Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Mosquito Vectors.

Microbial interactions are an underappreciated force in shaping insect microbiome communities. Although pairwise patterns of symbiont interactions have been identified, we have a poor understanding regarding the scale and the nature of co-occurrence and co-exclusion interactions within the microbiome. To characterize these patterns in mosquitoes, we sequenced the bacterial microbiome of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus caught in the field or reared in the laboratory and used these data to generate interaction networks. For collections, we used traps that attracted host-seeking or ovipositing female mosquitoes to determine how physiological state affects the microbiome under field conditions. Interestingly, we saw few differences in species richness or microbiome community structure in mosquitoes caught in either trap. Co-occurrence and co-exclusion analysis identified 116 pairwise interactions substantially increasing the list of bacterial interactions observed in mosquitoes. Networks generated from the microbiome of Ae. aegypti often included highly interconnected hub bacteria. There were several instances where co-occurring bacteria co-excluded a third taxa, suggesting the existence of tripartite relationships. Several associations were observed in multiple species or in field and laboratory-reared mosquitoes indicating these associations are robust and not influenced by environmental or host factors. To demonstrate that microbial interactions can influence colonization of the host, we administered symbionts to Ae. aegypti larvae that either possessed or lacked their resident microbiota. We found that the presence of resident microbiota can inhibit colonization of particular bacterial taxa. Our results highlight that microbial interactions in mosquitoes are complex and influence microbiome composition.

Efficacy of Deltagard®, Fyfanon®, and Evoluer™ 31-66 Against Culex quinquefasciatus In Harris County, Texas.

The comparative efficacy of ultra-low volume applications of deltamethrin, permethrin, and malathion against specific adult populations of Culex quinquefasciatus , a primary regional vector of Saint Louis encephalitis and West Nile virus, using field cage tests is described. At labeled application rates, the estimated mass median diameters for Fyfanon®, Evoluer™, and DeltaGard® were 18.32, 19.24, and 17.44 µm, respectively. No significant differences (P ≥ 0.01) in mean percent control were observed across all 3 wild populations and a susceptible Sebring strain tested with Fyfanon, DeltaGard, and Evoluer, providing 98.25%, 98.20%, and 97.24% mean percent control. Mean percent control for Sebring and 3 wild populations was 99.38%, 97.79%, 97.26%, and 97.15%, respectively, which were not significantly different from one another (P ≥ 0.01). Across all tests, highly significant differences (P ≤ 0.01) in mean percent control were obtained at 30.48, 60.96, and 91.44 m distances downwind, with mean percent control at 91.44 m being significantly less than that observed at 30.48 m, but not at 60.96 m. Individually, tests for DeltaGard, Fyfanon, and Evoluer resulted in no significant differences (P ≥ 0.01) in mean percent control between all mosquito populations tested, ranging from 96.47 to 100. However, Evoluer obtained significantly less (P ≤ 0.01) mean percent control at 91.44 m compared to DeltaGard and Fyfanon. Data suggest that the wild Cx. quinquefasciatus specimens collected were as susceptible as the laboratory Sebring strain. The use of Sebring as positive controls downwind strongly supported determination of the efficacy of all adulticides and susceptibility of wild populations tested.

Efficacy of Two Larvasonic™ Units Against Culex Larvae and Effects on Common Aquatic Nontarget Organisms in Harris County, Texas.

The Larvasonic™ Field Arm Mobile Wetlands Unit and SD-Mini were tested for efficacy against Culex larvae, and effects on aquatic nontarget organisms (NTO). The Field Arm provided 84.61% to 100% control of caged Culex larvae out to 0.91-m distance in shallow ditches and 60.45% control of Culex larvae at 0.61-m without any effects to caged NTO. Slow ditch treatment achieved 77.35% control compared to fast treatment (20.42%), whereas 77.65% control was obtained along edges of a neglected swimming pool, compared to near the middle (23.97%). In bucket tests, the SD-Mini provided >97% control of Culex and 85.35% reduction of immature giant water bugs, which decreased slightly (83.45%) over the monitoring period, which was not significantly different from cannibalistic damselflies (62.80%), with reduction of both being significantly higher than other NTO tested. There was a small (0.37%) reduction of dragonflies (naiads), due to cannibalism. Both Larvasonic units could effectively augment conventional larvicide operations in smaller areas without causing resistance within mosquito populations or harming NTO when used properly.

Continued evolution of West Nile virus, Houston, Texas, USA, 2002-2012.

We investigated the genetics and evolution of West Nile virus (WNV) since initial detection in the United States in 1999 on the basis of continual surveillance studies in the Houston, Texas, USA, metropolitan area (Harris County) as a surrogate model for WNV evolution on a national scale. Full-length genomic sequencing of 14 novel 2010-2012 WNV isolates collected from resident birds in Harris County demonstrates emergence of 4 independent genetic groups distinct from historical strains circulating in the greater Houston region since 2002. Phylogenetic and geospatial analyses of the 2012 WNV isolates indicate closer genetic relationship with 2003-2006 Harris County isolates than more recent 2007-2011 isolates. Inferred monophyletic relationships of these groups with several 2006-2009 northeastern US isolates supports potential introduction of a novel WNV strain in Texas since 2010. These results emphasize the need to maintain WNV surveillance activities to better understand WNV transmission dynamics in the United States.

Impact of phlebotomine sand flies on United State military operations at Tallil Air Base, Iraq: 6. Evaluation of insecticides for the control of sand flies.

We conducted a series of field experiments in 2003 and 2004 to evaluate the efficacy of a variety of insecticides and insecticide application technologies for the control of phlebotomine sand flies at Tallil Airbase, Iraq. During the experiments, 53,263 sand flies were collected. The experiments evaluated the following: (1) routine sand fly control operations using a variety of residual and area-wide insecticides; (2) a combination of five different insecticide application methods in and around tents; (3) residual application of lambda-cyhalothrin and ultra-low volume application of pyrethrins in houses; (4) carbaryl and lambda-cyhalothrin applied as barrier sprays; (5) a deltamethrin-impregnated fence; (6) lambda-cyhalothrin applied as a residual spray in concrete manholes; (7) deltamethrin-treated flooring in tents; and 8) ultra-low volume-applied malathion. Although some of the experiments resulted in limited reductions in the number of sand flies collected in light traps, in no instance did we completely eliminate sand flies or reduce populations for a sustained period. The implications of these findings are discussed.

Evolution of new genotype of West Nile virus in North America.

Previous studies of North American isolates of West Nile virus (WNV) during 1999-2005 suggested that the virus had reached genetic homeostasis in North America. However, genomic sequencing of WNV isolates from Harris County, Texas, during 2002-2009 suggests that this is not the case. Three new genetic groups have been identified in Texas since 2005. Spread of the southwestern US genotype (SW/WN03) from the Arizona/Colorado/northern Mexico region to California, Illinois, New Mexico, New York, North Dakota, and the Texas Gulf Coast demonstrates continued evolution of WNV. Thus, WNV continues to evolve in North America, as demonstrated by selection of this new genotype. Continued surveillance of the virus is essential as it continues to evolve in the New World.

Thermal fog efficacy tests against mosquitoes in storm drains in Harris County, Texas 2008-09.

In 2008 and 2009, our current thermal fogging methods and materials were evaluated in underground systems against feral and laboratory strains of adult Culex quinquefasciatus. Culex quinquefasciatus adults collected from storm drain systems the night before treatment were tested concurrently with adults from a susceptible laboratory strain (Sebring) in 10 separate tests. During 2008, there were no significant differences in the low mean percent control obtained between any of the feral populations (29.39%) and susceptible (Sebring) populations (56.04%) tested, whereas in 2009, application of mineral oil alone yielded extremely low, but significantly different mean percent control at 0.99% for ferals and 0.01% for Sebrings. During 2009 mineral oil evaluations, 45,677 droplets were collected in storm drains at distances of 99.1, 50.6, 57.9, 67.7, and 109.7 m from the application site, with 99% of the droplets below 3 microm in diam; additionally, we found no significant differences between mean percent control of Sebring and feral mosquito populations using the higher (3.2x) 1:10 application rate of Pyrocide. However, mean percent control between the feral and susceptible strain (Sebring) during 2009 was lower than in 2008 at 16.55% for ferals and 24.43% in Sebrings. Results indicated that control methodologies and/or chemicals used were ineffective at controlling Cx. quinquefasciatus in storm drains using the chosen experimental design. Based upon this information, thermal fog operations were discontinued due to lack of effectiveness.

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad.

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.

Biological and phylogenetic characterization of pigeon paramyxovirus serotype 1 circulating in wild North American pigeons and doves.

As part of West Nile virus surveillance programs in Rhode Island and eastern Texas between 2000 and 2007, brain tissue was collected from 5,608 dead birds representing 21 avian orders found in public places or reported by homeowners. Fifteen Newcastle disease virus isolates were recovered only from birds of the order Columbiformes and were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene and more specifically as pigeon paramyxovirus serotype 1 (PPMV-1) by hemagglutinin inhibition with monoclonal antibodies. Based upon partial genomic sequencing and phylogenetic analysis, the newly isolated viruses represent a distinct sublineage within class II genotype VIb. All of the viruses (15/15) were classified as virulent based upon their fusion cleavage site motif ((112)RRKKRF(117)) and intracerebral pathogenicity indices of >0.7 (ranging from 0.98 to 1.35); however, these viruses escaped detection by the fusion gene-based real-time PCR test for virulence. Modifications introduced to the probe site of the fusion gene-based assay allowed rapid virulence detection within this distinct sublineage.

