Abstract
Culex pipiens L. reared under diapause-inducing conditions (short daylength; 18°C) were more cold tolerant and desiccation resistant than their nondiapausing counterparts (long daylength; 18°C). Upon cold exposure (-5°C), diapausing mosquitoes reared at 18°C survived nearly twice as long as nondiapausing mosquitoes reared at 18°C and 10 times longer than nondiapausing mosquitoes reared at 25°C. Thus, rearing temperature provided partial protection against low temperature injury in nondiapausing mosquitoes, but maximum resistance to cold was attained by the diapause state. In this species, the supercooling point is not a good indicator of cold tolerance. Both diapausing and nondiapausing females had supercooling points of approximately -16°C, but diapausing as well as nondiapausing females died at temperatures well above the supercooling point, suggesting that low temperature mortality was due to indirect chilling injury. Diapause also conferred greater resistance to desiccation (1.6-2-fold increase in survival) compared with the nondiapause state. The gene encoding a 70-kDa heat shock protein, hsp70, was not up-regulated (i.e., more highly expressed) as a part of the diapause program, nor was it up-regulated by desiccation stress, but it was up-regulated during recovery from cold shock. Cx. pipiens thus differs from a number of other diapausing insect species that are known to developmentally up-regulate hsp70 during diapause.
The northern house mosquito, Culex pipiens L., is a major sylvatic vector of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in North America and has a wide distribution spanning much of the temperate zone of North America, Europe, and parts of Asia (Mattingly et al. 1951). Short daylength and low temperature received by fourth instars and early pupae program newly eclosed adult females to enter a reproductive diapause, characterized by an arrest in development of the primary ovarian follicles (Eldridge 1966, Sanburg and Larsen 1973, Spielman and Wong 1973). Before diapause, females feed only on carbohydrate sources, which they use to generate the lipid reserves needed for overwintering (Mitchell and Briegel 1989, Bowen 1992, Robich and Denlinger 2005). They then seek well-protected sites such as caves, culverts, or unheated basements that typically contain standing or running water (Vinogradova 2000), and survival is highest in sites that remain above 0°C with a stable humidity above 90% (Minar and Ryba 1971).
The ability of an insect to survive low temperatures through metabolic and physical change is referred to as cold hardiness (for reviews, see Lee 1991, Bale 2002, Sinclair et al. 2003). Two main strategies of cold hardiness have been well-described in the literature: freeze tolerance in which case the insect survives the internal formation of ice, and freeze avoidance that characteristically involves extensive supercooling that prevents ice formation. In this study, we provide evidence that Cx. pipiens is a freeze-avoiding species that relies on supercooling for low temperature survival.
We also examine the relationship between diapause and cold hardiness in Cx. pipiens. For cold hardiness to be considered a part of the diapause program, insects reared under diapause conditions should consistently be more cold tolerant than their nondiapausing counterparts reared at the same temperature (Denlinger 1991). In Aedes albopictus (Skuse), a mosquito that overwinters as a pharate first instar, both diapause and cold acclimation increase cold hardiness in the field and laboratory (Hanson and Craig 1994). This linkage has been well documented in several other insects, including, for example, diapausing pupae of the flesh flies Sarcophaga crassipalpis Macquart and Sarcophaga bullata Parker (Adedokun and Denlinger 1984) and the adult diapause of the Colorado potato beetle Leptinotarsa decemlineata (Say) (Lefevere et al. 1989).
Desiccation is another significant environmental stress that confronts overwintering insects. Dormant insects can resist desiccation by limiting water loss, by tolerating a low content of body water, or by mechanisms of water uptake; habitat choice is also an important feature for minimizing this form of stress (Danks 2000). One possible option for Cx. pipiens is to avoid desiccation by moving to a more humid location or by drinking water. Alternatively, they may be able to tolerate low relative humidities through some physiological adjustments.
One mechanism that could possibly play a role in both desiccation resistance and cold hardiness is the up-regulation of the molecular chaperone heat shock protein 70 (hsp70). Hsp70 is up-regulated during diapause in several insect species, and it is well-known to be up-regulated by temperature extremes and desiccation in nondiapausing individuals (Denlinger et al. 2001). For example, in the pupal diapause of the flesh fly S. crassipalpis, hsp70 is expressed immediately upon entry into diapause and remains up-regulated until diapause is terminated (Rinehart et al. 2000), and in nondiapausing flesh flies hsp70 is up-regulated in response to desiccation (Tammariello et al. 1999, Hayward et al. 2004). We thus test the possibility that hsp70 is expressed during diapause, in response to temperature or desiccation stress, or both.
In this study we show that 1) diapausing adults of Cx. pipiens are consistently more cold tolerant and desiccation resistant than their nondiapausing counterparts, 2) low temperature also enhances cold tolerance to some extent in nondiapausing individuals, and 3) hsp70 is not developmentally up-regulated during diapause nor responsive to desiccation stress, but it is up-regulated during recovery from cold shock.
Materials and Methods
Insect Rearing.
Anautogenous Cx. pipiens (Buckeye strain) were derived from mosquitoes collected in Columbus, OH, in September 2000. The colony was maintained at 25°C, 75% RH, and a photoperiod of 15:9 (L:D) h. Larvae were reared in 18- by 28- by 5-cm plastic containers in dechlorinated tap water, fed a daily diet of ground fish food (TetraMin), and held at a density of ≈250 mosquitoes per pan.
When larvae reached the second instar, the rearing containers were placed under one of three environmental conditions. Two nondiapause groups were created: the first group remained in the colony rearing room (nondiapause; 25°C), and a second nondiapause group was reared at 18°C, 75% RH, and a photoperiod of 15:9 (L:D) h (nondiapause; 18°C). By rearing nondiapausing mosquitoes at two temperatures, we were able to distinguish between the effects of photoperiod and rearing temperature. To induce diapause, a third group of mosquitoes was placed at 18°C, 75% RH, with a photoperiod of 9:15 (L:D) h (diapause; 18°C). The three rearing groups are hereafter referred to as ND25, ND18, and D18, respectively.
Adults, which also were maintained at ND18, ND25, or D18, were provided with water and honey-soaked sponges and kept in 30.5- by 30.5- by 30.5-cm screened cages. Honey sponges were removed from diapause cages 10-13 d after adult eclosion. None of the mosquitoes used in these experiments were offered a bloodmeal. Diapause status of adult females was confirmed by measuring the primary follicle and germarium lengths, and the stage of ovarian development was determined according to the methods described by Christophers (1911) and Spielman and Wong (1973). All experiments were conducted on females, 7-14 d after adult eclosion.
Monitoring Environmental Conditions at Field Sites.
We recorded temperature and relative humidity from 1 November 2003 to 27 March 2004 in three culverts located in Columbus, OH. Culverts were chosen based on the large number (>500) of diapausing Cx. pipiens females found in each site. Culverts were 1.5 m in diameter, constructed of cement, and extended ≥65 m in length, with a constant flow of running water. HOBO H8 Family Data Loggers (Onset Computer, Bourne, MA) were placed in each culvert at 25-40 m from the entrance, and data were recorded at hourly intervals.
Low Temperature Exposure.
To evaluate low temperature survival, groups of 15 laboratory-reared females, 7-10 d after adult eclosion, were aspirated into 50-ml polypropylene tubes that were submerged in a Lauda model RM20 circulating glycerol water bath that maintained a temperature of -5°C. ND18 and D18 mosquitoes were removed from the water bath at 24-h intervals until 100% mortality was attained. The ND25 group did not survive even 24 h at -5°C; thus, they were removed from the bath at 2-h intervals. Cold-treated mosquitoes were transferred to 750-ml plastic holding cages and returned to their original environmental chambers. Cages were supplied with a water source, and mosquito survival was assessed 24 h after exposure to -5°C. Survival was defined as the ability of the mosquito to right itself. Experiments for each of the three rearing groups were replicated six times.
Desiccation.
For each rearing group six replicates of 15 mosquitoes each were transferred by aspiration to 750-ml plastic holding cages and placed into 250-mm Nalgene plastic desiccators at 18°C under their respective light regimes. Six different relative humidities were attained by filling the bottom of the desiccators with one of the following solutions (Winston and Bates 1960): Drierite (0% RH), saturated MgCl2 (33% RH), saturated Ca(NO3)2 (50% RH), saturated NaCl (75% RH), saturated KCl (85% RH), and water-saturated towels (100% RH). Survivorship, defined as the ability of a mosquito to right itself, was assessed at daily intervals. All chambers were monitored until 100% mortality was attained.
Supercooling Points.
Supercooling points (SCPs) were determined for 20 females from each rearing group. A type-T 30-gauge copper-constantan thermocouple, coated with a thin film of petroleum jelly, was placed in contact with the female's abdomen. The mosquito was then lowered into a thin-walled 13- by 100-mm glass test tube that was plugged with cotton, and the tube was placed in a beaker containing 800 ml of isopropanol. The beaker was then placed in a -70°C freezer, thereby achieving a constant cooling rate of -1°C/min. The thermocouples were attached to an Omega HH506R datalogger (Omega Engineering, Stamford, CT), which recorded the temperature of the probe at 1-s intervals. The supercooling point was defined as the last temperature recorded before observing the temperature spike generated by the latent heat of crystallization.
Statistical Analysis of Survivorship Data.
Median lethal time (LT50) values were calculated for each replicate of low temperature and desiccation treatment groups from the resulting equation of the arcsine square root-transformed survival curve. The temperature data required square root transformation to accommodate the wide variance according to Sokal and Rohlf (1995) and was analyzed by one-way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) multiple comparison by using Statistica data analysis program (StafSoft, Tulsa, OK). Data from the desiccation experiments did not require further transformation and were therefore directly analyzed by two-way ANOVA with replication followed by Tukey's test of the least squares means. For both data sets, a P < 0.001 was considered statistically significant.
Before statistical analysis, the supercooling point data were square root-transformed to reduce heteroscedasticity and analyzed by one-way ANOVA. The lack of statistical differences precluded the use of post hoc testing.
Cloning and Sequencing of Hsp70.
Hsp70 was initially cloned from Cx. pipiens by reverse transcription-polymerase chain reaction (RT-PCR), by using RNA from mosquitoes that were incubated in a 38°C water bath for 30 min. RNA extraction was accomplished by grinding with 4.5-mm copper-coated spherical balls ("BBs") in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA). Insoluble material was removed by spinning at 12,404 × g at 4°C for 10 min, and the supernatant was used in RNA extraction following standard protocol (Chomczynski and Sacchi 1987). To obtain cDNA, random hexamers were used in reverse transcription followed by PCR consisting of a "hot start" at 94°C for 2 min and 35 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 2 min, followed by an additional 7 min at 72°C. The PCR reaction used primers based on conserved insect sequences that were designed to amplify a portion of the 5′ region of the open reading frame. The forward primer had a sequence of 5′-GAT GCA GTC ATC ACA GTT CCA GC-3′ and the reverse primer had a sequence of 5′-AAC AGA GAT CCC TCG TCG ATG GT-3′.
Full-length sequence was obtained using the SMART rapid amplification of cDNA ends (RACE) cDNA amplification kit (Clontech, Mountain View, CA) by 5′ and 3′ RACE by using the manufacturer's standard protocol. For 3′ RACE, a forward gene-specific primer (5′-AAG GAA ACT GCT GAG GCG TA-3′) was used in a PCR reaction consisting of 35 cycles with an annealing temperature of 58°C. The 5′ RACE was carried out using two gene-specific reverse primers: a reverse transcription primer (5′-GTA GAC GTC TCA GAG C-3′) and a PCR primer (5′-TTC GAC GAG ACG TCC TTC TT-3′). All PCR products were cloned using the TOPO TA cloning kit (Invitrogen), and the resulting clones were sequenced at The Ohio State University Plant-Microbe Genomics Facility on an Applied Biosystems 3730 DNA analyzer using BigDye Terminator Cycle Sequencing chemistry according to manufacturer's protocol (Applied Biosystems, Foster City, CA).
The 5′ and 3′ RACE products were edited and assembled using dnaLIMS (dnaTools, Ft. Collins, CO) and BioEdit Sequence Alignment Editor (Isis Pharmaceuticals, Carlsbad, CA). Similar sequences were identified by performing a BLASTn and BLASTx search in GenBank (http://www.ncbi.nlm.nih.gov/). The deduced amino acid sequences were assembled, analyzed, aligned, and percentage of identities were determined using BLASTp (National Center for Biotechnology Information, Bethesda, MD), the Baylor College of Medicine Search Launcher: Sequence Utilities (http://dot.imgen.bcm.tmc.edu/seq-util/seq-util.html), and Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html).
Northern Blot Analysis.
Total RNA was isolated from pools of 20 females, by using TRIzol reagent as described above. Twenty micrograms of denatured total RNA samples was separated by electrophoresis on a 1.4% agarose denaturing gel (0.41 M formaldehyde and 1X MOPS-EDTA-sodium acetate). The RNA was transferred onto a 0.45-μm MagnaCharge nylon membrane (GE Osmonics Inc., Minnetonka, MN) for 1.5 h by using downward capillary action in 3 M NaCl, 8 mM NaOH transfer buffer (Whatman Schleicher and Schuell, Keene, NH), neutralized in 1 M phosphate buffer solution, and crosslinked with UV irradiation. The crosslinked membrane was air-dried and either stored at –20°C or used immediately for hybridization.
Digoxigenin (DIG)-labeled hsp70 cDNA probe was prepared from the previously described RT-PCR product generated using the original hsp70 primers. Hsp70 cDNA was then labeled in an overnight DIG reaction by using 100 ng of template DNA and the Dig High Prime DNA labeling and detection starter kit II (Roche Diagnostics, Indianapolis, IN). Probes were stored at –20°C.
Overnight hybridization was carried out at 37°C by using the Dig High Prime DNA labeling and detection starter kit II. Stringency washes and immunological detection were done according to manufacturer's protocol, and the blots were subsequently exposed to chemiluminescence film (Kodak Biomax, Eastman Kodak, Rochester, NY). Northern blotting was performed in triplicate. To confirm equal loading of RNA, the membrane was stripped with 0.2 M NaOH/0.1% SDS and reprobed using DIG-labeled 28S cDNA, according to manufacturer's instructions.
Results
Environmental Conditions Recorded at Field Sites.
During the interval of December 2003 to April 2004, temperatures recorded in three Ohio culverts inhabited by diapausing females of Cx. pipiens ranged from –8.9°C to 16.4°C, with the lowest and highest temperatures recorded on 31 January 2004 and 25 April 2004, respectively. During the winter season, two of the three culverts had 4–6 d with temperatures below –5°C. The longest continuous stretches of days with temperatures dipping below –5°C were two 3-d periods from 23 January to 25 January 2004 and 30 January to 1 February 2004. Temperatures in the third culvert remained above –5°C for the entire winter season, with –2.9°C as the lowest temperature recorded. By late February, well before the termination of diapause, the warmest culvert had an abundance of mosquitoes remaining, few remained in the coldest culvert, and an intermediate number remained in the culvert with an intermediate mean temperature, thus suggesting that females in the coldest sites either died or moved to another location.
Relative humidity varied greatly in each site, fluctuating 40% or more during any given month. Relative humidity reached as high as 90% and dropped below 50% for each month recorded, with the lowest relative humidity (36%) recorded in January. This winter season included 23 d with a relative humidity lower than 50%. Mean relative humidity for the three sites was 68% in December, 64% in January, 74% in February, 80% in March, and 86% in April. By mid-April, few mosquitoes remained in any of the overwintering sites, suggesting that they had departed by this time.
Cold Tolerance.
Both diapause and low rearing temperature increased cold-tolerance (D18 > ND18 > ND25) (Fig. 1). One-way ANOVA showed significant variation in cold tolerance among mean LT50 values (F2, 15 = 231.7; P < 0.001), and analysis by the Tukey's HSD post hoc test revealed that all mean LT50 values were significantly different from one another (P < 0.001). Mosquitoes reared under long daylength at 25°C (ND25) died quickly when exposed to -5°C. Initial mortality was realized with as little as 2 h of exposure, with no individuals surviving >12 h of cold treatment. The calculated LT50 value for the ND25 mosquitoes was 4.9 ± 0.5 h (mean ± SE; n = 6 groups of 15 mosquitoes each). Lowering the rearing temperature to 18°C for nondiapausing mosquitoes significantly increased cold tolerance: nearly 60% of females survived a 24-h exposure to -5°C, and 100% mortality was not reached until 72 h of cold treatment. The calculated LT50 for the ND18 group was 28.9 ± 0.8 h, a five-fold increase in cold survival compared with the ND25 mosquitoes. Even greater cold tolerance was observed in mosquitoes reared under diapause-inducing conditions (D18): 86% of the females survived a 24-h exposure to –5°C, and 100% mortality was not attained until 120 h of cold treatment. D18 females exhibited a >10-fold increase in the LT50 value (50.3 ± 3.5 h) compared with ND25 mosquitoes and a two-fold increase compared with ND18 mosquitoes.
Percentage of survival (mean ± SE) of Cx. pipiens females after exposure to different durations of low temperature (−5°C). LT50 indicates the time (hours) when 50% of females were unable to right themselves. Nondiapausing females reared at 25°C (●), nondiapausing females reared at 18°C (▲), and diapausing females reared at 18°C (■). N = 6 groups of 15 individuals for each data point. 71
Desiccation Tolerance.
Because a >90% survival rate was observed for each rearing group placed at 100% RH, the 100% RH exposure was considered as our control and was not included in the statistical analysis. Within all experimental groups (ND25, ND18, and D18), a two-way ANOVA indicated significant variation (F4, 75 = 180.4; P < 0.001) among LT50 values as a function of relative humidity (Fig. 2). Increasing relative humidity resulted in an increase in survival of female mosquitoes for each rearing group tested. Nondiapausing mosquitoes reared at 25°C died quickly when placed in 0% RH, but survival increased progressively at higher humidities. More than 90% of the females placed in 100% RH survived the 24-d duration of our experiment, indicating that mortality in the other groups was due to desiccation stress rather than to factors such as lack of food or water.
Percentage of survival (mean ± SE) of Cx. pipiens females at various relative humidities. LT50 indicates the time (hours) when 50% of females were unable to right themselves. Nondiapausing females reared at 25°C (●), nondiapausing females reared at 18°C (▲), and diapausing females reared at 18°C (■). n = 6 groups of 15 individuals for each data point.
Contrary to what was observed for cold stress, nondiapausing Cx. pipiens reared at 18°C did not have an increased ability to withstand desiccative stress compared with nondiapausing mosquitoes reared at 25°C (Fig. 2). For all five of the relative humidities tested, there were no significant differences in survival to desiccation between the two nondiapausing groups reared at different temperatures (ND25 and ND18).
In contrast, females reared in diapause-inducing conditions had a significant increase in the ability to survive at all five relative humidities tested compared with nondiapausing mosquitoes reared at the same temperature. This is supported by a two-way ANOVA, which showed a significant difference between the three rearing groups in survival at each relative humidity (F2, 75 = 117.5; P < 0.001), and Tukey's test of the least squares means, which shows significant differences between the D18 LT50 and the ND18 LT50 at all five humidities (P < 0.001). D18 females showed LT50 survival with values 1.6–2.0-fold higher than the survival of the ND18 group at all five relative humidities. The two-way ANOVA also indicated a significant interaction (F8, 75 = 10.7; P < 0.001) between the diapause program and relative humidity exposure: as relative humidity increased, there was a widening gap in survivorship between nondiapausing (ND25 and ND18) and diapausing (D18) mosquitoes (Fig. 2).
Supercooling Point.
Neither low rearing temperature nor diapause status significantly affected the SCP of adult Cx. pipiens (F2, 57 = 1.55; P = 0.22). Females reared at ND25 had a mean SCP of –16.1 ± 0.5°C (mean ± SE; each n = 20), whereas those reared at ND18 and D18 had SCPs of –15.4 ± 0.2 and –16.0 ± 0.3°C, respectively. The SCPs for all three groups were substantially lower than the experimental temperature (–5°C) that caused mortality, thus indicating that the mortality observed at –5°C was due to indirect chilling injury, rather than the direct freezing of tissues.
Cloning and Analysis of the Deduced Amino Acid Protein Sequence for Hsp70.
Our initial hsp70 cDNA clone, attained using the universal primers in PCR, resulted in a 232-bp clone that aligned with 100% similarity to the Anopheles gambiae Giles hsp70 (AAM94344) amino acid sequence. To obtain full-length sequence, we used our hsp70 cDNA fragment to design gene-specific primers for 5′ and 3′ RACE. This resulted in 735 bp- (5′ end) and 1,725 (3′ end)-bp fragments, both of which overlapped the initial hsp70 clone and yielded a total product size of 2,274 bp. Conceptual translation showed that the Cx. pipiens hsp70 open reading frame is 638 residues, starting at nucleotide 178. The deduced amino acid sequence is highly conserved compared with those from Anopheles albimanus Wiedemann, Drosophila melanogaster Meigen, Ceratitis capitata (Wiedemann), and Manduca sexta (L.), sharing 89, 82, 80, and 76% identity, respectively (Fig. 3). The full-length nucleotide sequence for Cx. pipiens heat shock protein 70 was deposited in GenBank and assigned accession number AY974355.
Multiple sequence alignment of the deduced Cx. pipiens hsp70 with other hsp70 sequences retrieved from GenBank. Amino acids identical to Cx. pipiens are shaded. CpiHsp70: Cx. pipiens hsp70, AY974355; AalHsp70A2/B2: An. albimanus hsp70 A2, AAC41543 and B2, P41827; DmeHsp70Ba/b: D. melanogaster hsp70 Ba, AAG26905 and Bb, AAG26901; Ccahsp70: C. capitata Hsp70: CAA70153; MseHsp70: M. sexta Hsp70, AAO65964.
Expression of Hsp70.
Northern blot analysis revealed that diapausing Cx. pipiens does not express detectable levels of hsp70 transcript in early diapause (Fig. 4), nor at any other time during diapause (data not shown). In addition, the transcript was not up-regulated in response to desiccation (1 d at 0% RH and 4 d at 75% RH) in either diapausing or nondiapausing females (data not shown). Hsp70, however, was up-regulated after a 4-h cold shock at –5°C (Fig. 4). The transcript was detectable in all the experimental groups (ND25, ND18, and D18) 1 and 2 h after the mosquitoes were removed from the -5°C bath. Twenty-four hours later, the signal was once again undetectable. No apparent differences could be seen in the intensity of the signal in the three groups, but in all cases hsp70 was responsive to low temperature.
Northern blots showing expression of heat shock protein 70 in Cx. pipiens females at different times (hours) after a 4-h exposure to −5°C. A 28S cDNA probe was used to confirm equal loading. C, untreated females. HS, females heat shocked at 38°C for 30 min.
Discussion
Cold Hardiness.
Rearing Cx. pipiens at low temperature enhanced cold hardiness in nondiapausing individuals, but cold hardening was further increased if the mosquitoes reared at low temperature also were programmed for diapause by being reared under short daylength. Lowering the rearing temperature in nondiapausing mosquitoes from 25 to 18°C resulted in a greater than five-fold increase in the adult female's resistance to cold. Diapause status further increased cold hardiness: such females were able to withstand exposure to -5°C almost twice as long as their nondiapausing counterparts reared at 18°C and 10 times as long as those reared at 25°C. Cx. pipiens thus seems to be a species in which cold hardiness is a component of the diapause program, as noted for several other species (Denlinger 1991, Kostal et al. 2004), including pharate first instars of Ae. albopictus (Hawley et al. 1989, Hanson and Craig 1994).
Adults of Cx. pipiens died at subzero temperatures that were well above their supercooling point, indicating that lethality at -5°C was due to chilling injury rather than freezing. Nondiapausing mosquitoes reared at 25°C died within hours of exposure to low temperature and thus would be considered chill susceptible by Bale (1996): this category includes insects that die after a short exposure to temperatures well above the supercooling point. Cold acclimation and diapause, however, shift Cx. pipiens into the chill-tolerant category because these individuals have a much higher level of cold tolerance (i.e., can withstand days at -5°C).
The supercooling point of Cx. pipiens is not a good indicator of low temperature tolerance, an observation previously noted for other species (Lee and Denlinger 1985). Nondiapausing adults reared at 18 or 25°C and diapausing adults reared at 18°C all had similar SCPs around –16°C, yet even diapausing adults died after prolonged exposure to –5°C, a temperature well above the recorded supercooling point. Although some species can tolerate temperatures close to the supercooling point, many insects are like Cx. pipiens and are unable to do so (Lee 1991, Bale 1996). The supercooling points we noted for Cx. pipiens are similar to those recorded for diapausing adults of An. quadrimaculatus Say (–17.2°C) and An. punctipennis (Say) (–20.1°C) (Wallace and Grimstad 2002), both of which are frequently found in the same overwintering sites as Cx. pipiens in the American Midwest. Danks (1978) suggests that the SCP often approaches the climatic minima in the species habitat. The minimum temperature we observed in culverts used for overwintering in Ohio during winter 2003–2004 was –8.9°C. That particular winter had lower than average temperatures in January; thus, it is unlikely that temperatures in these protected sites ever drop as low as –16°C. Possibly Cx. pipiens that are in diapause can survive very brief exposure to temperatures just above the SCP, but this has not been tested.
Desiccation Resistance.
Diapausing Cx. pipiens were more resistant to desiccation than nondiapausing females reared at either high or low temperatures. Unlike some other insect species (Zachariassen 1991, Ring and Danks 1994, Block 1996), cold acclimation of nondiapausing females did not provide protection against exposure to dry conditions.
Although Cx. pipiens overwinters in sites that are well protected and often contain standing water, the relative humidity in such sites can fluctuate greatly over the course of a winter. Three Ohio culverts monitored during winter 2003-2004 showed large variation in relative humidity, ranging from a low of 36% to a high of 100%. Because Cx. pipiens diapauses in the adult stage, they have the ability to move within the hibernaculum, and our own unpublished observations confirm that the adults do indeed move around within the culverts during the winter. This behavior may allow the females to continually select optimum conditions by choosing a resting place with a high relative humidity, or they may even drink water during this time. Our unpublished laboratory observations suggest that it is critical for diapausing Cx. pipiens to have access to water: D18 survivorship was ≈3 wk at 85% RH with no access to water or food, whereas D18 females held at 75% RH with access to water but not to food survived >4 mo. Our results demonstrate that resistance to desiccation is a component of the diapause syndrome, but free access to water is essential for long-term survival. The factors contributing to desiccation resistance in diapausing adults of Cx. pipiens remain unknown.
A Role for Hsp70?
Heat shock proteins are best known for their role in the protection of normal cellular function during exposure or recovery from environmental stress (Feder and Hofmann 1999), but curiously they also are up-regulated in several insect species as a component of the diapause program (Denlinger et al. 2001). To test the possibility of diapause up-regulation of heat shock proteins in Cx. pipiens, we cloned hsp70 and determined whether it was up-regulated during diapause or by environmental stresses (high or low temperature, desiccation) during diapause.
The Cx. pipiens hsp70-deduced amino acid sequence is highly conserved compared with those from other insects. It is most similar (89% amino acid identity) to hsp70 from the mosquito An. albimanus (Benedict et al. 1993). In Cx. pipiens, hsp70 is not expressed as a component of diapause. Thus, Cx. pipiens is unlike several other species that up-regulate hsps upon entry into diapause, even in the absence of thermal stress. Diapause up-regulation of hsps has been well documented in the pupal diapause of the flesh fly S. crassipalpis (Yocum et al. 1998, Rinehart et al. 2000) and the pupal diapause of the solitary bee Megachile rotundata (F.) (Yocum et al. 2005) as well as in several other arthropods, including diapausing embryos of the brine shrimp Artemia franciscana Kellogg (McRae 2003) and diapausing adults of the Colorado potato beetle (Yocum 2001). But, up-regulation of hsps does not occur in all species. For example, none of the hsps seem to be up-regulated during the larval diapause of the blow fly Lucilia sericata (Meigen) (Tachibana et al. 2005) or during the adult diapause of Drosophila triauraria Bock & Wheeler (Goto et al. 1998, Goto and Kimura 2004). Although one of the hsp70 transcripts in diapausing adults of L. decemlineata is up-regulated (Yocum 2001), the up-regulation is rather modest in comparison to that observed in flesh fly pupae (Rinehart et al. 2000). Thus, the hsp70 expression pattern we observed in Cx. pipiens in association with diapause is very similar to what has been observed previously in other adult diapauses. Although relatively few species have been examined, it would seem that diapause up-regulation of hsp70 is less common in adult diapause than it is in diapauses occurring in preadult stages of development.
Although hsp70 is not expressed as a component of the diapause program in Cx. pipiens, hsp70 does remain responsive to cold shock (4 h at -5°C) during diapause. As in S. crassipalpis (Joplin and Denlinger 1990, Rinehart et al. 2000), expression was not observed during the cold shock, but only after a 1-2-h recovery period from cold shock, and the signal again returned to undetectable levels after 24 h. There were no major differences in the intensity of response by all three groups tested (ND25, ND18, and D18), indicating that hsp70 expression elicited by cold shock was not affected by the diapause state or cold acclimation. The signal detected by northern blotting after cold shock was also weak compared with the heat shock response. In diapausing embryos of the gypsy moth, Lymantria dispar (L.), hsp70 is expressed strongly in response to low temperature and expression and then persists for the duration of diapause (Yocum et al. 1991, Denlinger et al. 1992), but such is not the pattern observed in Cx. pipiens. In Cx. pipiens, the gene is turned on briefly in response to a cold shock, but expression does not persist. Together, the weak intensity of the response, coupled with the brevity of expression, suggests that hsp70 is most likely not the major factor contributing to the increased cold resistance seen in diapausing females.
Acknowledgements
We appreciate helpful comments on the manuscript provided by Phil Lounibos (University of Florida, Vero Beach, FL). This work was supported in part by grants from USDA-National Research Initiative (98-35302-6659), National Science Foundation (IBN-0416720), National Institutes of Health (R01 AI058279), and by a grant-in-aid from the Ohio Mosquito Control Association.
References Cited
