US Department of Agriculture, Agriculture Research Service, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, FL 32604, USA1
West Virginia University, Morgantown, WV 26506, USA2
Author for correspondence: James Becnel. Fax +1 352 374 5966. e-mail jbecnel{at}gainesville.usda.ufl.edu
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Abstract |
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Introduction |
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Methods |
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Larvae were concentrated at the field sites (number of dips and water volumes not standardized) by straining water through 60 mesh sieves and transferred to containers with 1530 litres of field water for transport to the laboratory. At the laboratory, larvae were strained through a series of 10, 18, 35 and 80 mesh sieves (2·0, 1·0, 0·5 and 0·18 mm openings respectively) and the contents of each sieve washed into 300800 ml of water. Third- and fourth-instar larvae were retained in the 35 and 18 mesh sieves while the first- and second-instar larvae remained in the 80 mesh sieve. The total volume of the sample (300800 ml) was measured from the 18 and 35 mesh sieves. After agitation, a sample of 10100 ml was removed, measured and the number of larvae counted to estimate the total number of larvae in each of the 18 and 35 mesh sieves. Larvae from the 18 mesh sample were examined with a stereo microscope at 20xmagnification for signs of infection and for species identification and provided the information for percentage infection by species. Infected larvae were identified by the presence of highly refractile nuclei in midgut epithelial cells, which could be viewed through the cuticle (accompanying paper: Moser et al., 2001 ). Larvae from the 35 mesh were examined for signs of infection (larvae from this mesh size were too small to accurately identify the species). The total number of larvae, the proportion of each species (from the 18 mesh only) and the percent infection in Culex spp. were estimated from the samples of the two sieves. The population index reflected the total number of third- and fourth-instar Culex spp. larvae collected.
Field water analysis.
An estimate of the total dissolved salts was determined by recording conductivity from each field water sample. Samples of the field water were prepared for detailed chemical analysis by the following methods. Water strained through 400 mesh (38 µm openings) was centrifuged at 4800 g for 10 min and the supernatant frozen. In addition, supernatant and field water were digested in 1 M HNO3 by heating a 1:1 mixture of water1 M HNO3 just below the boiling point until the volume had been reduced to 12 ml. The residue was reconstituted in 1 M HNO3 and held at room temperature. In addition, whole water was filtered through a no. 41 Whatman filter and stored frozen. Water samples were submitted for elemental analysis to the University of Florida Analytical Research Laboratory. Cation concentrations were obtained by ICAP (inductive coupled argon plasma, Thermal Garrel Ash, mfg).
Laboratory bioassay.
Tissue culture production methods for the virus were unavailable and all studies were done in vivo. A colony of C. nigripalpus was unavailable until recently and therefore insects from a C. quinquefasciatus colony (Alachua County, Florida, strain 5 years in culture) were used for the majority of bioassays. Standard bioassays were done with groups of 100 3 to 4-day-old C. quinquefasciatus larvae (second-instars) exposed in 3·5 oz plastic cups in 100 ml of water with 2 ml of 2% alfalfa and potbelly pig chow mixture (2:1).
Because of the small size of the occlusion bodies (<0·5 µm), CuniNPV concentrations were usually based on larval equivalents (LE; 10 infected third-instar larvae/ml=10 LE) but purified virus was used in some tests and obtained and quantified according to procedures described in Moser et al. (2001) . A laboratory culture of the baculovirus was initiated by amplifying field-collected virus from C. nigripalpus in colony-reared C. quinquefasciatus. Approximately 3000 3-day-old C. quinquefasciatus larvae were exposed to 100 LE virus in 14 mM MgSO4 and harvested 48 h p.i. Groups of 50 infected larvae were frozen in deionized water and held at -80 °C. These infected larvae were used for additional amplifications or for laboratory bioassay. On the day of the assay, individual vials were removed and held at room temperature to thaw. The infected larvae were then homogenized in a glass tissue grinder and a concentration of 58 LE was used per exposure group. After 48 h, the larvae were removed and examined microscopically for signs of infection. Only those larvae with hypertrophied nuclei in midgut epithelial cells (Moser et al., 2001) were scored as positive.
Field water assays.
Larvae were exposed to CuniNPV in whole field water strained through a 400 mesh sieve (collected from August 1996 through December 1997) and deionized water. Field water samples were held for a maximum of 5 days at 5 °C before testing. Controls without the addition of virus were included in all assays. The increase in percent infection was calculated based on paired tests with and without virus in deionized water, swine wastewater and dairy wastewater. Data were analysed by SAS general linear model and comparison among mean differences by DuncanWaller.
Enhancement assays.
Alkali pretreatment of CuniNPV prior to exposure, reported as important for the transmission of AesoNPV in the mosquito Aedes triseriatus (Federici & Lowe, 1972 ), was performed according to the previously described procedure. After neutralization, treated and untreated virus were assayed in deionized water in paired tests. Also, virus exposures were made with the addition of the optical brightener Calcofluor M2R (Sigma), which has been shown to enhance the transmission of certain baculoviruses (Shapiro & Robertson, 1992
).
Deletion analysis of the principle cations present in the field water was used to determine if salts were critical for transmission of CuniNPV. A salt mixture of 1·8 mM MgCl2, 0·5 mM CaCl2, 6·0 mM KCl, 1·8 mM NaCl and 3 mM NH4Cl (based on the initial swine wastewater chemical analysis) was used as an exposure medium. Bioassays were done in the complete salt mixture, mixtures with one salt deleted and in each salt individually. Based on the results of these tests, additional assays with CuniNPV were conducted in 63% serial dilutions of 20 mM MgCl2 or MgSO4 to determine the effect of cation concentration on infection levels. To determine possible inhibition of transmission, assays were done in 10 mM MgCl2 with 50% serial dilutions of 20 mM CaCl2, KCl and NaCl. Data were analysed by SAS general linear model and comparison among mean differences by DuncanWaller.
To determine the activation potential of other cations on transmission of CuniNPV, exposures were made in the highest sublethal concentrations of BaCl2, CoCl2, CuCl2, FeCl2, KCl, MnCl2, NaCl, NiCl2, SnCl2, SrCl2 and ZnCl2 (n=3). The cations that did not result in increased transmission of CuniNPV were tested (at the highest sublethal concentration) to determine if they would inhibit transmission when exposures were made in the presence of 10 mM Mg2+ (n=3).
The role of magnesium in transmission and development of CuniNPV was investigated by exposing larvae to Mg2+ and virus for 2, 4, 6, 8, 12, 24 and 48 h periods. All exposures were made in 10 mM Mg2+ and CuniNPV concentrations of 5 LE or purified virus at an estimated concentration of 4·5x108 occlusion bodies (OBs)/ml. After each exposure time, larvae were either removed from the exposure media and transferred to deionized water (n=5) or 10 mM CaCl2 was added to the exposure mixture to stop the infection process (n=4). Percent infections were determined for each exposure group at 48 h p.i. and the percent infection was evaluated by log probit analysis.
Assays were also done with EDTA (Sigma), a strong and nonspecific divalent ion chelator and metalloprotease inhibitor and EGTA (Sigma) a chelator of divalent cations with a significantly higher affinity for calcium than magnesium. Larvae were exposed according to the protocols for the virus assay experiments (described above) with the addition of 40% serial dilutions of 1 mM EDTA or 40% serial dilutions of 1 mM EGTA (total of five concentrations).
Electron microscopy.
