Non-diapausing Females Research Articles

Elevated couch potato transcripts associated with adult diapause in the mosquito Culex pipiens

The couch potato ( cpo) cDNA that we cloned from the northern house mosquito, Culex pipiens, encodes the C-terminus containing a highly conserved RNA recognition motif (RRM). Protein structure prediction indicates a canonical RRM structure with a βαββαβ topological structure. Northern blots indicate a single mRNA band over 9.49 kb, and Southern blot analysis suggests that the cpo gene contains large introns. Highest expression was noted in first instar larvae and pupae. A comparison of nondiapausing (long daylength) and diapausing (short daylength) adult females showed no difference immediately at adult eclosion, but by day 7 and thereafter, expression of cpo was much higher in diapausing adults. When 2-month old diapausing females were transferred from short daylength to diapausing-terminating conditions of long daylength and high temperature, expression of cpo declined. Similarly, when a topical application of JH III was used to terminate diapause abundance of the cpo transcript declined. Consistent with observations in Drosophila melanogaster and several other species levels of cpo in C. pipiens are influenced by the diapause program, although the direction of change is not the same in all species.

Molecular structure of the prothoracicotropic hormone gene in the northern house mosquito, Culex pipiens, and its expression analysis in association with diapause and blood feeding

We cloned the gene that encodes prothoracicotropic hormone (PTTH) in the northern house mosquito, Culex pipiens, and investigated its expression profile in short-day (diapause-destined) and long-day (nondiapause-destined) individuals from the fourth-instar larval stage to 2 months of adulthood, as well as after a blood meal. The deduced C. pipiens PTTH (Cupip-PTTH) amino acid sequence contains seven cysteines with a specific spacing pattern. Sequence alignment suggests that Cupip-PTTH is 23% identical to Drosophila melanogaster PTTH, but is ≥59% identical to the PTTHs of other mosquitoes. Cupip-PTTH has structural characteristics similar to those of Bombyx mori PTTH and some vertebrate nerve growth factors with cysteine-knot motifs. PTTH transcripts exhibit a daily cycling profile during the final (fourth) larval instar, with peak abundance occurring late in the scotophase. The fourth-larval instar stage is one day longer in short-day larvae than in long-day larvae, resulting in larger larvae and adults. This additional day of larval development is associated with one extra PTTH cycle. No cycling was observed in pupae, but PTTH transcripts were slightly higher in short-day pupae than in long-day pupae throughout much of the pupal stage. PTTH expression persisted at a nearly constant level in diapausing adult females for the first month but then dropped by ∼50%, while expression decreased at the beginning of adulthood in nondiapausing females and then remained at a low level as long as the females were denied a blood meal. However, when nondiapausing females were offered a blood meal, PTTH transcripts rose approximately 7 fold in 2 h and remained elevated for 24 h. A few diapausing females (∼10%) will take a blood meal when placed in close proximity to a host, but much of the blood is ejected and such meals do not result in mature eggs. Yet, elevated PTTH mRNA expression was also observed in diapausing females that were force fed. Our results thus point to several distinctions in PTTH expression between short-day and long-day mosquitoes, but both types of females responded to a blood meal by elevating levels of PTTH mRNA.

Repeated bouts of dehydration deplete nutrient reserves and reduce egg production in the mosquito Culex pipiens

In this study of the mosquito, Culex pipiens, we examined the impact of multiple bouts of dehydration and rehydration on survival, depletion of metabolic reserves and egg production in both non-diapausing and diapausing females. Mosquitoes provided with access to sugar during rehydration survived longer than those allowed to rehydrate without sugar, and their survival was similar to that of mosquitoes of the same age that were not dehydrated. Among mosquitoes not provided with sugar, each dehydration bout reduced the mosquito's dry mass - an effect likely to be due to the utilization of carbohydrates and lipid reserves. The toll on glycogen and lipid reserves is likely to be especially costly for diapausing mosquitoes that are dependent on these stored reserves for winter survival. Egg production in both non-diapausing and post-diapausing C. pipiens was also reduced in response to multiple bouts of dehydration. Although egg quality was not compromised, the number of eggs produced was reduced. Both non-diapausing and diapausing females can compensate for the nutrient loss due to dehydration by sugar feeding but the opportunity to feed on sugar is likely to be rarely available in the overwintering habitat of diapausing females, thus the impact of dehydration may be especially pronounced in overwintering populations of C. pipiens.

