in plants. The rate of bacterial infection of leafhoppers fed upon plants that had previously been exposed to BEV-infected leafhoppers declined with an increase in the time that infected leafhoppers had been off rye grass. Transmission of BEV also occurred on sugar beet and barley but not celery. The bacterium was also transmitted to and acquired from membrane-encased artificial diets. There was no evidence that the bacterium was transmitted via plant surfaces, but transmission and direct culture assays from plants indicated that the bacterium did not multiply or move within plants. This parasite-host relationship may represent a primitive stage in either the evolution of intracellular symbiosis with its insect host or to alternative parasitization of plant and insect hosts via insect transmission, as is the case for insect-vectored plant pathogens.Correspondence to: A.H. Purcell.     

Ultrastructure of the digestive system of the Le fhopper Euscelidius variegatus Kirshbaum (Homoptera : Cicadellidae), with and without congenital bacterial infections

In the digestive system of Euscelidius variegatus Kirshbaum (Homoptera : (Cicadellidae), the close apposition of the anterior midgut with its posterior tabular midgut forms a filter chamber, which shunts excess water in the imbibed plant sap to the hindgut. Leafhoppers congenitally infected with a parasitic enteroform bacterium (designated BEV) had slightly atrophied digestive systems. There were numerous bacteria within the cells of the filter chamber, conical segment, and tubular midgut. Bacteria within the epithelium cells were usually enclosed within lysosomes. Epithelium cells swollen with large numbers of bacteria, had deteriorated cell membranes, and bacteria had erupted into the gut lumen. Leafhoppers not infected by BEV, harbored bacteria in the gut lumen, but not intracellularly within gut cells.     

Alternative evolutionary outcomes following endosymbiont‐mediated selection on male mating preference alleles

In many arthropods, intracellular bacteria, such as those of the genus Wolbachia, may spread through host populations as a result of cytoplasmic incompatibility (CI). Here, there is sterility or reduced fertility in crosses between infected males and uninfected females. As the bacterium is maternally inherited, the reduced fertility of uninfected females increases the frequency of the infection. If the transmission fidelity of the bacterium is less than 100%, the bacterium cannot invade from a low frequency, but if its frequency exceeds a threshold, it increases to a high, stable, equilibrium frequency. We explore the expected evolutionary dynamics of mutant alleles that cause their male bearers to avoid mating with uninfected females. For alleles which create this avoidance behaviour conditional upon the male being infected, there is a wide zone of parameter space that allows the preference allele to drive Wolbachia from the population when it would otherwise stably persist. There is also a wide zone of parameter space that allows a joint stable equilibrium for the Wolbachia and a polymorphism for the preference allele. When the male's avoidance of uninfected females is unconditional, the preference allele's effect on Wolbachia frequency is reduced, but there is a narrow range of values for the transmission rate and CI fertility that allow an unconditional preference allele to drive Wolbachia from the population, in a process driven by positive linkage disequilibrium between Wolbachia and the preference allele. The possibility of the evolution of preference could hamper attempts to manipulate wild populations through Wolbachia introductions.     

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression

Context: Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization.

Objective: We aimed to determine whether BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US) and whether the released BEV inhibits VEGF expression by endothelial cells in vitro.

Materials and methods: BEV-ELIP samples were subjected to 6?MHz color Doppler ultrasound (MI?=?0.4) for 5?min. We assessed release of BEV with a direct ELISA and with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method. Human umbilical vein endothelial cell (HUVEC) cultures were stimulated to express VEGF by 10?nM phorbol-12-myristate 13-acetate (PMA). Cell-associated VEGF levels were determined using a cell-based ELISA.

Results: Overall, US caused an additional 100?µg of BEV to be released or exposed per BEV-ELIP aliquot within 60?min BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US. Also, US-treated BEV-ELIP inhibited HUVEC proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US.

Discussion and conclusion: We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody.     

Human Probing Behavior of Aedes aegypti when Infected with a Life-Shortening Strain of Wolbachia

Background

Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations.

Methodology/Principal Findings

Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied.

Conclusions/Significance

As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

The endosymbiotic bacterium <Emphasis Type="Italic">Wolbachia</Emphasis> enhances the nonspecific resistance to insect pathogens and alters behavior of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

To determine biologically important effects of the cytoplasmic endosymbiont Wolbachia, two substrains of the same Drosophila melanogaster strain have been studied, one of them infected with Wolbachia and the other treated with tetracycline to eliminate the bacterium. Females of D. melanogaster infected with Wolbachia are more resistant to the fungus Blauveria bassiana (an insect pathogen) than uninfected females; infected females also exhibited changes in oviposition substrate preference. Males infected with the bacterium are more competitive than uninfected males. The possible role of Wolbachia in the formation of alternative ecological strategies of D. melanogaster is discussed.     

