Fate of the blood meal in force-fed, diapausing Culex pipiens (Diptera: Culicidae)

Diapausing Culex pipiens L. do not display host-seeking behavior and can be induced to take blood only by being placed in contact with or in proximity to a host for prolonged periods. Such "force-fed" females do not use the blood for lipogenesis, and only some of them use the blood to initiate vitellogenesis. Diapausing Cx. pipiens that are induced to feed eject an average of 4.2-4.6 microliters of blood during overnight feeding periods compared with an average of 0.1 microliters for nondiapausing controls. The reduced avidity of diapausing females for blood, even under optimum conditions, and the ejection by fed females of blood volumes in excess of volumes usually retained indicate that such females are not physiologically programmed for taking and retaining blood. Data for uric acid and hematin excretion and bloodmeal volumes retained by diapausing females are positively correlated with diapause termination and yolk deposition. The occurrence of gonotrophic dissociation need not be invoked to explain the failure of some diapausing females to initiate vitellogenesis following a blood meal. Instead, this is explained by retention of small quantities of blood followed by incomplete digestion and is the expected result of a dose-dependent phenomenon determined by threshold blood volumes. Our data support the concept that the overwintering strategy of Cx. pipiens is limited to gonotrophic concordance in which overwintering females in nature do not take blood or develop eggs until diapause is terminated.     

Resistance to deltamethrin in Culex pipiens pallens (Diptera: Culicidae) from Zhejiang, China.

Resistance to deltamethrin, a pyrethroid insecticide, was found in 7 Culex pipiens pallens L. populations collected in Zhejiang (southeast China). Larval populations exhibited resistance ratios of 5.9-24.73 at LC50 and of 95.8-294.5 at LC95. Mortality ratios between field-collected adults and susceptible laboratory adults exposed to the discriminating dosage (0.01%/20 min) were 44.7-87.3. Resistance levels varied among populations as follows: Wenzhou > Ningbo > Hangzhou > Zhoushan > Jiaxing > Jinhua > Taizhou. Heavy use of pyrethroid insecticides for controlling mosquitoes in some areas has increased pyrethroid resistance in their regional populations. After selecting larvae from each population with deltamethrin for 12-15 generations, the resistance ratio increased by 103.3-342.5 at LC50 and by 79.7-203.1 at LC95. These results indicated that a large-scale Cx. p. pallens control with pyrethroid insecticides would induce a further increase of pyrethroid resistance in Zhejiang.     

Reproductive activity and survival of Culex pipiens pallens and Culex quinquefasciatus (Diptera: Culicidae) in Japan at high temperature

The egg hatchability, insemination, and longevity of Japanese Culex pipiens pallens Coquillett and Japanese Culex quinquefasciatus Say were compared at 25 and 30 degrees C. Egg hatchability was high in Cx. p. pallens at 25 degrees C, but it was very low at 30 degrees C because almost no females were inseminated at this temperature. In Cx. quinquefasciatus, the egg hatchability and insemination rates were very high, even at 30 degrees C. The longevity of adult females and males was generally shorter in Cx. p. pallens than in Cx. quinquefasciatus at both temperatures. Because high temperatures may restrict the spread of Cx. p. pallens, we suggest that even if this species spreads to Okinawa, the possibility of it becoming established is very low.     

Attraction of Culex pipiens pipiens (Diptera: Culicidae) to flower volatiles.

Methylene chloride extracts of flowers from 4 plants (common milkweed, Asclepias syriaca L; ox-eye daisy, Leucanthemum vulgare Lamarck; Canada goldenrod, Solidago canadensis L.; and yarrow, Achillea millefolium L.) were tested in a dual-port olfactometer for attractiveness to female northern house mosquitoes, Culex pipiens pipiens L. Fifty milligrams of extract was used for each test, which was equivalent to the amount of extract from flowers of 0.58 common milkweed inflorescences, 1.75 ox-eye daisy flower heads, 0.33 Canada goldenrod plants, or 3.14 yarrow plants. Significantly more Cx. pipiens responded to the common milkweed flower extract than to a control. Ox-eye daisy, Canada goldenrod and yarrow flower extracts were not significantly attractive to this species. A headspace sample of the common milkweed extract was collected by solid phase microextraction and analyzed by gas chromatography-mass spectrometry. The most abundant compounds in the headspace sample were 2-phenylethanol and benzyl alcohol. Analysis of the common milkweed extract and standards of 2-phenylethanol and benzyl alcohol by gas chromatography-flame ionization detection confirmed that these compounds were present in the extract. Cx. pipiens did not respond to synthetic blends of these 2 compounds when tested in a dual-port olfactometer.     

Genetic differences between Culex pipiens f. molestus and Culex pipiens pipiens (Diptera: Culicidae) in New York

The definition and phylogenetic placement of the autogenous molestus form of Culex pipiens has puzzled entomologists for decades. We identified genetic differences between Cx. p. pipiens (L.) and Cx. pipiens f. molestus Forsk?l in the SH60 fragment described previously. Single-strand conformation polymorphism analysis, cloning, and sequencing of this fragment demonstrated high polymorphism within and among individual Cx. p. pipiens, with common SH60 variants shared among individuals from distant locations. In contrast, Cx. pipiens f. molestus from New York City each contained a single SH60 variant, which was not identified in any other Cx. p. pipiens specimens analyzed. Supporting microsatellite analysis demonstrated significant but reduced gene flow between Cx. p. pipiens and Cx. pipiens f. molestus in New York relative to Cx. p. pipiens populations in New York and California. Results are discussed in the context of two contrasting hypotheses regarding the origin of Cx. pipiens f. molestus populations.     

Overproduced esterases in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Vietnam

The electrophoretic polymorphism of loci encoding for 10 enzymes was studied in Culex p. quinquefasciatus Say from six localities of Vietnam. The analysis of 11 "neutral genes" showed that differentiation among samples was low, but significant (Fst = 0.06), and significantly related to geographic distance between sample sites. These results are similar to those observed in other countries (Europe and west Africa). A single type of overproduced esterases (A2-B2) was observed, and its frequency was high (60-100%) in all samples. This situation is in sharp contrast with that observed in other countries of South East Asia (China, South Korea and Japan), where two or more types of overproduced esterases have been reported. A map summarizing the geographic distribution of Asian Cr. p. quinquefasciatus with overproduced esterases is provided.     

Enhanced toxicity of binary mixtures of larvicidal constituents from Asarum heterotropoides root to Culex pipiens pallens (Diptera: Culicidae)

The toxicity of pellitorine alone or in combination with (-)-asarinin, alpha-asarone, methyleugenol, or pentadecane (1:1, 1:2, 1:3, 2:1, and 3:1 ratios) to third instars from an insecticide-susceptible KS-CP strain and -resistant DJ-CP colony of Culex pipiens pallens Coquillett was evaluated using a direct-contact mortality bioassay. The binary mixture of pellitorine and (-)-asarinin (3:1 ratio) was significantly more toxic against KS-CP larvae (0.95 mg/liter) and DJ-CP larvae (1.07 mg/liter) than either pellitorine (2.08 mg/liter for KS-CP and 2.33 mg/liter for DJ-CP) or (-)-asarinin (11.45 and 12.61 mg/liter) alone. The toxicity of the other binary mixtures (1:1, 1:2, 1:3, and 2:1 ratios) and pellitorine did not differ significantly from each other. Based on the co-toxicity coefficient (CC) and synergistic factor (SF), the three binary mixtures (1:3, 2:1, and 3:1) operated synergistically (CC, 250-390 and SF, 1.4-2.2 for KS-CP; CC, 257-279 and SF, 1.1-2.1 for DJ-CP). The binary mixtures of pellitorine and (-)-asarinin merit further study as potential larvicides for the control of insecticide-resistant mosquito populations.     

Fumigant activity of eleven essential oil compounds and their selected binary mixtures against Culex pipiens pallens (Diptera: Culicidae)

To seek natural products for the development of environment friendly mosquito control agents, fumigant activity of eleven essential oil compounds and the joint action of the active compounds were evaluated against Culex pipiens pallens adults. Fumigant bioassay demonstrated that carvacrol exhibited the highest fumigant activity followed by thymol and l-perillaldehyde, with LC₅₀ values of 0.26, 0.28, and 0.34 mg/L air, respectively. Among the binary mixtures of four compounds with preferable performance, only the binary mixture of carvacrol and thymol (1:1, w/w) displayed a synergistic effect with the co-toxicity coefficient (CTC) value of 174.1 and LC₅₀ value of 0.16 mg/L air. Furthermore, the actual efficacy of the binary mixture at 300 mg/mat (KT₅₀?=?7.9, 15.8, and 22.0 min after 0, 2, and 4 h of preliminary heating, respectively) was comparable with that of d-allethrin at 30 mg/mat (KT₅₀?=?8.7, 17.9, and 21.2 min after 0, 2, and 4 h of preliminary heating, respectively) tested in vaporizing mats by the glass chamber method (70?×?70?×?70 cm). These results revealed that carvacrol, thymol, and their binary mixture have potential for the development of natural fumigants for adult mosquito control.     

