Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysine residues in antimicrobial properties.

Human apolipoprotein A-I (apoA-I) is a 28kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. In the present study the potential protective role against bacterial pathogens was explored. ApoA-I suppressed bacterial growth of Escherichia coli and Klebsiella pneumoniae. The protein was able to bind lipopolysaccharides and showed a strong preference for bilayer vesicles made of phosphatidylglycerol over phosphatidylcholine. Lysine side chains of apoA-I were acetylated to evaluate the importance of electrostatic forces in the binding interaction with both membrane components. Electrophoresis properties, dot blot analysis, circular dichroism, and fluorescence spectroscopy to probe for changes in protein structure indicated that the acetylated protein displayed a strongly reduced lipopolysaccharide and phosphatidylglycerol binding. A mutant containing only the N-terminal domain of apoA-I also showed a reduced ability to interact with the membrane components, although to a lesser extent. These results indicate the potential for apoA-I to function as an antimicrobial protein and exerts this function through lysine residues.

Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin.

Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected with AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released.

Mr. Mister: Rockin' the Aedes of the San Francisco Bay Salt Marshes.

Mist sprayers (MS) are being rapidly adopted nationwide for applying larvicides to control peridomestic Aedes aegypti. Because MS can loft large quantities of larvicide over relatively long distances, we examined its efficacy in a tidal marsh habitat for controlling Ae. dorsalis. Liquid Vectobac 12AS larvicide, containing Bacillus thuringiensis israelensis, was applied at 1.16 liter/ha using a MS. Cards that change color when exposed to liquid were placed perpendicular to the path of the MS showed that the larvicide mist traveled up to 60 m from the MS and did not extend to 90 m. Use of the MS enabled a 4-fold increase in total hectares treated during 2020-21 relative to the prior 2 years without an increase in staff time. Notably, there was 83% reduction in the quantity of Ae. dorsalis larvae at 5 days posttreatment. Similarly, there was 63% reduction in adult female Ae. dorsalis that were collected in encephalitis virus surveillance traps from nearby communities relative to the prior 2 years. There were 2.3-fold fewer requests for service to address a mosquito problem from residents of communities that abut the tidal marshes, suggesting the applications had a positive impact on these communities. The MS offer an attractive alternative to hand treatments in tidal marshes where the use of unmanned aircraft or all-terrain vehicles is prohibited by national wildlife refuge managers.

Quantitative reverse transcription PCR assay to detect a genetic marker of pyrethroid resistance in Culex mosquitoes.

Pyrethroid insecticides are widely used to control mosquitoes that transmit pathogens such as West Nile virus (WNV) to people. Single nucleotide polymorphisms (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene in Culex mosquitoes are associated with knockdown resistance to pyrethroids. RNAseq was used to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. The cDNA sequences were used to develop a quantitative reverse transcriptase PCR assay that detects the L1014F kdr mutation in the Vgsc. Because this locus is conserved, the assay was used successfully in six Culex spp. The resulting Culex RTkdr assay was validated using quantitative PCR and sequencing of PCR products. The accuracy of the Culex RTkdr assay was 99%. The L1014F kdr mutation associated with pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and was more prevalent in mosquitoes collected near farmland. The Culex RTkdr assay takes advantage of the RNA that vector control agencies routinely isolate to assess arbovirus prevalence in mosquitoes. We anticipate that public health and vector control agencies may employ the Culex RTkdr assay to define the geographic distribution of the L1014F kdr mutation in Culex species and improve the monitoring of insecticide resistance that will ultimately contribute to effective control of Culex mosquitoes.

Culex erythrothorax (Diptera: Culicidae): Activity periods, insecticide susceptibility and control in California (USA).

The mosquito Culex erythrothorax Dyar is a West Nile virus (WNV) vector that breeds in wetlands with emergent vegetation. Urbanization and recreational activities near wetlands place humans, birds and mosquitoes in close proximity, increasing the risk of WNV transmission. Adult Cx. erythrothorax abundance peaked in a wetland bordering the San Francisco Bay of California (USA) during the first 3 hours after sunset (5527 ± 4070 mosquitoes / trap night) while peak adult Culex tarsalis Coquillett abundance occurred during the subsequent 3 h period (83 ± 30 Cx. tarsalis). When insecticide resistance was assessed using bottle bioassay, Cx. erythrothorax was highly sensitive to permethrin, naled, and etofenprox insecticides compared to a strain of Culex pipiens that is susceptible to insecticides (LC50 = 0.35, 0.71, and 4.1 µg/bottle, respectively). The Cx. erythrothorax were 2.8-fold more resistant to resmethrin, however, the LC50 value was low (0.68 µg/bottle). Piperonyl butoxide increased the toxicity of permethrin (0.5 µg/bottle) and reduced knock down time, but a higher permethrin concentration (2.0 µg/bottle) did not have similar effects. Bulk mixed-function oxidase, alpha-esterase, or beta-esterase activities in mosquito homogenates were higher in Cx. erythrothorax relative to the Cx. pipiens susceptible strain. There was no difference in the activity of glutathione S-transferase between the two mosquito species and insensitive acetylcholine esterase was not detected. Larvicides that were applied to the site had limited impact on reducing mosquito abundance. Subsequent removal of emergent vegetation in concert with larvicide applications and reduced daily environmental temperature substantially reduced mosquito abundance. To control Cx. erythrothorax in wetlands, land managers should consider vegetation removal so that larvicide can efficiently enter the water. Vector control agencies may more successfully control adult viremic Cx. erythrothorax that enter nearby neighborhoods by applying adulticides during the 3 h that follow sunset.

Assessing Mosquito Breeding Sites and Abundance Using An Unmanned Aircraft.

An unmanned aircraft system (UAS; i.e., drone) with an attached multispectral camera was used to quantify accumulated surface water on a 0.54-km2 tidal marsh that abuts San Francisco Bay, CA, USA. The results of the survey showed unequal accumulation of surface water and provided information for focused inspections of potential mosquito breeding areas and identified areas where existing ditches needed improvement for increasing water circulation in the marsh to reduce mosquito breeding. The UAS was also outfitted with a high-magnification zoom video camera and piloted at varying heights to measure the video camera's ability to visualize immature mosquitoes in 2 small containers of contrasting colors during simulation tests in a marsh habitat. Immature mosquitoes could be seen clearly in white or black containers at heights up to 14 and 8 m, respectively. An artificial intelligence algorithm identified mosquito larvae and pupae in videos of the white tray with 94.1% and 52.8% accuracy, respectively. Together, our studies show that an UAS equipped with multispectral and zoom cameras provides a means for vector control agencies to rapidly and quantitatively assess the landscape for the presence of surface water and mosquito larvae.

Field Evaluation of a Redesigned Oviposition Trap to Monitor Gravid Invasive Aedes Mosquitoes in a Suburban Environment.

Oviposition cup traps (OCT) are commonly used to detect gravid invasive Aedes mosquitoes. Employing OCT during hot summer months or over broad geographic areas is labor intensive because the water in these small-volume traps must be frequently replenished to maintain their attractiveness to mosquitoes. We developed low-cost and simple-to-build oviposition bucket traps (OBT) that attract mosquitoes for more than 1 wk. Comparison of adjacently placed OCT and OBT in the city of Madera, CA, showed OBT captured significantly more Ae. aegypti eggs per trap-night relative to the OCT (8.8 ± 2.6 and 4.1 ± 1.1, respectively; paired t-test, P = 0.0076), and a significantly greater proportion of OBT contained Ae. aegypti eggs relative to OCT (83% of OBT and 65% of OCT; Fisher's exact test, P = 0.0214). The results suggest that OBT can collect larger quantities of Ae. aegypti eggs relative to OCT while potentially offering greater flexibility in scheduling trap inspections.

Pathogenesis of Autographa californica multiple nucleopolyhedrovirus in fifth-instar Anticarsia gemmatalis larvae.

We have investigated infection and pathogenesis of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Anticarsia gemmatalis (velvetbean caterpillar) larvae using a lacZ recombinant virus (AcMNPV-hsp70/lacZ) to track the temporal progression of infection in the midgut intestine and haemocoel. A. gemmatalis was highly resistant to fatal infection by occlusion bodies (OBs; LD(50)>5.5 x 10(5) OB) and budded virus (BV; LD(50)>3 x 10(5) BV) administered via oral and systemic routes, respectively. Orally administered occlusion-derived virus (ODV) efficiently attached and fused to midgut cells; however, high levels of infection-induced apoptosis limited infection in the midgut. Transcriptional analysis of AcMNPV genes expressed in the midgut of OB-inoculated A. gemmatalis larvae showed high levels of mRNA encoding the major capsid protein VP39 in the absence of immediate-early transactivator 1 (ie-1) expression. In the midgut, virus was efficiently transferred from infected midgut epithelial cells to nearby tracheolar cells and circulating haemocytes to initiate systemic infection in the haemocoel. However, haemocoelic BV did not efficiently disseminate infection and only cuticular epidermal cells displayed high levels of viral infection. Flow cytometry analysis of haemocytes isolated from BV-inoculated A. gemmatalis larvae showed low-level expression of the BV envelope protein GP64 on the cell surface, suggesting that A. gemmatalis haemocytes have a limited capacity for amplifying virus. These results show that AcMNPV is not an effective biological control agent for limiting crop damage caused by A. gemmatalis larvae.

Candida albicans modulates host defense by biosynthesizing the pro-resolving mediator resolvin E1.

Candida albicans is an opportunistic fungal pathogen of humans that resides commensally on epithelial surfaces, but can cause inflammation when pathogenic. Resolvins are a class of anti-inflammatory lipids derived from omega-3 polyunsaturated fatty acids (PUFA) that attenuate neutrophil migration during the resolution phase of inflammation. In this report we demonstrate that C. albicans biosynthesizes resolvins that are chemically identical to those produced by human cells. In contrast to the trans-cellular biosynthesis of human Resolvin E1 (RvE1), RvE1 biosynthesis in C. albicans occurs in the absence of other cellular partners. C. albicans biosynthesis of RvE1 is sensitive to lipoxygenase and cytochrome P450 monoxygenase inhibitors. We show that 10nM RvE1 reduces neutrophil chemotaxis in response to IL-8; 1nM RvE1 enhanced phagocytosis of Candida by human neutrophils, as well as intracellular ROS generation and killing, while having no direct affect on neutrophil motility. In a mouse model of systemic candidiasis, RvE1 stimulated clearance of the fungus from circulating blood. These results reveal an inter-species chemical signaling system that modulates host immune functions and may play a role in balancing host carriage of commensal and pathogenic C. albicans.

Spodoptera frugiperda resistance to oral infection by Autographa californica multiple nucleopolyhedrovirus linked to aberrant occlusion-derived virus binding in the midgut.

Spodoptera frugiperda larvae are highly resistant to oral infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (LD(50), approximately 9200 occlusions), but extremely susceptible to budded virus within the haemocoel (LD(50), <1 p.f.u.). The inability of AcMNPV occlusion-derived virus (ODV) to establish primary infections readily within midgut cells accounts for a major proportion of oral resistance. To determine whether inappropriate binding of AcMNPV ODV to S. frugiperda midgut cells contributes to lack of oral infectivity, the binding and fusion properties of AcMNPV ODV were compared with those of the ODV of a new isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) obtained from a field-collected larva (oral LD(50), 12 occlusions). By using a fluorescence-dequenching assay conducted in vivo, it was found that AcMNPV ODV bound to the midgut epithelia of S. frugiperda larvae at approximately 15 % of the level of SfMNPV ODV, but that, once bound, the efficiencies of fusion for the two ODVs were similar: 60 % for AcMNPV and 53 % for SfMNPV. Whilst the difference in binding efficiencies was significant, it could not account entirely for the observed differences in infectivity. Competition experiments, however, revealed that, in S. frugiperda larvae, SfMNPV ODV bound to a midgut cell receptor that was not bound by AcMNPV ODV, indicating that ODV interaction with a specific receptor(s) was necessary for productive infection of midgut columnar epithelial cells. Fusion in the absence of this ligand-receptor interaction did not result in productive infections.

P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens Larvae.

P74, an envelope protein of the occlusion-derived virus (ODV) of Autographa californica M nucleopolyhedrovirus (AcMNPV), is critical for oral infection of Trichoplusia ni larvae. The role of P74 during primary infection, however, is unknown. Here we provide evidence that P74 facilitates binding of AcMNPV ODV to a specific receptor within the larval midgut epithelia of another host species, Heliothis virescens. We adapted a fluorescence dequenching assay to compare binding, fusion, and competition of wild-type AcMNPV ODV in vivo with itself and with the ODV of a p74-deficient AcMNPV mutant. We found that relative to wild-type ODV, binding and fusion of ODV deficient in P74 were both qualitatively and quantitatively different. Unlike wild-type ODV, an excess of P74-deficient ODV failed to compete effectively with wild-type ODV binding, and the overall binding level of the mutant ODV was one-third that of the wild type. These results implicated P74 as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut.

Pathogenesis of Autographa californica M nucleopolyhedrovirus in fifth instar Spodoptera frugiperda.

We have characterized infection and pathogenesis of an Autographa californica M nucleopolyhedrovirus recombinant, AcMNPV-hsp70/lacZ, carrying the lacZ reporter gene, in penultimate (fifth) instar Spodoptera frugiperda. Bioassays revealed that while <0.1 p.f.u. of budded virus was required to generate an LD(50) by intrahaemocoelic injection, approximately 6000 occlusions were required orally to achieve the same mortality in newly moulted fifth instar (5(0)) larvae. In pathogenesis experiments, 78 % of the 5(0) larvae inoculated orally with 6000 occlusions of AcMNPV-hsp70/lacZ were LacZ-positive at 8 h post-inoculation (p.i.) and 50 % had LacZ signals in tracheal cells indicating that in these larvae infection had been transmitted from the midgut to secondary target cells. At 24 h p.i., maximum numbers of midgut and midgut-associated tracheal foci were observed (mean of 35 foci per infected larva), and 88 % of the larvae were LacZ-positive. The extremely low foci-per-occlusion ratio (0.006) indicated that successful infection of midgut cells was the primary barrier to fatal infection. A second barrier involved the loss of infected tracheal cells associated with the midgut. At 24 h p.i., 88 % of the inoculated larvae had a systemic infection, but in bioassays only 51 % succumbed to polyhedrosis disease. The absence of melanized tracheal cells in the insects throughout the time-course suggested that the larvae that cleared their infections (38 %) did so by a mechanism other than a classical immune response.