Characterizing the Aedes aegypti Population in a Vietnamese Village in Preparation for a Wolbachia-Based Mosquito Control Strategy to Eliminate Dengue

Background

A life-shortening strain of the obligate intracellular bacteria Wolbachia, called wMelPop, is seen as a promising new tool for the control of Aedes aegypti. However, developing a vector control strategy based on the release of mosquitoes transinfected with wMelPop requires detailed knowledge of the demographics of the target population.

Methodology/Principal Findings

In Tri Nguyen village (611 households) on Hon Mieu Island in central Vietnam, we conducted nine quantitative entomologic surveys over 14 months to determine if Ae. aegypti populations were spatially and temporally homogenous, and to estimate population size. There was no obvious relationship between mosquito (larval, pupal or adult) abundance and temperature and rainfall, and no area of the village supported consistently high numbers of mosquitoes. In almost all surveys, key premises produced high numbers of Ae. aegypti. However, these premises were not consistent between surveys. For an intervention based on a single release of wMelPop-infected Ae. aegypti, release ratios of infected to uninfected adult mosquitoes of all age classes are estimated to be 1.8–6.7∶1 for gravid females (and similarly aged males) or teneral adults, respectively. We calculated that adult female mosquito abundance in Tri Nguyen village could range from 1.1 to 43.3 individuals of all age classes per house. Thus, an intervention could require the release of 2–78 wMelPop-infected gravid females and similarly aged males per house, or 7–290 infected teneral female and male mosquitoes per house.

Conclusions/Significance

Given the variability we encountered, this study highlights the importance of multiple entomologic surveys when evaluating the spatial structure of a vector population or estimating population size. If a single release of wMelPop-infected Ae. aegypti were to occur when wild Ae. aegypti abundance was at its maximum, a preintervention control program would be necessary to ensure that there was no net increase in mosquito numbers. However, because of the short-term temporal heterogeneity, the inconsistent spatial structure and the impact of transient key premises that we observed, the feasibility of multiple releases of smaller numbers of mosquitoes also needs to be considered. In either case, fewer wMelPop-infected mosquitoes would then need to be released, which will likely be more acceptable to householders.     

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Diatom Communities and Water Quality Assessment in Mountain Rivers of the Upper Segre Basin (La Cerdanya,Oriental Pyrenees)

Epilithic diatoms of mountain rivers from the upper Segre catchment (Oriental Pyrenees) were studied in 1998, during three different seasons: March, July and September. Four rivers, the river Segre and its three most important tributaries, Duran, Molina and Querol, were sampled in upstream and downstream stretches. The diatom communities were comparable in all upstream stretches of these mountain rivers draining siliceous substrates. Dominant taxa were Achnanthidium subatomus, Diatoma mesodon, Encyonema cf. minutum, E. silesiacum, Fragilaria arcus, F. capucina, Gomphonema calcifugum, G. pumilum, Meridion circulare and Nitzschia pura. Changes in water quality in the downstream stretches lead to the appearance of pollution tolerant taxa, such as Eolimna minima, Gomphoneis minuta, Navicula gregaria, and Nitzschia inconspicua. As a result, the values obtained with the diatom water quality indices (IPS Specific Polluosensitivity Index, CEE and IBD Biological Diatom Index) decreased. The diatom community composition and the derived water quality values did not change in the upstream stretches over the year. In contrast, significant changes were observed in the downstream stretches with best water␣quality in July, during high flows due to melting snow, and worst values in September, during low␣discharge. The diatom indices, especially the IPS, showed a good performance in these mountain rivers.     

Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli

Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin. Release of periplasmic proteins into the culture medium was observed throughout the entire cultivation of recombinant or non-recombinant cells. Leakage was intensified, however, in the final part of high-cell-density cultures (>100 g L(-)(1) dry cell mass) or when a temperature upshift was used for induction of recombinant protein synthesis. After temperature upshift, formation rates and residual cellular concentrations of periplasmic proteins declined with individual rates; e.g., the cellular content of the large periplasmic dipeptide binding protein DppA (57.4 kDa) started to decline about 4 h after the temperature upshift, whereas the smaller periplasmic d-galactose/d-glucose binding protein MglB (33.4 kDa) was already lost during the first hour after the upshift. In addition to periplasmic proteins, the osmotic-shock-sensitive heat-shock protein DnaK was found in significantly higher proportion in the cell-free medium of the temperature-challenged culture than other cytoplasmic proteins. Cell lysis was not observed even after prolonged cultivation. Thus, loss of a subset of cellular proteins of mainly periplasmic origin ordinarily occurs during cultivation and is intensified through stressful conditions in high-cell-density cultures. The selective release of cellular proteins of periplasmic origin offers the opportunity to simplify the downstream processing of recombinant proteins directed to the periplasm of E. coli.     

Mouse natural killer (NK) cells express the nerve growth factor receptor TrkA, which is dynamically regulated

Background

Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75^NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.

Methodology/Principal Findings

We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75^NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.

Conclusions/Significance

We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.     

Effects of pre-adult and adult experience on host acceptance in choice and non-choice tests in two strains ofTrichogramma

The effect of adult learning through an oviposition experience, and pre-adult learning through development inside a host was investigated in two strains of an Australian egg parasitoid,Trichogramma nrivelae (Hymenoptera: Trichogrammatidae). Host response was measured in two types of laboratory preference tests. In single host tests, females reared on three lepidopteran hosts (Heliothis punctigera (Noctuidae),Papilio aegeus (Papilionidae), andHypolimnas bolina (Nymphalinae)) were presented host eggs individually, and allowed two ovipositions. Pre-adult experience affected host preference in only one strain, while acceptance of a host was increased in both strains when they had previously oviposited in this host species. An oviposition experience had a stronger effect on host preference than pre-adult experience. In choice tests, pairwise combinations of hosts were arranged in an alternating grid. The ratio of host acceptances to host contacts was computed for the whole test and up to the first oviposition. These indicated that effects of rearing host were weak or absent, but the host chosen initially had a strong effect on host preference. Relative size of the hosts had a strong effect on choice of the first host. The implications of learning inTrichogramma are discussed in relation to host preference testing procedures and the selection of candidate strains for mass rearing and inundative release.     

Characterization of a novel fibroblast‐like cell line from rainbow trout and responses to sublethal anoxia

A novel fibroblast‐like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e . anoxia, activation of the stress activated member of the mitogen‐activated protein kinase family, p38^MAPK and induction of heat shock protein (Hsp70) were evident. The time‐course of the p38^MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38^MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38^MAPK‐specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38^MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress.