James L. Hardy

Western equine encephalomyelitis virus: In vivo infection and morphogenesis in mosquito mesenteronal epithelial cells

The infection and morphogenetic events associated with the replication of Western equine encephalomyelitis (WEE) virus within the mesenterons of Aedes dorsalis and three strains of Culex tarsalis are compared and contrasted. WEE virus apparently penetrates mesenteronal epithelial cells in vivo through membrane fusion. Profiles of apparent membrane fusion events were observed between virus particles and the microvillar surface of the mesenteron and naked nucleocapsids are observed intracellularly along the apical margin of the mesenteronal epithelial cell within 3 h of ingestion of the bloodmeal. Further, no viral particles were found in association with endocytotic nor lysosomal vacuoles during the initial phases of infection. In those strains of Cx. tarsalis that supported viral replication and in Ae. dorsalis, accumulations of nucleocapsids and maturation of WEE virus were evident along basolateral membranes of the mesenteron by 22-24 h after ingestion of the blood-meal. Maximal extracellular nascent virus occurred between 30-36 hrs. The Knights Landing strain of Cx. tarsalis revealed no subcellular morphological alteration in response to infection throughout the period of study. However, distinct morphological structures associated with the infection were observed in strains or species with enhanced susceptibility compared to Knights Landing (i.e., Cx. tarsalis WS-3 and Ae. dorsalis). In both, apical accumulations of nucleocapsids were apparent by 29 h post infection. These nucleocapsids were most often embedded in a rather amorphous matrix and occasionally in association with membrane profiles; presumably endoplasmic reticulum. Ae. dorsalis also demonstrated some alterations in response to WEE viral infection that were unique relative to Cx. tarsalis and some of these may be considered cytopathological. First, progeny virions were observed repeatedly within lysosomal figures. Second, extensive cytoplasmic vacuolization was noted and occasionally it appeared that these vacuolated cells were being sloughed off into the lumen of the mesenteron.

Alkaline Phosphatases of the Mosquito, Culex tarsalis Coquillett,

Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the alkaline phosphatase (ALKP) of the mosquito, Culex tarsalis, are presented. With p-nitrophenylphosphate (Pnp) as substrate, ALKP was optimally active at 37 degrees C, pH 8.0, 30 mM MgCl2, Vmax was 35.8 mumoles/10 min and the Km was 5.7 mM, with no demonstrable requirement for Zn2+. The spectrophotometric enzyme(s) was stimulated by dithiothreitol, 2-mercaptoethanol, and poly-vinylpyrollidone (PVP); inhibited by NaF, several alternative cations (Ca2+, Ba2+, Fe2+, Cu2+), and EDTA. ALKP activity was cyclic during the 15 day post-adult emergence period of the study. No significant differences were noted between the specific activities of males and females. IEF electrophoresis revealed 6 ALKP isozymes detected with alpha-naphthylphosphate within the pH range 4.0-5.5, with a second group of 3 rather indistinct species in the pH 6.0-7.0 range. IEF ALKP isozymes were stimulated by Mg2+ and PVP and inhibited by EDTA (except ALKP5.0) and cysteine; partial inhibition with phenylalanine. IEF detection of ALKP activity with Pnp indicated that the majority of the activity was localized in the pH 4.0-5.5 range, in close agreement with the alpha-naphthylphosphate results.

Binding of western equine encephalomyelitis virus to brush border fragments isolated from mesenteronal epithelial cells of mosquitoes

Brush border fragments (BBF) were isolated from mesenteronal epithelial cells of mosquitoes that are either susceptible (WS Culex tarsalis) or refractory (WR Cx. tarsalis; Culex pipiens) to peroral infection by western equine encephalomyelitis (WEE) virus. The isolated BBF were combined with radiolabeled WEE virus in a binding assay to compare the amount of virus bound by BBF from susceptible and refractory mosquitoes. BBF and WEE virus were mixed in a microcentrifuge tube, incubated for 1 h and centrifuged at 27,000 X g-30 min to pellet WEE virus bound to BBF. Optimal binding occurred at pH 7.2, 20 degrees C and there was no requirement for divalent cations. BBF isolated from perorally susceptible mosquitoes (WS Cx. tarsalis) bound significantly greater amounts of radiolabeled WEE virus, compared to BBF isolated from refractory mosquitoes (WR Cx. tarsalis; Cx. pipiens), in all experiments. The binding of WEE virus to BBF from WS Cx. tarsalis appears to be specific, based on saturation and competitive binding studies; binding to BBF from WR Cx. tarsalis and Cx. pipiens is nonspecific. Scatchard analysis of the binding data for BBF from WS Cx. tarsalis yields an estimated 1.8-3.5 X 10(6) binding sites per mesenteronal epithelial cell with an affinity constant of (Ka) of 2.2 X 10(11) M-1.

Midgut cellular responses to bloodmeal digestion in the mosquito, Culex tarsalis Coquillett (Diptera : Culicidae)

Abstract The cellular responses observed within the midgut epithelium of the mosquito, Culex tarsalis (Diptera : Culicidae), during digestion of a bloodmeal are discussed. The gross morphological changes are similar to those reported for other mosquito species. However, some changes are unique and appear to be related to the relatively large quantities of blood ingested by this mosquito. These changes are specifically the virtual obliteration of midgut epithelial microvilli and the presence of bloodmeal material within intercellular junctional spaces. The process of bloodmeal digestion can be broken down into 5 distinct phases: 0–4-hr postbloodmeal (PBM)-osmoregulation; 4–8-hr PBM-induction of the synthesis of presumed secretory materials; 8–24-hr PBM-synthesis and secretion of synthesized materials (i.e. peritrophic membrane and digestive enzymes); 24–36-hr PBM-digestion and transport of bloodmeal materials across the midgut epithelium into the hemocoel and finally beginning at 48-hr PBM-resting stage until next bloodmeal.

INITIATION AND CHARACTERIZATION OF A DIPLOID CELL LINE FROM LARVAL TISSUES OF AEDES DORSALIS (MEIGEN)

SummaryMosquito cell cultures were initiated from the minced tissues of newly hatchedAedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n=6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (includingMycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation.

Electrophoretic characterization of the nonspecific esterases of the mosquito, Culex tarsalis: conventional and isoelectric focused acrylamide gels

1. The nonspecific esterases of the mosquito, Culex tarsalis, were examined through conventional and isoelectric focusing acrylamide gel electrophoresis. 2. Conventional acrylamide gel electrophoresis resolved five components. These were characterized as: three carboxylesterases, one acetylcholinesterase and one acetylesterase. 3. Isoelectric focusing resolved 18 components. These were characterized as: 14 carboxylesterases, two acetylcholinesterases, one acetylesterase and one arylesterase. 4. The reproducibility and reliability of isoelectric focusing is discussed and compared to conventional acrylamide gel electrophoresis for the examination of multi-component isozyme systems such as non-specific esterases.

Host-dependent mutants of Sindbis virus whose growth is restricted in cultured Aedes albopictus cells produce normal yields of virus in intact mosquitoes.

Two host-dependent (hd), temperature-sensitive (ts) mutants of Sindbis virus (SV), clones 35 and 58, which are restricted in their ability to grow at 34 degrees in cultured Aedes albopictus cells [K.J. Kowal and V. Stollar (1981). Virology 114, 140-148] were not restricted in their ability to grow at 28-34 degrees after intrathoracic inoculation of Ae. albopictus, Ae. dorsalis, Ae. epactius, Anopheles freeborni, and Culex tarsalis mosquitoes. Compared to standard SV (SVSTD), the growth of SV clone 35 was somewhat restricted in parenterally infected C. pipiens females. Titers of SVSTD and the two mutants were modulated to relative low levels by some Ae. albopictus and Ae. dorsalis females after 192 hr incubation at 34 degrees but not at 28 degrees, suggesting that under some conditions mosquitoes have the ability to control the replication of SV. The hd and ts phenotypes of SV mutants were retained during growth at 31 degrees for 36 hr and 72 hr in Ae. epactius and Ae. dorsalis females, respectively, and at 28 degrees or 34 degrees for 192 hr in Ae. albopictus females. The demonstration that mutant viral clones, which are restricted in cultured mosquito cells, can produce normal yields of virus in intact mosquitoes indicates that considerable caution must be exercised in extrapolating results obtained with cultured cells to the level of the intact organism.

Further characterization of the nonspecific esterases of the mosquito, Culex tarsalis coquillett

Abstract The effects of various parameters on the nonspecific esterase electropherograms of the Knights Landing strain of the mosquito Culex tarsalis were determined by isolelectric focusing electrophoresis in acrylamide gels. The parameters examined were substrate preference, isolation/incubation media composition, tissue distribution, aging, deoxycholate stimulation and acetylthiocholine hydrolysis. The results corroborate the identity of two major acetylcholinesterases and document a single lipase in this mosquito. The nonspecific esterases of C. tarsalis can now be classified as: one acetylesterase, one arylesterase, one lipase, two acetylcholinesterases and thirteen carboxylesterases.

P-Nitrophenylacetate Hydrolysis by Nonspecific Esterases of the Mosquito Culex Tarsalis Coquillett,

The activities of esterases, hydrolyzing p-nitrophenylacetate (Acnp), of several strains of the mosquito, Culex tarsalis, are examined spectrophotometrically. Michaelis-Menten constants were determined to be 49.5 μmole/10 min (Vmax) and 1.4 mM (Km). Mg2+ ions stimulate the reaction much above other divalent cations (Ba, Ca, Co, Cu, Mn and Fe) with a maximal activity at 33 mM. The optimal temperature is 30°C, with a range of approximately 1°C depending on the mosquito strain. The efficacy of adding deoxycholate to the isolation medium is examined and compared to H2O and buffer. Inhibition data and isoelectric focusing electrophoresis indicate that, with Acnp as the substrate, 50–55% of the measured activity is attributable to an arylesterase.

Histochemical staining of the complex carbohydrates of the midgut of the mosquito, Culex tarsalis coquillett

Abstract Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic -glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic -glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic -glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed.