Artificial Membrane Feeding System Research Articles

Investigation of Hepatitis B Virus in the Body of the Bed Bug Cimex hemipterus (Hemiptera: Cimicidae) Fed on Infected Human Blood by RT-PCR Method

Background Bed bugs (Hemiptera: Cimicidae) are insects found in abundance among urban and rural communities. Both male and female bed bugs thrive on human blood. The main aim of this study was to detect Hepatitis B Virus (HBV) in the body of the bed bug, Cimex hemipterus (Hemiptera: Cimicidae), fed on naturally infected patient blood by Reverse Transcription Polymerase Chain Reaction (RT-PCR) Methods In this experiment, C. hemipterus bugs nourished once with HBV-positive blood were examined by RT-PCR at 1, 2, 3, 7- and 10 days post-engorgement. Bloodsucking was performed using an artificial membrane feeding system. RNA was extracted from infected bed bugs, and cDNA was then synthesized to monitor the HBV mRNA. Results The RT-PCR test results for infected specimens were positive on the first to third days post-bloodsucking. Moreover, the sample on days 7-10 was negative for HBV. This study also revealed that the breeding of bed bugs under laboratory conditions lasted for one week from the first to fifth nymph stages. Conclusion Since HBV mRNA was detected in infected bed bugs until the third-day post-engorgement, it is likely that they potentially transmitted HBV. It is thus indispensable to conduct more thorough research in the future.

Differential susceptibility of geographically distinct Ixodes ricinus populations to tick-borne encephalitis virus and louping ill virus.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi

Obtaining Plasmodium vivax sporozoites is essential for in vitro culture of liver stage parasites, not only to understand fundamental aspects of parasite biology, but also for drug and vaccine development. A major impediment to establish high-throughput in vitro P. vivax liver stage assays for drug development is obtaining sufficient numbers of sporozoites. To do so, female anopheline mosquitoes have to be fed on blood from P. vivax-infected patients through an artificial membrane-feeding system, which in turns requires a well-established Anopheles colony. In this study we established conditions to provide a robust supply of P. vivax sporozoites. Adding a combination of serum replacement and antibiotics to the membrane-feeding protocol was found to best improve sporozoite production. A simple centrifugation method appears to be a possible tool for rapidly obtaining purified sporozoites with a minimal loss of yield. However, this method needs to be better defined since sporozoite viability and hepatocyte infection were not evaluated.

Topical, contact, and oral susceptibility of adult Culicoides biting midges (Diptera: Ceratopogonidae) to fluralaner

BackgroundCulicoides biting midges (Diptera: Ceratopogonidae) are economically important blood-feeding pests closely associated with livestock production. They are the principal vectors of two hemorrhagic disease viruses affecting both wild and domestic ruminants within the US: bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). BTV impacts the US agriculture sector through direct commodity loss and strict international livestock trade restrictions. Yet, despite posing a considerable threat to US livestock, Culicoides are understudied, and management strategies are lacking. Current control tools for Culicoides are limited to synthetic chemicals, predominantly pyrethroids. With limited products available for livestock producers, proper pesticide rotation is difficult. The present study investigates the efficacy of fluralaner, an isoxazoline insecticide, beyond its current labeled use as an ectoparasiticide in anticipation of adding a new class of pesticides into rotation for use against biting midges.MethodsThe efficacy of fluralaner was evaluated by conducting contact, topical, and oral toxicity bioassays on adult female Culicoides sonorensis. Contact toxicity was assessed by using a modified WHO cone assay, which simulates exposure through landing on an insecticide-treated surface. A modified WHO topical toxicity assay, in which fluralaner dilutions were administered to the lateral thorax, was used to assess topical toxicity. For evaluation of oral toxicity, females were offered a blood meal spiked with fluralaner in an artificial membrane feeding system to simulate a systemic insecticide.ResultsContact exposure of fluralaner did not cause extensive or consistent mortality. Even the highest concentration tested (100 mg/ml) resulted in an average of only 24.3% mortality at 24 h, and mortality did not significantly differ between exposed and control midges at any concentration. One hundred percent mortality was consistently achieved at concentrations of 1 mg/ml when fluralaner was applied topically. The LC50 for topical exposure to fluralaner at 24 h was estimated to be 0.011 mg/ml. Oral exposure to fluralaner through ingestion of a spiked blood meal proved to be the most effective exposure method, significantly increasing mortality in a dose-dependent manner at 1 h post-exposure. The LC50 at 24 h following ingestion was 14.42 ng/ml.ConclusionOur results suggest that fluralaner is a viable candidate for use as an insecticide against adult biting midges if exposed orally, such as in a systemic given to livestock. As withdrawal period requirements for meat animals present unique yet definitive challenges, pharmacokinetic studies of isoxazoline drugs need to be pursued and finalized for livestock before fluralaner may be used as a management strategy in this manner. Alternatively, livestock not raised for consumption, such as hair sheep, would directly benefit from administering oral fluralaner as a component of a BTV disease management program.Graphical abstract

Optimisation of laboratory-rearing parameters for Anopheles funestus larvae and adults

Anopheles funestus is one of the major malaria vectors in Africa. As with the other main vectors, insecticide resistance in this species threatens existing vector control strategies. Unfortunately, scientific investigations, which could improve understanding of this vector species or lead to the development of new control strategies, are currently limited by difficulties in laboratory rearing of the species. In an attempt to optimise laboratory-rearing conditions for An. funestus, the effect of an artificial blood-feeding system for adults, different larval diet doses, and a range of other rearing conditions on the life history traits of an existing colony were investigated. Firstly, fecundity and fertility in An. funestus adult females fed on either live guinea pigs or bovine blood supplied through an artificial membrane feeding system were assessed. Secondly, a life-table approach was used to assess the impact of larval food dose (mg/larvae), larval density (larvae/cm2), and the depth of water used for larval rearing on life history traits. Fecundity was significantly higher when females were blood-fed on live anaesthetised guinea pigs than when fed on defibrinated bovine blood. However, the fertility of these eggs did not differ significantly between the two feeding methods or blood meal sources. Mosquitoes fed on defibrinated bovine blood using the artificial membrane feeding system showed an increase in egg production when the blood-feeding frequency was increased, but this difference was not statistically significant. The quantity of larval food influenced both time-to-pupation and pupal production. Increasing the larval densities resulted in reduced both time-to-pupation and pupal productivity. An optimal larval density of 0.48 larvae/cm2 was vital in preventing overcrowding. Increased water depth in the larval trays, was associated with significantly lower pupal production and reduced pupal weight. In conclusion, these results show that An. funestus can be reared using defibrinated bovine blood delivered via an artificial membrane feeding system. The quantity of larval food, optimal larval density, and depth of water used for larval rearing are critical factors influencing colony productivity. These findings can be used to improve current guidelines for rearing An. funestus under insectary conditions.

Use of a cost-effective artificial feeding system to evaluate the effect of blood meal source and its role in feeding success, reproductive parameters and larval growth of laboratory-reared Aedes Aegypti (L.)

Five different blood sources, human, cattle, rabbit, sheep and dog were delivered to laboratory-reared Aedes Aegypti to determine the effect of blood meal source on reproductive performance and larval growth characteristics of the mosquitoes. The blood meal was delivered with the use of a cost-effective artificial membrane feeding system. The mosquitoes displayed a significant feeding success for human blood over the other given blood meal sources. Thus, significantly a higher fecundity for human blood followed by cattle blood over other given blood meal sources was observed. The mean percentage of egg hatching for human blood and cattle blood was more or less the same, while the values were significantly different from that of other given blood meal sources rabbit, sheep and dog. Furthermore, larval growth parameters did not show any significant relationship with the blood meal source. These findings agree with established literature and facilitate the rearing and maintaining of Ae. Aegypti mosquitoes in a cost-effective manner. Hence the findings of the study aid in empowering laboratory research on Ae. Aegypti cost-effectively while enabling studies on its biology in in vitro conditions which are crucial in implementing disease control strategies.

Kinetics of tick infection by the relapsing fever spirochete Borrelia hermsii acquired through artificial membrane feeding chambers

The relapsing fever agent Borrelia hermsii is transmitted by the tick Ornithodoros hermsi. To study the B. hermsii-tick interactions required for pathogen acquisition and transmission we developed an artificial membrane feeding system for O. hermsi nymphs and adults that results in a high percentage of engorgement. This system provides the nutritional requirements necessary for the tick to develop, mate, and produce viable eggs. By inoculating the blood with B. hermsii, we were able to obtain infected ticks for quantitative studies on pathogen acquisition and persistence. These ticks subsequently transmitted the spirochetes to mice, validating this system for both acquisition and transmission studies. Using this feeding method, a mutant of the antigenic variation locus of B. hermsii (Vmp–) that is incapable of persisting in mice was acquired by ticks at equivalent densities as the wild-type. Furthermore, Vmp is not required for persistence in the tick, as the mutant and wild-type strains are maintained at similar numbers after ecdysis and subsequent feeding. These results support the theory that Vmp is an adaptation for mammalian infection but unnecessary for survival within the tick. Interestingly, B. hermsii numbers severely declined after acquisition, though these ticks still transmitted the infection to mice. This procedure reduces animal use and provides a safe, highly controlled and well-contained alternative method for feeding and maintaining O. hermsi colonies. Importantly, this system permits quantitative studies with B. hermsii strains through ingestion during the blood meal, and thus more closely recapitulates pathogen acquisition in nature than other artificial systems.