RNA interference directed against ribosomal protein S3a suggests a link between this gene and arrested ovarian development during adult diapause in Culex pipiens

Arrested ovarian development is a key characteristic of adult diapause in the mosquito Culex pipiens. In this study we propose that ribosomal protein S3a (rpS3a), a small ribosomal subunit, contributes to this shutdown. RpS3a is consistently expressed in females of C. pipiens that do not enter diapause, but in females programmed for diapause, expression of the rpS3a transcript is dramatically reduced for a brief period in early diapause (7-10 days after adult eclosion). RNA interference directed against rpS3a in nondiapausing females arrested follicle development, mimicking the diapause state. The effect of the dsRNA injection faded within 10 days, allowing the follicles to grow again, thus the suppression of rpS3a caused by RNAi did not permanently block ovarian development, implying that a brief suppression of rpS3a is not the only factor contributing to the diapause response. The arrest in development that we observed in dsRNA-injected females could be reversed with a topical application of juvenile hormone III, an endocrine trigger known to terminate diapause in this species. Though we speculate that many genes contribute to the diapause syndrome in C. pipiens, our results suggest that a shut down in the expression of rpS3a is one of the important components of this developmental response.

A potential role for ribosomal protein S2 in the gene network regulating reproductive diapause in the mosquito Culex pipiens

We propose that a shut-down in expression of ribosomal protein S2 (rpS2) contributes to regulation of diapause in adult females of Culex pipiens. While this gene is expressed continuously in nondiapausing females reared under long-day conditions, it is strongly down-regulated 5-18 days after adult eclosion in females reared under the short-day conditions that induce diapause. The possibility that this shut-down in expression of rpS2 contributes to the arrest in ovarian development characteristic of diapause is bolstered by the diapause-like arrest in follicle growth observed when nondiapausing females are injected with dsrpS2. A control gene encoding another ribosomal protein, L19, is expressed equally in nondiapausing and diapausing females, and RNA interference directed against rpL19 did not arrest follicle growth, thus indicating that the response we observed in knocking down expression of rpS2 is not common to all ribosomal proteins. Diapause in C. pipiens is readily terminated with juvenile hormone (JH), and in this study we demonstrated that an exogenous application of JHIII can rescue the arrest in follicle growth caused by dsrpS2. Together, these results suggest that rpS2 plays a critical role in arresting the ovarian development associated with diapause in this mosquito.

Transcription profiling and regulation of fat metabolism genes in diapausing adults of the mosquito Culex pipiens

Culex pipiens, the mosquito that vectors West Nile virus in North America, overwinters in an adult diapause (dormancy) that is programmed by the short day length and low temperatures of autumn. In response to these environmental signals, females cease feeding on blood and instead seek sources of nectar used to generate the huge lipid reserves required for winter survival. To identify regulatory networks that regulate fat accumulation and fat consumption during diapause, we compared expression of fat-related genes from nondiapausing females with expression of those same genes in early and late diapause and at diapause termination. Among the 31 genes we examined, 4 were expressed more highly in early diapause than in nondiapause, while 14 genes were downregulated in early diapause. In the transition from early to late diapause, 19 genes related to fat metabolism were upregulated. As reported previously, fatty acid synthase, identified as fas-1 in this study, was upregulated in early diapause. Numerous fat metabolism genes, including multiple kinetic classes and genes involved in beta-oxidation, an energy-generation step, were suppressed in early diapause but were highly expressed in late diapause and at diapause termination. RNA interference (RNAi) analysis revealed that the fas-1 gene and others (fas-3 and fabp) have important roles in fat storage during early diapause. When expression of these genes is suppressed, female mosquitoes fail to sequester the lipids needed for overwintering.

Diapause-inducing signals prolong nymphal development in the two-spotted spider miteTetranychus urticae

Female two-spotted spider mite Tetranychus urticae are grown under different photoperiods and the photoperiodic regulation of diapause is examined. The photoperiodic response curve for diapause induction was of the long day–short day type, with critical day lengths (CDLs) of 2 and 12.5 h; diapause was induced between these CDLs. The preimaginal period is significantly longer in diapausing females than in non-diapausing females; moreover, a significant positive correlation is detected between diapause incidence and deutonymphal period. Diapause incidence is high when long-night photoperiods are applied against a background of continuous darkness in the stages including the deutonymph; this stage appears to be the most sensitive to photoperiod. These observations suggest that diapause-inducing conditions inhibit nymphal development, particularly in the deutonymphal stage when photoperiodic time measurement for determination of reproduction or diapause is carried out.

Decrease in expression of beta‐tubulin and microtubule abundance in flight muscles during diapause in adults of Culex pipiens

The cDNA encoding beta-tubulin in the mosquito Culex pipiens has high similarity with the beta-tubulins reported in other insects. In this study, we examine expression of this gene and microtubule abundance in relation to diapause and low temperature. While non-diapausing mosquitoes express beta-tubulin highly in their thoracic muscles, expression is quite low during adult diapause. The abundance of microtubules was also much lower in flight muscles of diapausing adults than in flight muscles from non-diapausing individuals, as confirmed by laser confocal microscopy of tubulins stained using indirect immunofluorescence. Low temperatures decreased microtubule abundance in midguts of non-diapausing mosquitoes, but microtubule abundance in diapausing mosquitoes was already low and remained unchanged by low temperature exposure. Overall, pixel intensity averages were higher in the flight muscles than in the midguts, and again low temperatures decreased microtubule abundance in the flight muscles of non-diapausing females, while levels remained consistently low in diapausing females. These results clearly indicate that a decrease in microtubule abundance is evoked both by the programming of diapause and, in non-diapausing females, by exposure to low temperatures. Quite possibly the reduced microtubule abundance in the flight muscles and reduced expression of beta-tubulin are functionally correlated to the reduction in flight activity that is associated with low temperature and diapause.

UV tolerance in the two-spotted spider mite, Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae was exposed to UV-C (250 nm), UV-B (300 nm), and UV-A (350 nm). In non-diapausing females, the median effective doses for 50% mortality plus escape incidence (ED 50) were 21 (UV-C) and 104 kJ m −2 (UV-B); those for 50% oviposition rate in continuous darkness-treated mites were 6.2 (UV-C) and 41 kJ m −2 (UV-B). No significant effects of UV-A on mortality and oviposition rate were observed. The ED 50 values for UV-B were similar to the natural UV-B observed for 2–5 days in summer when T. urticae inhabits the undersides of leaves. Therefore, T. urticae possibly uses leaves as a filter to avoid the deleterious effects of UV-B. In diapausing females, low mortality was observed even at high doses of UV radiation, but more than half escaped even at low doses. The orange body color of diapausing females results from accumulation of carotenoids, a scavenger for UV-induced reactive oxygen species; this may explain the low mortality of diapausing females. Diapausing females may overcome the deleterious effects of UV-B during winter in the absence of leaves by emigrating to UV-free environments and by accumulating carotenoids.

Suppression of water loss during adult diapause in the northern house mosquito,Culex pipiens

One of the major challenges of overwintering in the mosquito, Culex pipiens, is prevention of dehydration. In this study, we compare the water balance requirements of nondiapausing and diapausing adult females of C. pipiens. Although their percentage water content is lower, diapausing females contain both higher initial and dry masses than nondiapausing individuals. Both nondiapausing and diapausing females tolerate a loss of up to 40% of their water mass before dying, but diapausing female C. pipiens reach this point after a longer period due to their lower rate of water loss. Males, which do not overwinter in diapause, showed no differences in their water balance characteristics when reared under diapausing or nondiapausing conditions. Likewise, no changes were noted in the water balance of pupae, indicating that diapause-related changes do not occur prior to adult eclosion. This mosquito does not replenish internal water stores by generating metabolic water or by absorbing vapor from the atmosphere, but instead relies on drinking liquid water (or blood feeding in the case of nondiapausing females). The critical transition temperature, a point where water loss increases rapidly with temperature, was the highest for females, then males, then pupae, but was not influenced by the diapause program. Females in diapause did not utilize common polyols (glycerol, trehalose and sorbitol) to retain water, but instead the presence of twice the amount of cuticular hydrocarbons in diapausing compared with nondiapausing females suggests that the deposition of hydrocarbons contribute to the reduced rates of water loss. The laboratory results were also verified in field-collected specimens: mosquitoes in the late fall and winter had a lower percentage water content and water loss rate, higher initial mass, dry mass and more cuticular hydrocarbons than individuals collected during the summer. Thus, the major features of diapause that contribute to the suppression of water loss are the large size of diapausing females (reduction of surface area to volume ratio lowers cuticular water loss), their low metabolic rate and the deposition of extra cuticular hydrocarbons.

Enhanced Cold and Desiccation Tolerance in Diapausing Adults of <I>Culex pipiens</I>, and a Role for <I>Hsp70</I> in Response to Cold Shock but Not as a Component of the Diapause Program

Culex pipiens L. reared under diapause-inducing conditions (short daylength; 18 degrees C) were more cold tolerant and desiccation resistant than their nondiapausing counterparts (long daylength; 18 degrees C). Upon cold exposure (-5 degrees C), diapausing mosquitoes reared at 18 degrees C survived nearly twice as long as nondiapausing mosquitoes reared at 18 degrees C and 10 times longer than nondiapausing mosquitoes reared at 25 degrees 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 degrees 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.

Photoperiodic induction and termination of summer diapause in adult Epilachna admirabilis (Coleoptera: Coccinellidae) from a warm temperate region

In warm temperate and subtropical regions an adult summer diapause regulates the timing of oviposition. Epilachna admirabilis is a univoltine phytophagous lady beetle ranging from south to north Japan, Taiwan, China and Myanmar. In Japan the species hibernates in the full-grown larval stage. In cool temperate Sapporo (43°04´N) the adults never enter summer diapause but pass a second winter in diapause. This study revealed that in Kyoto (35°01´N), the adults had a summer diapause induced by an intermediate photoperiodic response; they had a critical photoperiod of approximately 14.5 h light per day. All females entered reproductive diapause under a long photoperiod of 16L (light) : 8D (dark) and 6.7% of them did so under shorter photoperiods of 13L : 11D and 12L : 12D. However, diapause incidence was 40% at 8L : 16D, suggesting the existence of a second critical photope- riod at a photophase slightly less than 8 h. At photophases of 12-15 h, non-diapausing females laid eggs on average between 27.1 to 39.0 days after emergence. Photoperiod reversibly regulated the induction, maintenance and termination of the adult diapause. Tem- peratures of 20-30°C did not affect the incidence and termination of diapause at 16L : 8D. Adult winter diapause at the higher lati- tude Sapporo might have originated from adult summer diapause at middle latitude regions such as Kyoto. Selection at increasing latitudes would have forced northerly populations of the species to lose the adult summer diapause in the range of natural day- lengths. Subsequent selection pressure should have favored adults that avoided futile oviposition in late summer or autumn, survived cold winters in diapause and commenced an additional oviposition in the second summer. Thus, a latitudinal difference in photoperi- odic responses might have developed.

Life history of Nippoleucon hinumensis (Crustacea: Cumacea: Leuconidae) in Seto Inland Sea of Japan. II. Non-diapausing subpopulation

The great majority of Nippoleucon hinumensis (Gam6) in the Seto Inland Sea of Japan enters a long-term summer diapause from May to November. We identified a minor subpopulation of N. hinumensis (0.7% of the total population) that became adults in a short period without diapause. Small-sized ovigerous females (15% shorter than the ordinary-sized ovigerous females) appeared from January to early February, a month earlier than the appearance of the ordinary-sized ovigerous females that produce diapausing offspring. These precociously ovigerous females released offspring from early February to early March. The offspring developed from 1st manca larvae to 2nd manca larvae, 1st juvenile stage and 2nd juveniles stage in that order, and in mid-April they reached the adult stage (5th instar) without diapause. In the non-diapausing subpopulation, sexual dimorphism was apparent from the 1st manca instar and the sex ratio (males:females) was 2 to 4:1. The molts, occurring at intervals of ca. 10 d, were not highly synchronous among individuals. The non-diapausing females mostly disappeared in April without producing offspring, but many non-diapausing males survived until the ordinary-sized ovigerous females started incubation of the second brood in late April. Possibly, the non-diapausing subpopulation is maintained through mating of non-diapausing males with ordinary-sized ovigerous females. The non-diapausing mode of life in N. hinumensis may represent the ancestral life mode before this species acquired a summer diapause mechanism.

Preliminary observations of sperm storage in Adalia bipunctata (Coleoptera: Coccinellidae): sperm size and number

Sperm storage in Adalia bipunctata (L.) (Coleoptera, Coccinellidae) was examined and compared between diapausing and non-diapausing females. We observed that non-diapausing females store on average 13,688 (±722, S.E.) sperm in their spermatheca and that every female examined had sperm in storage (n=16). In contrast, most diapausing females had empty spermatheca and only two had sperm in storage (n=15). Mean sperm number in diapausing females was 250 (±112, S.E.). The mean length of the spermatozoa stored by non-diapausing females was 2,068.5 μm (±9.8, S.E.), ranging from 1,530.8 to 3,171.7 μm. Sperm length in diapausing females was only measurable in one female and was of 2,102.9 μm (±22.4, S.E.).

Effects of temperature on the establishment potential of the predatory mite Amblyseius californicus McGregor (Acari: Phytoseiidae) in the UK

Amblyseius californicus was introduced into the UK in the early 1990s as a biocontrol agent against glasshouse red spider mite Tetranychus urticae. This study investigated the effects of temperature on the establishment potential of A. californicus in the UK in the light of recent reports of their successful overwintering outside of glasshouse environments. The developmental thresholds were 9.9 and 8.6 °C respectively using simple and weighted linear regression. Using the day-degree requirement per generation calculated by weighted regression (143 day-degrees) in combination with climate data, it was estimated that up to seven generations would be possible annually outdoors in the UK. Non-diapausing adult females froze at −22 °C, with 100% mortality after reaching their freezing temperature. Up to 90% of mites died before freezing after short exposures to low temperatures. Significant acclimation responses occurred; 90% of acclimated individuals survived 26 days exposure at 0 °C and 11 days at −5 °C (acclimated mites were reared at 19 °C, 6L:18D followed by 1 week at 10 °C, 12L:12D). Non-diapausing adult females survived over 3 months outdoors in winter under sheltered conditions and oviposition was observed. The experimental protocol used in this study is discussed as a pre-release screen for the establishment potential of other Amblyseius species, and similar non-native biocontrol agents.

Regulation of corpora allata in females of Pyrrhocoris apterus (Heteroptera) (a mini-review).

Mechanisms for the transduction of photoperiodic and food signals to the corpus allatum (CA) are described. The focus of this paper is on the control of the CA by the brain in adult females of the firebug, Pyrrhocoris apterus. By using surgical interventions to the neuroendocrine complex of brain-subesophageal ganglion-corpora cardiaca-CA (BR-SG-CC-CA) in vivo and in vitro we were able to identify two regulatory pathways. (1) Slow regulation of the CA activity (stimulation or inhibition) needs a relatively long period of time to be accomplished (several d) in vivo and is associated with changes of the gland cell volume and ultrastructure. The stimulated or inhibited activity of the CA is maintained during short-term incubation of the isolated CA in vitro. (2) Fast inhibition of the CA activity is reversible during short-term incubation in vitro; the CA can be switched from lower to higher activity and vice versa, depending on the presence or absence of the BR-SG in the medium. Both slow and fast regulatory factors originate in the pars intercerebralis of the brain and in intact neuroendocrine complex they reach the CA via nerves. A slow inhibitor, induced by short d, causes reproductive diapause. A fast inhibitor prevents ovarian maturation in starved nondiapausing females. A slow stimulator, induced by feeding under long d, overcomes the fast inhibition of the CA, thereby stimulating vitellogenesis. Food signals are transmitted to the brain via humoral pathways.

Juvenile hormone biosynthesis in diapausing and nondiapausing Culex pipiens (Diptera: Culicidae).

The synthesis of juvenile hormone in vitro by diapausing and nondiapausing Culex pipiens L. was measured by radiochemical assay. Paired corpora allata from diapausing females synthesized < 18 fmols of juvenile hormone per hour during the first 3 wk after emergence. In contrast, juvenile hormone synthesis in nondiapausing females increased rapidly reaching a peak of 87.3 +/- 21 (mean +/- SE) fmol/h 3 d after emergence. By 3 wk, juvenile hormone synthesis had decreased in both groups of females, but corpora allata from nondiapausing mosquitoes still were 3 times more active than those from diapausing mosquitoes. By 16 wk after diapause induction, females maintained at 8:16 (L:D) h and 15 degrees C produced levels of juvenile hormone similar to 3-wk-old nondiapausing females. When females were held in diapause conditions for up to 22 wk, follicles gradually grew longer and by 15 wk were significantly longer than in the previous 14 wk. Blood feeding also increased in older females, indicating that over time, juvenile hormone synthesis gradually stimulated blood-feeding behavior. When 21-d-old diapausing mosquitoes were moved to a long-day photoperiod of 16:8 (L:D) h and 26 degrees C, juvenile hormone synthesis increased rapidly and peaked 5 d later while the ovarian follicles grew to the resting stage. Allatectomy of young diapausing females prevented follicle growth and blood feeding when diapause was terminated prematurely, demonstrating that the physiological events associated with diapause termination were associated with juvenile hormone biosynthesis.

Hexameric haemolymph protein related to adult diapause in the red firebug, Pyrrhocoris apterus (L.) (Heteroptera)

A hexameric storage protein was electrophoretically isolated from the haemolymph of diapausing adults of the red firebug, Pyrrhocoris apterus. This protein ( M rmr ~ 480,000, glycoprotein) was found to be a monohexamer. During the penultimate and last larval instar (both in diapausing and non-diapausing bugs), its concentration increased from about 1 mg/ml up to 8 and 13 mg/ml, respectively, and declined to almost zero value during ecdysis. In diapausing adults, the titre of the protein rose steeply from day 3 and reached a level of about 21 mg/ml in females and 24 mg/ml in males. In non-diapausing adult females there was an initial rise in the protein titre during the first 6 days but subsequently it stabilized to a level of about 3 mg/ml. In males the hexameric protein content reached only 1 mg/ml after imaginal ecdysis; an increase on day 21 and 30 was observed. In 14-day-old diapausing and non-diapausing bugs the hexameric protein constitutes about 15 and 4% of all haemolymph proteins, respectively. An amino acid analysis showed that the hexameric protein is neither an arylphorin nor a methionine-rich protein.

A reappraisal of the hormonal regulation of larval fat body histolysis in female Drosophila melanogaster.

The histolysis of larval fat body cells in adult female Drosophila melanogaster was examined in wild type and mutant animals. The fat body cells of wild type (Canton-S), apterous56f homozygotes, apterous78jts homozygotes and heterozygotes, apterous4/+, ecdysoneless1 homozygotes and heterozygotes all underwent histolysis normally during the 72 h following adult eclosion. Only in the case of ap4/ap4 adults did the cells fail to histolyze normally. The fat body cells of both diapausing and non-diapausing wild type females underwent histolysis at the same rate. Attempts to demonstrate histolysis in vitro were unsuccessful, even in the presence of juvenile hormones (JHs), larval ring glands, or adult ovaries. In all strains other than the ap4 homozygotes, a significant proportion of larval fat body cells were dead at any time while the ap4/ap4 animals, almost all cells remained viable. It is postulated that fat body cell lysis following eclosion is not a JH-mediated event, but is elicited by an as yet unidentified factor(s), possibly originating in the ovary.

Patterns of Sugar Feeding in Diapausing and Nondiapausing Culex pipiens (Diptera: Culicidae) Females

In the laboratory, diapausing Culex pipiens L. females fed on a continuously available sugar source more frequently and longer after adult emergence than did non-diapausing females. They also showed an extended nocturnal pattern of sugar feeding compared with that of non-diapausing females. Differences between diapausing and non-diapausing females with respect to sugar feeding and host-seeking behavior also were related to responses to volatiles from fruit and vertebrate hosts. In this Colorado strain of Cx. pipiens, increased sugar feeding by diapausing females during the first few weeks of adult life may be an adaptation that enhances overwintering survival.