To investigate early events in the infection process of CuniNPV, larval guts were dissected and fixed (as described below) at time intervals of 30 min, 1, 2 and 4 h p.i. Exposures were made to groups of larvae (as described above) with virus alone and with virus in the presence of 10 mM MgCl2. Exposed larvae were prepared for ultrastructural examination by fixing dissected guts in 2·5% gluteraldehyde for 2 h, post-fixing in 2% osmium tetroxide, dehydrating in an ethanol series and embedding in EponAraldite. Thin sections were stained in uranyl acetate and lead citrate and examined and photographed at 75 kV.
Virus host-range.
CuniNPV for these exposures was produced in C. quinquefasciatus as described above and a dose of 10 LE was used for all tests. Mosquitoes tested were from laboratory colonies of Aedes aegypti, A. albopictus, A. triseriatus, A. taeniorhynchus, Anopheles albimanus, A. quadrimaculatus and field-collected Culex restuans, C. salinarius and Culiseta melanura. Groups of 100 48-h-old larvae were counted into cups containing 100 ml 10 mM MgCl2 and susceptibility was determined with paired tests, one exposed group and one unexposed control. Positive controls of 100 C. quinquefasciatus larvae were exposed with each test to verify the infectivity of the virus. All larvae were examined for infection at 48 h p.i. To test the susceptibility of the predacious mosquito, Toxorhynchites ambionensis, 20 second-instar larvae were set up individually in well plates with 10 mM MgCl2 and fed three live C. quinquefasciatus larvae infected with CuniNPV. Similar groups of T. ambionensis were fed healthy C. quinquefasciatus larvae and served as a control. Larvae were examined for signs of infection at 48, 72 and 96 h p.i. and mortality was calculated.
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Results |
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Larvae were collected at the field sites and then separated into two categories first- and second-instars, and third- and fourth-instars which were examined microscopically for signs of viral infection and for species identification. The two predominant mosquito species present at the swine wastewater site were C. nigripalpus during the warmer months (MayNovember) and C. quinquefasciatus during the cooler months (DecemberApril). C. salinarius and C. restuans were also present during the winter but at low levels. Epizootics of CuniNPV were found in all instars of the Culex spp. larvae collected in the swine wastewater site throughout the study (Fig. 1). The highest infection levels in third- and fourth-instar larvae were found during the spring and fall months with the lowest infection levels found during the winter (Fig. 1
).
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C. quinquefasciatus was the dominant species collected at the dairy wastewater site with C. nigripalpus collected infrequently. A total of 16 larval collections was made during the sampling period. Although high larval populations of C. quinquefasciatus were present (average of 19000±5000) larvae infected with CuniNPV were collected on only five occasions and never at epizootic levels (0·08±0·06% infection).
Water analysis
The conductivity of the swine wastewater averaged 1·8 mS/cm, and the pH averaged 7·8 (Table 1). In contrast, the dairy lagoon wastewater showed a higher conductivity, (3·9 mS/cm), but the pH was similar (8·0). Although in both ponds K+, Na+, Mg2+ and Ca2+ accounted for 90% of the cations, there were significant differences in their concentrations. Whereas Na+ levels were similar at 3·23·6 mM, the concentration of K+ was almost twice as high in the swine pond (10·7 vs 5·8 mM) but the Mg2+ and Ca2+ concentrations were significantly lower (Mg2+=1·9 vs 3·7 mM; Ca2+ =0·8 vs 3·0 mM).
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Virus host-range
CuniNPV infected C. salinarius (infection rate 32·9±9·8%, n=3) but not C. restuans. Species of Aedes, Anopheles, Culiseta and Toxorhynchites were refractory to infection.
Electron microscopy
Examination of the larval gut contents 30 min p.i. revealed intact OBs regardless of the presence or absence of Mg2+ (Fig. 4a). Release of virions from OBs was first found 1 h p.i. and did not depend on the presence of Mg2+. Release of virions occurred prior to complete dissolution of the OBs leaving ghosts in the OBs indicating the former location of virions (Fig. 4b
, c
). Numerous free virions were observed in the gut lumen 1, 2 and 4 h p.i. and were often found in close proximity to the peritrophic matrix (PM) of the insect midgut (Fig. 4d
). This preliminary investigation failed to demonstrate virions crossing the PM but when Mg2+ was present some virions were observed embedded in the internal layer of the PM (Fig. 4e
).