Transmission of Bartonella henselae within Rhipicephalus sanguineus: Data on the Potential Vector Role of the Tick.

Bartonella henselae is a fastidious intraerythrocytic, gram-negative bacteria that causes cat scratch disease in humans. Ixodes ricinus has been confirmed to be a competent vector of B. henselae, and some indirect evidences from clinical cases and epidemiological studies also suggested that some other tick species, including Rhipicephalus sanguineus, may transmit the bacteria. B. henselae has been detected in R. sanguineus but no experimental investigations have been performed to evaluate the vector competency of this tick species regarding B. henselae transmission. To this end, this work aimed to assess the transstadial transmission of B. henselae between larvae and nymphs of R. sanguineus as well as transmission by nymphs infected at the larval stage. Four hundred B. henselae negative larvae were fed with B. henselae-infected blood by using an artificial membrane feeding system. After five days of feeding, B. henselae was detected by PCR in 57.1% (8/14) of engorged larval pools, 66.7% (4/6) of semi-engorged larval pools, and 66.7% (2/3) of larval feces pools. After molting, B. henselae DNA was also detected in 10% (1/10) of nymph pools, but not in tick feces. After a pre-fed step of nymphs infected at the larval stage on non-infected blood meal, B. henselae was detected by PCR in blood sample from the feeder, but no Bartonella colonies could be obtained from culture. These findings showed that B. henselae could be transstadial transmitted from R. sanguineus larvae to nymphs, and also suggest that these nymphs may retransmitted the bacteria through the saliva during their blood meal. This is the first study that validated the artificial membrane feeding system for maintaining R. sanguineus tick colony. It shows the possibility of transstadial transmission of B. henselae from R. sanguineus larvae to nymphs.

Optimization of Aedes albopictus rearing procedures for combined sterile insect techniques (SIT) and Wolbachia-based laboratory studies in Sri Lanka

The establishment of a laboratory colony is an essential first step for conduction of laboratory studies on the biology and control of Aedes albopictus mosquitoes. Therefore, with the objective of generating high quality mosquitoes as research material for utilization in ongoing vector control studies and to disseminate authenticated, high-quality Ae. albopictus mosquito rearing information to the research community, maintenance of an Ae. albopictus mosquito colony was initiated at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. A self-mating colony was established from eggs of the F1 progeny of individuals collected as free-living larvae in Narahenpita (Western Province), Sri Lanka. The mean temperature of 28 °C (± 2 °C) and relative humidity of 80% (± 5%) was constantly maintained inside the insectary. Lighting was provided by fluorescent lights, regulated with 12:12 h continuous dark and light period. Pest insects were controlled manually. Mosquitoes were maintained on bovine blood provided via an artificial membrane feeding system and a continuous supply of 10% sugar supplements. Larvae were maintained in deoxygenate water and fed with International Atomic Energy Agency (IAEA) recommended diet of tuna meal, bovine liver powder, and brewery yeast in a ratio of 37.5:27:10.5 in 1 L. Data on fecundity, fertility, larvae death, pupation, adult emergence, adult mosquito longevity were recorded. Adhering to bio-safety, all discarded materials were boiled thoroughly and incinerated if required. This report on the establishment and maintenance of a laboratory colony of Ae. albopictus will be of value for identifying the critical requirements essential under artificial conditions.

Effect of the Topical Repellent para-Menthane-3,8-diol on Blood Feeding Behavior and Fecundity of the Dengue Virus Vector Aedes aegypti.

Dengue fever is an acute disease caused by the dengue virus and transmitted primarily by the mosquito Aedes aegypti. The current strategy for dengue prevention is vector control including the use of topical repellents to reduce mosquito biting. Although N,N-diethyl-m-methylbenzamide (DEET) is the most common active ingredient in topical repellent products, para-menthane-3,8-diol (PMD) is also used commercially. Studies have indicated PMD reduced biting by 90–95% for up to 6–8 h, similar to the efficacy of DEET, depending on the testing environment. The purpose of this study was to evaluate the behavioral effects of PMD on Ae. aegypti blood feeding and fecundity to explore the potential impact of PMD on downstream mosquito life-history traits. Two experiments were performed. In both experiments, cohorts of female Ae. aegypti (Belize strain) were exposed to 20% PMD or ethanol for 10 min in a closed system and introduced to an artificial membrane feeding system. Following a 30min feed time, mosquitoes of Experiment 1 were killed and weighed as a proxy measure of blood meal, whereas mosquitoes of Experiment 2 were monitored for oviposition, a measure of fecundity. Results showed a statistically significant reduction (p < 0.001) in the percentage of Ae. aegypti that blood-fed when exposed to PMD (38%) compared to those non-exposed (49%). No significant difference in fecundity between test populations was indicated. These findings suggest that exposure of Ae. aegypti to 20% PMD may influence the probability of subsequent blood feeding but of those mosquitoes that do blood feed, egg-lay density is not affected. Further studies are warranted to investigate the full range of effects of PMD exposure on other Ae. aegypti life-history traits such as mating, to continue characterizing the potential effects of PMD to impact overall vector population dynamics.

Borrelia burgdorferi surface protein Lmp1 facilitates pathogen dissemination through ticks as studied by an artificial membrane feeding system

In its natural infection cycle, the pathogen of Lyme borreliosis transits between a tick vector and a mammalian host. As relatively a minor fraction of spirochetes transits between the host and the vector precluding their reliable detection at early infection, artificial membrane feeders emerged as useful tools to study roles of spirochete proteins in pathogen entry, persistence, and exit through ticks. Here we report the development of a modified membrane feeder to study the role of a Borrelia burgdorferi surface protein called Lmp1 in spirochete transitions between the murine host and ticks. We show that our membrane feeder supports the blood meal engorgement process where ticks can acquire spirochetes from the feeder containing extremely low levels of pathogens (102 cells/ml of blood). Our data revealed that in comparison to wild-type spirochetes, lmp1 deletion mutants are significantly impaired for acquisition in naïve ticks as well as transmission from infected ticks. Taking together, our data suggest that Lmp1 plays an essential role in spirochete transitions between hosts and the vector. These studies also underscore the usefulness of artificial membrane feeding system as a valuable tool to study the role of B. burgdorferi gene-products in pathogen persistence in and passage through vector ticks.

Blood feeding of Ornithodoros turicata larvae using an artificial membrane system.

An artificial membrane system was adapted to feed Ornithodoros turicata (Ixodida: Argasidae) larvae from a laboratory colony using defibrinated swine blood. Aspects related to larval feeding and moulting to the first nymphal instar were evaluated. A total of 55.6% of all larvae exposed to the artificial membrane in two experimental groups fed to repletion and 98.0% of all fed larvae moulted. Mortality rates of first instar nymphs differed significantly depending on the sorting tools used to handle engorged larvae (χ2 = 35.578, P < 0.0001): engorged larvae handled with featherweight forceps showed significantly higher mortality (odds ratio = 4.441) than those handled with a camel-hair brush. Differences in the physical properties of the forceps and camel-hair brush may affect the viability of fragile soft tick larvae even when care and the same technique are used to sort them during experimental manipulations. The current results represent those of the first study to quantify successful feeding to repletion, moulting and post-moulting mortality rates in O. turicata larvae using an artificial membrane feeding system. Applications of the artificial membrane feeding system to fill gaps in current knowledge of soft tick biology and the study of soft tick-pathogen interactions are discussed.

Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum)

The Lone Star tick, Amblyomma americanum, is endemic to the southeastern United States and capable of transmitting pathogenic diseases and causing non-pathogenic conditions. To remain firmly attached to the host, the tick secretes a proteinaceous matrix termed the cement cone which hardens around the tick’s mouthparts to assist in the attachment of the tick as well as to protect the mouthparts from the host immune system. Cement cones collected from ticks on a host are commonly contaminated with host skin and hair making analysis of the cone difficult. To reduce the contamination found in the cement cone, we have adapted an artificial membrane feeding system used to feed long mouthpart ticks. Cones collected from in vivo and membrane fed ticks are analyzed to determine changes in the cone morphology. Comparisons of the cement cones using light microscopy shows similar structures and color however using scanning electron microscopy the cones have drastically different structures. The in vivo cones contain fibrils, sheets, and are heavily textured whereas cones from membrane fed ticks are remarkably smooth with no distinct structures. Analysis of the secondary protein structures using FTIR-ATR show both in vivo and membrane fed cement cones contain β sheets but only in vivo cement cones contain helical protein structures. Additionally, proteomic analysis using LC–MS/MS identifies many proteins including glycine rich proteins, metalloproteases, and protease inhibitors. Proteomic analysis of the cones identified both secreted and non-secreted tick proteins. Artificial membrane feeding is a suitable model for increased collection of cement cones for proteomic analysis however, structurally there are significant differences.

A cold snap! Diffusion tensor imaging of a pig׳s brain under controlled cooling

Artificial membrane feeding systems are effective tools for both tick rearing and studying tick-borne pathogen transmission. In order to compare the effects of the type of feeding system on tick engorgement, Ixodes ricinus ticks were either fed on an artificial membrane feeding system or on live mice. Sheep and chicken blood were used with the membrane system to assess the effects of blood origin on tick engorgement. To investigate the effects of blood meal infection on tick engorgement, ticks were either fed with Bartonella-infected or uninfected blood, both via membrane feeding and on mice. The proportion of engorged ticks, the duration of tick feeding, and the weight of engorged ticks were assessed. Feeding on the artificial system led to a longer duration of tick feeding and a lower proportion of engorged ticks than when fed on mice, however, the weight of engorged ticks was unaffected. The proportion and weight of engorged ticks, as well as the duration of feeding were not affected by blood origin. Feeding on an infected blood meal or on infected mice decreased the proportion and the weight of engorged ticks, but did not affect tick feeding duration.

Impact of feeding system and infection status of the blood meal on Ixodes ricinus feeding

Artificial membrane feeding systems are effective tools for both tick rearing and studying tick-borne pathogen transmission. In order to compare the effects of the type of feeding system on tick engorgement, Ixodes ricinus ticks were either fed on an artificial membrane feeding system or on live mice. Sheep and chicken blood were used with the membrane system to assess the effects of blood origin on tick engorgement. To investigate the effects of blood meal infection on tick engorgement, ticks were either fed with Bartonella-infected or uninfected blood, both via membrane feeding and on mice. The proportion of engorged ticks, the duration of tick feeding, and the weight of engorged ticks were assessed. Feeding on the artificial system led to a longer duration of tick feeding and a lower proportion of engorged ticks than when fed on mice, however, the weight of engorged ticks was unaffected. The proportion and weight of engorged ticks, as well as the duration of feeding were not affected by blood origin. Feeding on an infected blood meal or on infected mice decreased the proportion and the weight of engorged ticks, but did not affect tick feeding duration.

B22 人工膜吸血装置によるコロモジラミへのBartonella quintana感染実験(一般講演,演題要旨,第58回日本衛生動物学会大会)

B22 人工膜吸血装置によるコロモジラミへのBartonella quintana感染実験(一般講演,演題要旨,第58回日本衛生動物学会大会)

Rediocides B-E, potent insecticides from Trigonostemon reidioides.

Four new congeners, rediocides B-E (2-5), of the previously reported rediocide A (1) were isolated from a methanol extract of the roots of the plant Trigonostemon reidioides. The structures of these minor analogues were elucidated by comparison of their NMR and mass spectral data with those of rediocide A and confirmed by extensive 2D NMR spectral analysis. They all possess potent activity against fleas (Ctenocephalides felis) in an artificial membrane feeding system and exhibited LD(90) values ranging from 0.25 to 0.5 ppm.

Lack of manipulation of Rhodnius prolixus (Hemiptera: Reduviidae) vector competence by Trypanosoma cruzi.

Behavioral implications of Trypanosoma cruzi Chagas infection in Rhodnius prolixus Stål were observed. Feeding and defecation behaviors of infected versus uninfected insects were assessed on an artificial membrane-feeding system and on live guinea pigs. Based on a defecation index, fifth instars were the most efficient vectors, followed by adult females, fourth instars, and adult males. Bugs fasted for longer periods (5-6 mo) took smaller blood meals but defecated significantly earlier than bugs fasted for shorter periods (2-3 mo). Multiple blood feeding, degree of fasting, life stage, T cruzi infection, and gender affected the vector potential of R. prolixus. Our data indicate that T. cruzi and R. prolixus have not coevolved to facilitate the transmission of T. cruzi, which suggests that this parasite-host relationship may be relatively recent.