Effect of Temperature on Attachment,Development, and Interactions of Pasteuria penetrans on Meloidogyne incognita

The effect of temperature (10, 20, 25, 30, and 35 C) on attachment and development of Pasteuria penetrans on Meloidogyne arenaria race 1 was elevated in growth chambers. The greatest attachment rate of endospores of P. penetrans occurred on second-stage juveniles at 30 C. The bacterium developed more quickly within its host at 30 and 35 C than at 25 C or below. The development of the bacterium within the nematode female was divided into nine recognizable life stages, which ranged from early vegetative thalli to mature sporangia. Mature sporangium was the predominant life stage observed after 35, 40, 81, and 116 days at 35, 30, 25, and 20 C, respectively. The body width and length of M. arenaria females infected with P. penetrans were smaller initially than the same dimensions in uninfected females, but became considerably larger over time at 25, 30, and 35 C. This isolate of P. penetrans also parasitized and completed its life cycle in males of M. arenaria.     

Spiroplasma Symbiont of the Pea Aphid, Acyrthosiphon pisum (Insecta: Homoptera)

From a laboratory strain of the pea aphid, Acyrthosiphon pisum, we discovered a previously unknown facultative endosymbiotic bacterium. Molecular phylogenetic analysis based on 16S ribosomal DNA revealed that the bacterium is a member of the genus Spiroplasma. The Spiroplasma organism showed stable vertical transmission through successive generations of the host. Injection of hemolymph from infected insects into uninfected insects established a stable infection in the recipients. The Spiroplasma symbiont exhibited negative effects on growth, reproduction, and longevity of the host, particularly in older adults. Of 58 clonal strains of A. pisum established from natural populations in central Japan, 4 strains possessed the Spiroplasma organism.     

Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics

The endosymbiotic bacterium Wolbachia shows viral blocking in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11–19°C and 22–30°C. Eggs stored at 11–19°C had higher hatch proportions than those stored at 22–30°C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22–30°C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected A. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.     

Removing endosymbiotic <Emphasis Type="Italic">Wolbachia</Emphasis> specifically decreases lifespan of females and competitiveness in a laboratory strain of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

To understand specific symbiotic relationships ensuring stable existing of the bacterium Wolbachia in laboratory strains of Drosophila melanogaster, the imago lifespan and senescence rate, as well as competitiveness, have been evaluated as components of fitness in females from the following laboratory strains: (1) inbred strain 95 infected with Wolbachia; (2) two uninfected strains obtained by tetracycline treatment that were genetically similar to strain 95; and (3) two control, uninfected, wild-type laboratory strains that were used to assess the possible effects of the antibiotic on the studied characters in the absence of Wolbachia. The results have shown that infected females have longer lifespan and competitiveness than females with the same genotype uninfected with Wolbachia. The increase in the senescence and mortality rates with age was also slower in infected females. It is noteworthy that tetracycline does not affect the lifespan of females from the two control, uninfected, wild-type strains. Therefore, the antibiotic is not the cause of the positive changes in fitness that were observed in infected females. The obtained results are the first direct evidence that the relationships in the Wolbachia-D. melanogaster symbiotic system are mutualistic rather than parasitic, at least in micropopulations adapted to laboratory conditions.     

Parasite-induced alteration of odour responses in an amphipod–acanthocephalan system

Odour-related behaviours in aquatic invertebrates are important and effective anti-predator behaviours. Parasites often alter invertebrate host behaviours to increase transmission to hosts. This study investigated the responses of the amphipod Hyalella azteca when presented with two predator chemical cues: (i) alarm pheromones produced by conspecifics and (ii) kairomones produced by a predatory Green Sunfish (Lepomis cyanellus). We compared the responses of amphipods uninfected and infected with the acanthocepalan parasite Leptorhynchiodes thecatus. Uninfected amphipods reduced activity and increased refuge use after detecting both the alarm pheromones and predator kairomones. Infected amphipods spent significantly more time being active and less time on the refuge than uninfected amphipods, and behaved as if they had not detected the chemical stimulus. Therefore, L. thecatus infections disrupt the amphipods’ anti-predator behaviours and likely make their hosts more susceptible to predation.     

Influence of piperonyl butoxide on the toxicity of organophosphate insecticides to three species of freshwater benthic invertebrates

Toxicity tests were conducted with amphipods (Hyalella azteca), chironomids (Chironomus tentans) and oligochaetes (Lumbriculus variegatus) exposed to a series of organophosphate insecticides in the absence or presence of piperonyl butoxide (PBO), an inhibitor of cytochrome(s) P450. Piperonyl butoxide effectively reduced the toxicity to H. azteca and C. tentans of three organophosphates (diazinon, chlorpyrifos, azinphos-methyl) which undergo metabolic activation by cytochrome(s) P450. Coadministration of PBO with another organophosphate (dichlorvos) which is not activated by cytochrome(s) P450, did not reduce the toxicity to the two species. Lumbriculus variegatus was relatively insensitive to the organophosphates, and PBO did not reduce their toxicity to the oligochaete. These data indicate that both H. azteca and C. tentans possess cytochrome P450-mediated MOs capable of metabolizing organic xenobiotics. However, the degree to which L. variegatus might be capable of the oxidative metabolism of organic xenobiotics is uncertain.     

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5–6 months old Assaf lambs using Al(OH)₃ or Escherichia coli lipopolysaccharide + inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%–85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.     

Effects of Schistosomatium douthitti infection on the growth, survival, and reproduction of Lymnaea catascopium

Lymnaea catascopium snails infected with Schistosomatium douthitti grew faster than uninfected control snails during the first 2 months postexposure, but thereafter grew more slowly, and by 8 months postexposure were significantly smaller. When reared in isolation, uninfected snails survived significantly longer (mean survival time, 515 days) than snails exposed to three miracidia each (400 days), which in turn survived longer than snails exposed to 10 miracidia per snail (223 days). When maintained in aquaria in contact with other snails, snails exposed to three miracidia each survived longer (227 days), but not significantly longer, than control snails (198 days). Production of large numbers of eggs by control snails grown under the latter conditions may account for their reduced survival. The ovotestes and accessory genitalia of snails infected with S. douthitti were much reduced in size in comparison with uninfected control snails. These effects were most pronounced in snails which had been infected for over 100 days. Egg production was normally totally inhibited if snails were infected before the onset of sexual maturity. If infected after the onset of maturity, eggs were produced only during the prepatent period.     

Characterizing the host specificity of Ischnodemus variegatus (Signoret) (Hemiptera: Blissidae) on two congeneric grass species

Life history parameters of the sap-feeding bug Ischnodemus variegatus (Signoret) (Hemiptera: Blissidae) were compared between the invasive Neotropical grass, Hymenachne amplexicaulis (Rudge) Nees and the Australian native Hymenachne acutigluma (Steudel) Guilliland. Development of immatures, adult longevity, ovipositional preference and population growth of I. variegatus were compared on H. amplexicaulis and H. acutigluma under choice and no-choice conditions. Third-instar nymphs developed on both hosts but individuals reared on H. acutigluma had a 40% lower survival rate to the adult stage, 40% longer developmental time, a 9% reduction in length and a 30% reduction in weight of females compared to those reared on H. amplexicaulis. The no-choice adult longevity and oviposition study indicated that individuals feeding on H. acutigluma had half the lifespan, 62% as many ovipositing females, 52% longer time to first oviposition and only 14% as many eggs per female. Under choice conditions, females laid only 37% as many eggs on H. acutigluma as on H. amplexicaulis. Population growth of I. variegatus was slower on H. acutigluma compared to H. amplexicaulis over a 60 day period resulting in negative population increase on H. acutigluma but positive increase on H. amplexicaulis. These results reflect an overall poorer performance of I. variegatus on H. acutigluma for most life stages.     

Interaction and Transmission of Two Borrelia burgdorferi Sensu Stricto Strains in a Tick-Rodent Maintenance System

In the northeastern United States, the Lyme disease agent, Borrelia burgdorferi sensu stricto, is maintained by enzoonotic transmission, cycling between white-footed mice (Peromyscus leucopus) and black-legged ticks (Ixodes scapularis). B. burgdorferi sensu stricto is genetically variable and has been divided into three major genotypes based on 16S-23S ribosomal DNA spacer (RST) analysis. To better understand how genetic differences in B. burgdorferi sensu stricto may influence transmission dynamics in nature, we investigated the interaction between an RST1 and an RST3 strain in a laboratory system with P. leucopus mice and I. scapularis ticks. Two groups of mice were infected with either BL206 (RST1) or B348 (RST3). Two weeks later, experimental mice were challenged with the opposite strain, while control mice were challenged with the same strain as that used for the primary infection. The transmission of BL206 and B348 from infected mice was then determined by xenodiagnosis with uninfected larval ticks at weekly intervals for 42 days. Mice in both experimental groups were permissive for infection with the second strain and were able to transmit both strains to the xenodiagnostic ticks. However, the overall transmission efficiencies of BL206 and B348 were significantly different. BL206 was more efficiently transmitted than B348 to xenodiagnostic ticks. Significantly fewer double infections than expected were detected in xenodiagnostic ticks. The results suggest that some B. burgdorferi sensu stricto strains, such as BL206, may be preferentially maintained in transmission cycles between ticks and white-footed mice. Other strains, such as B348, may be more effectively maintained in different tick-vertebrate transmission cycles.