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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study was therefore undertaken to investigate the effect of various higher temperatures during larval stages on the susceptibility of Ae. aegypti to chikungunya (CHIK) virus. Initial experiments showed an increase in the susceptibility of mosquitoes to CHIK virus following temperature stress. Therefore, it was essential to determine 1) if this effect was a temporary phenomenon associated with some kind of selection of a temperature-tolerant strain and 2) if exposure to heat leads to up-regulation and down-regulation of immunoresponsive (IR) genes affecting the susceptibility of mosquitoes to virus. Some of the biologic parameters such as larval duration, mortality, and adult survival at various temperatures were also studied in both the normal and temperature-tolerant strains.
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MATERIALS AND METHODS |
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In some of the experiments the normal larvae were divided into five batches and were exposed to temperatures ranging from 42°C to 46°C. The larval mortality was recorded after 24 hours. Probit analysis4 was applied to the proportions of mortalities obtained for the heat shock of various temperatures to calculate the 50% lethal temperature (LT50) for different mosquito strains. Similarly, in the separate experiments adults obtained from the larvae exposed to various higher temperatures were fed on fresh chicken blood and virus mixture.5
To determine the expression of various IR genes, larvae were exposed to a temperature of 41°C for 60 minutes. This temperature was chosen because in an earlier study this temperature was found to eliminate gregarine parasites from the larvae, but the host could sustain this heat shock,6 and with the view that a prolonged period of exposure of 60 minutes to this temperature would ensure generation of sufficient level of heat shock to determine up-regulation and down-regulation of IR genes in the mosquitoes.
Virus. The Kolkata strain (634029) of CHIK virus was used throughout the study. In an earlier study, Ae. aegypti mosquitoes were found to be highly susceptible to this strain.7 The virus stock was prepared in mice and used at the seventh mouse passage level.
Oral infection of mosquitoes. Four- to five-day-old female mosquitoes from different batches were orally infected through an artificial membrane (Parafilm®; American National Can, Greenwich, CT) as described by Harada and others.8 Post-feeding virus titers in the mosquito feeding suspensions were calculated in mice. Orally infected mosquitoes were maintained at a temperature of 28°C ± 2°C and relative humidity of 80 ± 5%. On 10th day post-infection, the mosquito positivity was determined by an indirect immunofluorescence test on head squashes as previously described.9
Extraction of RNA and reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNAs were extracted from surviving larvae at 1, 4, 8 and 24 post-heat shock hours (PHSH) using Trizol reagent (Gibco-BRL, Gaithersburg, MD) according to the manufacturer’s protocol. The RNA from five larvae was dissolved in 100 µL of diethylpyrocarbonate-treated water. cDNA was prepared using Moloney murine leukemia virus-reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
The following PCR cycling programs were used: for defensin and nitric oxide synthetase (NOS), a five-minute denaturation at 94°C; five 30-second steps at 94°C, 57°C, and 72°C; five 30-second steps at 94°C, 56°C, and 72°C; five 30-second steps at 94°C, 55°C, and 72°C; five 30-second steps at 94°C, 54°C, and 72°C; 25 one-minute steps at 94°C, 57°C, and 72°C; and a final 10-minute extension at 72°C; for cecropin and lectin, a five-minute denaturation at 94°C; five 30-second steps at 94°C, 52°C, and 72°C; five 30-second steps at 94°C, 51°C, and 72°C; five 30-second steps at 94°C, 50°C, and 72°C; five 30-second steps at 94°C, 49°C, and 72°C; 25 one-minute steps at 94°C, 52°C, and 72°C; and a final 10-minute extension at 72°C; for serine proteases and heat shock protein 70 (Hsp70), a five-minute denaturation at 94°C; five 30-second steps at 94°C, 55°C, and 72°C; five 30-second steps at 94°C, 54°C, and 72°C; five 30-second steps at 94°C, 53°C, and 72°C; five 30-second steps at 94°C, 51°C, and 72°C; 25 one-minute steps at 94°C, 55°C, and 72°C; and a final 10-minute extension at 72°C.