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Discussion |
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A cascade of events is required for infection of a host with a baculovirus. This involves: (i) release of virions from OBs in the insect gut, (ii) the movement of virions across the PM, (iii) the attachment of virions to the midgut epithelial cells and the transfer of the nucleocapsids to the nucleus where they undergo replication, (iv) budding of virions from the midgut cell and spread of infection within the insect. To produce high levels of infection, Mg2+ must be present during the first 812 h of CuniNPV exposure indicating that the activity of Mg2+ occurs early in the process with either entry into the cells or nuclei. EM observations of OBs in the midgut lumen revealed that Mg2+ was not required for dissolution. Other possible targets are receptors on midgut epithelial cells or PM or enzymes associated with the virion or midgut that are required for initiating infectivity. Receptors may require divalent cations for the attachment to and/or passage of virions through the PM, midgut cells or entry into nuclei.
Our observations of virions embedded in the PM indicated that specific receptors on the inner layer of the PM might be crucial for attachment but it is not known if this process is Mg2+ dependent. However, attachment alone is insufficient for the virion to pass through the PM as the maximum size of pores in the inner PM layer is 20 nm (Werner, 1979 ) while the width of the virion is approximately 40 nm (Moser et al., 2001
). This implies that proteins in the PM must be broken down for the virions to cross. Mg2+ may be involved in this process. In addition, Mg2+ could be required for the interaction of CuniNPV with receptors on cells in the posterior midgut and the gastric caeca (Moser et al., 2001
).
This is only the second documented epizootic of a baculovirus in a naturally occurring mosquito population. A mixed epizootic involving a baculovirus and a cytoplasmic polyhedrosis virus (CPV) in Aedes sollicitans occurred over a 3 week period in the summer of 1971 in Louisiana (Clark & Fukuda, 1971 ). Although many insects were infected (56·7% at the peak), the infections were apparently due to a mixture of both viruses and therefore it was difficult to evaluate the role of each. Attempts to transmit the A. sollicitans baculovirus in the laboratory resulted in less than 15% infection, but transmission in field water or with the addition of salts was not investigated (Clark & Fukuda, 1971
).
Because C. nigripalpus is an important vector of St Louis and Eastern equine encephalitis (Nayar, 1982 ), our investigations demonstrating the role of Mg2+ and Ca2+ in facilitating the infectivity of the CuniNPV baculovirus have direct and important implications for utilizing baculoviruses for control of these insects. It may be possible to develop mosquito baculoviruses as a new type of biopesticide by microencapsulating the virus and Mg2+ into formulations that would be effective regardless of the water quality. In addition, this new insight into transmission may facilitate the discovery and development of additional baculoviruses for the control of other mosquitoes species.
In contrast to employing baculoviruses for insect control, there are a number of situations where baculoviruses can cause economic harm. For example, viruses resembling baculoviruses are responsible for annual economic losses in cultured penaeid shrimp in the Americas and Hawaii due to mass mortalities that occur in the hatchery phase of production (Stuck & Overstreet, 1994 ). A method that could easily and economically inhibit transmission of these viruses in the shrimp farming industry would have positive and important implications. Similar methods could be employed by the silk industry to inhibit the spread of baculoviruses among silkworm larvae.
Future studies will focus on development of CuniNPV as a biopesticide and to characterize the entire genome of this baculovirus. It is hoped that this characterization will provide the knowledge necessary for manipulation of the virus to enhance desirable properties and to identify novel genes and proteins. These new constructs and proteins may prove useful in the development of improved strategies and tools for the control of mosquitoes worldwide that vector diseases of man and animals.
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Acknowledgments |
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References |
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Received 12 June 2000;
accepted 26 September 2000.