Genetic similarity among Egyptian populations of Culex pipiens (Diptera: Culicidae)

Geographic variation among Culex pipiens L. populations was studied along a north-south transect across Egypt. Horizontal starch gel electrophoresis of 10 enzyme loci permitted estimates of adult polymorphism for 10 demes of Cx. pipiens. Levels of genetic variability, as indicated by a mean of 1.25 alleles per locus, a mean expected heterozygosity of 0.023, and a proportion of 0.25 of the loci being polymorphic, indicated limited genetic heterogeneity within and among demes. Nei's estimates of genetic divergence indicated considerable genetic exchange among demes (average genetic distance, 0.013). Thus, Cx. pipiens in Egypt was not differentiated into genetically isolated demes but, rather, seemed to consist of a single panmictic population.     

Seasonal abundance of Culex tarsalis and Culex pipiens complex mosquitoes (Diptera: Culicidae) in California

Large-scale patterns in the seasonal abundance profiles of the arboviral mosquito vectors Culex tarsalis Coquillett and the species of the Culex pipiens complex were described based on a decade of counts from 868 New Jersey light traps located throughout the urban and agricultural areas of California. Mean seasonal abundance profiles varied markedly among hydrologic regions. For all species, abundance increased earlier and declined later in drier, warmer southern regions, but variation could not be explained fully by latitude. The observed patterns may be driven by temperature, availability of larval habitats, and for the Cx. pipiens complex, the taxonomic composition of local populations.     

Tissue localization of overproduced esterases in the mosquito Culex pipiens (Diptera: Culicidae).

We have investigated the tissue distribution of overproduced esterases A (A1 and A2) and B (B1 and B2) in strains of Culex pipiens L. by immunocytochemistry. S-LAB mosquitoes, lacking overproduced esterases, were used as reference. Tissues showing a strong specific reaction (fluorescence) were observed with anti-esterase A1 antiserum in S54 (with A1) and BOUAKE (with A2) strains, and with anti-esterase B1 antiserum in TEM-R and EDIT (with B1) and BOUAKE (with B2) strains. Overproduction of esterases A and B was tissue-specific. The most constant pattern for the two types of esterases was their overproduction in the alimentary canal and Malpighian tubes, although fluorescence varied in intensity depending on strains and developmental stages. There was no difference in the tissue distribution of esterases Al and A2. In contrast, esterases B pattern was highly variable among strains. Differences between TEM-R and EDIT were explained by the different overall overproduction and number of copies of the amplified gene (10-fold higher in TEM-R). The most striking difference in esterase B1 and B2 tissue localization concerned the nervous system where neurons were intenisely fluorescent in TEM-R and EDIT (B1), but not in BOUAKE (B2). All esterase B positive tissues in TEM-R contained large quantities of esterase B1 mRNA (in situ hybridization), indicating that at least part of the protein revealed by immunochemistry was produced in the tissues where it was observed. Our results are discussed in terms of the protection that the different esterases can confer during exposition to organophosphorous insecticides.     

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.     

First record of a mosquito iridescent virus in Culex pipiens L. (Diptera: Culicidae)

The mosquito iridescent viruses (MIVs) are large icosahedral DNA viruses that replicate and assemble in the cytoplasm of the host. Paracrystalline arrangements of virions that accumulate in the cytoplasm produce an iridescent color that is symptomatic of acute infections. In August 2010, we found larvae of Culex pipiens with these symptoms in suburban ditches around the city of La Plata, Argentina. Electron microscope studies, DNA sequencing, and phylogenetic analysis of the major capsid protein confirmed this as the first record of an MIV in C. Pipiens.     