The following primers were used: NOS1F 5'-GCG CCG TTC AGG TGG TTC TGG CA-3' and NOS1R 5'-CCG AAG ATA TCT TCATGATGGTATCG-3' (500 basepairs [bp]); DEFF 5'-GAT GAA CTG CCG GAG GAA AC-3' and DEFR 5'-CGG CAG ACG CAC ACC TTC TT-3'(180 bp); CECROF 5'-ATG AAC TTC ACG AAG TTA TTT CTC C-3' and CECROR 5'-CTT TCT TAG AGC TTT AGC CCC-3' (180 bp); LECTF 5'-ACA GCG ACA ACA GAT TCT GCT TCC-3' and LECTR 5'-CCT GCA ATT TAC GCC TGT TCG CAC-3' (400 bp); SERF 5'-TAC AGT TAC AGG ATG GGG TAA G-3' and SERR 5'-ATC CTC TGA TTG CTG GGT GTG C-3' (290 bp); and HSP70F 5'-CCC GTC CTA CGT GGC GTT CA-3' and HSP70R 5'-GGT GGC CTG ACG TTG CGA GT-3' (342 bp).
Sequencing analysis. The amplified products were analyzed after electrophoresis on 2% agarose gels in Tris-acetate-EDTA buffer (0.04 M Tris-acetate, 1 mM EDTA), stained with ethidium bromide, and visualized on an ultraviolet transilluminator at 302 nm. The bands of interest were recovered using the StrataPrep DNA Gel Extraction Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Direct sequencing of the amplified product was performed using an ABI 310 automated DNA sequencer using the Big Dye terminator kit (Applied Biosystems, Inc., Foster City, CA).
Larval duration and adult survival experiment. Approximately 200 first instar larvae of each strain were placed in 250 mL of water in a 500-mL beaker. The larvae were fed with 25 mg of feed/day/beaker for four days, followed by 50 mg for two days, and 100 mg for the remaining period. Fresh feed was added to each beaker after changing the water every day. A daily record of larval and pupal mortality and emergence of adults was maintained. The emerging adults were kept in small cages and maintained on a 10% glucose solution in cotton pads. The adult mortality was also recorded every day.
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RESULTS |
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). There was a sharp increase in the mortality above 43°C; however, even at this temperature the TT strain showed some tolerance. There was an approximately 0.14°C difference in the LT50 values in the TT strain larvae compared with the unstressed control strain larvae (
2= 33.7; degrees of freedom = 3).
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). It is interesting to note that when these strains were not subjected to a higher temperature stress, there were no differences in the susceptibility of the mosquitoes to the virus. It appears that continued selection is required to maintain differences in the susceptibility of mosquitoes to virus.
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). Expression of this heat shock marker changed with the PHSH. However, defensin showed no difference in expression between heat shock and control batches.
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Life table parameters studied in these strains showed that when both the strains were maintained at 28 ± 1°C, there was no difference in the larval duration and mortality in the immature stages, but the mean survival of the female mosquitoes in the TT strain was 5–6 days longer. However, when mosquitoes were maintained throughout at 37°C, the mean survival of the mosquitoes was lowered drastically in both the strains, but the mean survival of females in the TT strain was 5–6 days longer compared with the unstressed controls. However, fecundity of the TT strain was lower than that in the control mosquitoes (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An increase in the susceptibility of mosquitoes to virus was noticed above 39°C. In Anopheles mosquitoes, it has been reported that temperatures greater than 39°C induced heat shock proteins.10 In a study of Ae. aegypti and Ae. albopictus, heat shock proteins have been demonstrated in vitro and in vivo.11 Other reports have demonstrated a correlation between heat shock and virus multiplication in vitro.12,13 Our earlier studies with this mosquito species have shown that susceptibility to CHIK virus is governed by a major recessive gene located on the linkage group-III.14 Other studies have shown that the major factor in the mesentronal barrier seems to be the presence of a larger amount of the CHIK virus-specific receptor proteins in the brush border membrane of the gut epithelial cells.15 However, it is known that cecropin and other IR genes are expressed at high levels in fat tissues and in hemolymph. Their higher expression at this level probably results in more resistance to the virus during dissemination from the midgut to the salivary glands. At this juncture, it is difficult to conclude what exactly was the reason for increased susceptibility in the mosquitoes that were exposed to higher temperatures in their developmental stages. Based on our results, it appears that there is no selection pressure due to heat shock since the TT strain shows similar IR gene expression compared with the controls. In addition, after the heat shock was ended, the TT strain showed susceptibility similar to the normal control. This suggests that the factor that has a modifying effect on susceptibility is also affected by temperature.
The cecropins are 4-kD cationic peptides that show lytic activity against both Gram-negative and Gram-positive bacteria.16 In Drosophila melanogaster, three cecropin genes (CecA1, CecA2, and CecB) are known. The fat body is shown to be the major site of synthesis, although some transcription has been demonstrated in hemocytes.17 It was interesting to note that 180-bp cecropin gene product was obtained only from RNA extracted from larvae after 8 PHSH. This suggested that the transcript originated from genes expressed due to heat shock. Such heat shock responses are supported by the expression of heat shock marker Hsp70. A second major group of membrane-active peptides induced in challenged insects are defensins. These are 4-kD peptides with activity against Gram-positive bacteria,18 but they kill bacteria at slower rates. It has been suggested that defensins act by a mechanism different from that of the cecropins.18 Lectins are a class of glycoproteins that bind specifically to carbohydrates moieties. They are often observed after wounding or infection. One of the proposed functions of lectin in hemolymph is as an immune molecule that mediates adherence and phagocytosis by hemocytes. Lectins bind to biologically relevant foreign particles including bacteria and protozoan and metazoan parasites, thus supporting this hypothesis. It has also been reported that injury response, injection of saline, or carbohydrate enhances encapsulation of a parasite.19 In our study, we showed that the lectin response deceased after heat exposure compared with control mosquitoes. Nayar and Knight19 studied hemagglutinins from different organs of Anopheles quadrimaculatus and found that they were heat labile and lost their activity when subjected to a temperature of 60°C for 30 minutes. The majority of these studies have been conducted with bacteria and the precise role of these peptides in defenses against viruses is not known. It is suggested that they are active against enveloped viruses.
It has been shown that entry of dengue virus into the host cells is through adsorption and penetration, leading to infection within two hours, and carbohydrate residues may contribute to binding and penetration of the virus into mosquito cells.20 However, when mosquitoes take a blood meal, formation of the peritrophic membrane begins within 20–30 minutes, which subsequently surrounds the blood bolus. Therefore, it is presumed that the entry of virus particles into mid-gut epithelial cells should take place before the formation of peritrophic membrane. In orally infected mosquitoes soon after the blood meal, proteolytic enzymes are activated that destroy virus particles present in the blood bolus. After initial multiplication in midgut cells, the dissemination of virus to the salivary gland takes at least four days. However, the virus probably encounters many defensive responses. It has been shown that the susceptibility of Ae. aegypti to CHIK virus is polygenic and the mesentronal barrier is believed to play an important role.14 Several intrinsic and extrinsic factors may affect gene controlling virus susceptibility. Our data suggest that cecropin and lectin can affect this susceptibility. Particularly in the case of cecropin and lectin, expression in normal mosquitoes and its up-regulation and down-regulation after infection and heat shock suggest that the synthesis of these peptides probably provides a barrier to virus multiplication once the virus has crossed the midgut barrier.
In tropical regions, the environmental temperature is often quite high, although the temperature of the mosquito breeding container may not always reach 41°C. A temperature higher than the ambient temperature for a few hours might produce a similar effect. We do not know if such a phenomenon exists in nature that affects mosquito susceptibility to dengue virus. It is surmised that an increase in temperature above the average temperature in an endemic area would not only enhance the selection of temperature-tolerant mosquitoes in a population having greater longevity, but would also affect both intrinsic and extrinsic factors by reducing extrinsic incubation period and increasing susceptibility of mosquito to viruses due to affected expression of IR genes. It would be worthwhile to carry out retrospective analysis of the correlation between epidemics and temperatures of specific areas in the summer before the epidemics have occurred with average temperatures of the previous 3–5 years. Additional detailed laboratory and field studies are required to affirm that the stress of environmental temperature on the developmental stages may have effects on vector competence.
Received September 29, 2003. Accepted for publication December 18, 2003.
Acknowledgments: We thank to Dr. V. A. Arankalle (National Institute of Virology) for critical comments and suggestions on the manuscript.
Authors’ addresses: D. T. Mourya, Microbial Containment Complex, Sus Road, Pashan, Pune 411 021, India, Telephone: 91-20-589-3640, Fax: 91-20-588-3595, E-mail: mouryadt{at}vsnl.net. P. Yadav and A. C. Mishra, National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune 411 001, India.
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