Culex pipiens (Diptera: Culicidae): a bridge vector of West Nile virus to humans

Host-feeding patterns of Culex pipiens L. collected in southwest suburban Chicago in 2005 were studied using polymerase chain reaction (PCR) and DNA sequencing techniques. Culex spp. mosquitoes, most identified to Cx. pipiens and the remainder to Cx. restuans by PCR, had fed on 18 avian species, most commonly American robin (Turdus migratorious), house sparrow (Passer domesticus), and mourning dove (Zenaida macroura). Additional blood meals were derived from four mammal species, primarily humans and raccoons (Procyon lotor). During a West Nile virus (WNV) epidemic in 2005, West Nile virus (WNV) RNA was detected in heads and thoraces of five Cx. pipiens (n = 335, 1.5%) using quantitative PCR. The hosts of these virus-infected, blood-fed mosquitoes included two American robins, one house sparrow, and one human. This is the first report of a WNV-infected Cx. pipiens mosquito collected during an epidemic of WNV that was found to have bitten a human. These results fulfill a criterion for incrimination of Cx. pipiens as a bridge vector.     

Naive and conditioned responses of Culex pipiens pipiens biotype molestus (Diptera: Culicidae) to flower odors

Flower odors are important signals for chemical communication between plants and flower visitors. Here, we studied the naive responses of Culex pipiens pipiens biotype molestus Forskal 1775 (Diptera: Culicidae) to typical flower odors and assessed the learning capacity of mosquitoes to floral volatiles. The odor compounds used in the bioassay, phenyl acetaldehyde, veratrole, and 2-methoxyphenol, are typically found in the floral odor of Silene otites (L.) Wibel, a plant that is pollinated by nectar-drinking mosquitoes and moths, and/or in other closely related Silene species. Wind tunnel bioassays with a mixture of these compounds revealed that attraction of mosquitoes to odors was positively correlated with time passed since the last feeding. In single component bioassays, mosquitoes showed strong innate responses to phenyl acetaldehyde and only moderate or weak responses to veratrole and 2-methoxyphenol. Furthermore, in comparison with naive mosquitoes, conditioned mosquitoes were significantly more attracted to the mixture and single volatiles. These results indicate that naive mosquitoes are effectively attracted by appropriate floral scent compounds and that learning can increase the attractiveness of these compounds.     

Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae)

Despite the detection of West Nile (WN) virus in overwintering Culex pipiens L. in New York in February 2000, the mechanism by which this virus persists throughout the winter to initiate infections in vertebrate hosts and vectors the following spring remains unknown. After a blood meal, parous mosquitoes generally do not survive until spring and gonotrophic dissociation occurs in only a small percentage of the population. To investigate vertical transmission as a means of viral survival during interepizootics, we intrathoracically inoculated Cx. pipiens and Aedes albopictus (Skuse) with WN virus and subsequently tested their F1 progeny for the presence of virus. Among the Cx. pipiens, we recovered virus from two of 1,417 adult progeny that had been reared at 18 degrees C for a minimal filial infection rate (MFIR) of approximately 1.4/1,000 and four of 1,873 adult progeny reared at 26 degrees C (MFIR = 2.1/1,000). The mean titer of the positive pools was 10(5.6) plaque-forming units (PFU)/ml (=10(5.9) PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was approximately 1.8/1,000 for Cx. pipiens. Although reports indicate that Ae. albopictus vertically transmit various viruses in the Japanese encephalitis virus complex, we did not detect WN virus in any of > 13,000 F1 progeny of WN virus-inoculated specimens. Female Cx. pipiens that are vertically infected during the late summer season and then survive the winter could serve as a source of WN virus to initiate an infection cycle the following spring.     

Interspecific competition between larval Culex restuans Theobald and Culex pipiens L. (Diptera: Culicidae) in Michigan

Many invasive species succeed in becoming established in new locations because of their competitive superiority to native species. This has been shown in several examples involving mosquitoes. In this study, we examined the interspecific competition between mosquito larvae of a well-established, non-native species, Culex pipiens, and those of its ecologically similar, native congener Culex restuans. Small but significant differences in survival, growth, and development rates were found in Cx. restuans as a response to varying proportions of Cx. pipiens, suggesting that Cx. restuans is a slightly superior competitor. However, the overall differences between the species were small, and they may be nearly ecological equivalents as larvae. Nevertheless, the observed seasonal pattern of feeding and oviposition activity suggests some phenological avoidance of competition, thus demanding further study of the interaction of these two species.