Associations between two mosquito populations and West Nile virus in Harris County, Texas, 2003-06.

Associations between Culex quinquefasciatus, Aedes albopictus and West Nile virus (WNV) activity, temperature, and rainfall in Harris County, Texas 2003-06 are discussed. Human cases were highly correlated to Cx. quinquefasciatus (r = 0.87) and Ae. albopictus (r = 0.78) pools, blue jays (r = 0.83), and Ae. albopictus collected (r = 0.71), but not Cx. quinquefasciatus collected (r = 0.45). Human cases were associated with temperature (r = 0.71), not rainfall (r = 0.29), whereas temperature correlated with Ae. albopictus and Cx. quinquefasciatus collections (r = 0.88 and 0.70, respectively) and Cx. quinqueftsciatus pools (r = 0.75), but not Ae. albopictus pools (r = 0.55). Both species (collections and pools) and blue jays were weakly correlated (r 5 0.41) with rainfall, but blue jays were better correlated with Cx. quinquefasciatus pools (r = 0.87), compared with Ae. albopictus pools (r = 0.67), Ae. albopictus collections (r = 0.69), and Cx. quinquefasciatus collections (r = 0.46). Peak minimum infection rate for Cx. quinquefasciatus (4.55), and Ae. albopictus (4.41) was in August with highest human cases (17.87), blue jays (55.58), and temperature (29.01 degrees C). Between both species, blood meal analysis indicated 68.18% of Cx. quinquefasciatus mammalian hosts were dog, while 22.72% were human, whereas Ae. albopictus had higher human (44.44%) but fewer dog hosts (22.22%). Ten bird species were identified as hosts for Cx. quinquefasciatus, with northern cardinal and blue jay representing 26.66% and 20.00%, respectively. No bird feeding activity was observed in Ae. albopictus. The earliest and latest human blood meal occurred in May (Ae. albopictus) and November (Cx. quinquefasciatus); 66.66% of human host identifications between both species occurred in October-November, after the seasonal human case peak. Based upon our data, WNV activity in both mosquito species warrants further investigation of their individual roles in WNV ecology within this region.

Description and use of the Harris County gravid trap for West Nile virus surveillance 2003-06.

The Harris County Gravid Trap (HCGT) used for West Nile virus (WNV) surveillance from 2003-06 is described. The HCGT collected 1,918,483 Culex quinquefasciatus females (over 36,627 periods), with 466,296.37 (8,452.74) yearly. Only 48,094 Aedes albopictus females (over 15,139 periods) were collected, with 10,277.79 (3,260.33) yearly. The highest mosquito production occurred in July for Cx. quinquefasciatus (63,851.25 +/- 0.13), and for Ae. albopictus (2,981.80 +/- 0.09). Integrating gravid traps within our program has increased our ability to detect WNV efficiently in both species.

Host feeding pattern of Culex quinquefasciatus (Diptera: Culicidae) and its role in transmission of West Nile virus in Harris County, Texas.

The vertebrate hosts of 672 blood-engorged Culex quinquefasciatus Say, collected in Harris County, Texas, during 2005, were identified by nucleotide sequencing PCR products of the cytochrome b gene. Analysis revealed that 39.1% had acquired blood from birds, 52.5% from mammals, and 8.3% were mixed avian and mammalian blood meals. Most frequent vertebrate hosts were dog (41.0%), mourning dove (18.3%), domestic cat (8.8%), white-winged dove (4.3%), house sparrow (3.2%), house finch (3.0%), gray catbird (3.0%), and American robin (2.5%). Results are interpreted in conjunction with concurrent avian and mosquito West Nile virus (WNV) surveillance activities in Harris County. We conclude that Cx. quinquefasciatus is an opportunistic feeder and principal mosquito vector of WNV in this metropolitan area; however, transmission by other mosquito species or by other modes of infection, such as ingestion, must account for the high WNV infection rates among local blue jays and American crows.

Genetic stasis of dominant West Nile virus genotype, Houston, Texas.

The accumulation and fixation of mutations in West Nile virus (WNV) led to the emergence of a dominant genotype throughout North America. Subsequent analysis of 44 isolates, including 19 new sequences, from Houston, Texas, suggests that WNV has reached relative genetic stasis at the local level in recent years.

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 3. Evaluation of surveillance devices for the collection of adult sand flies.

We evaluated the effectiveness of commercially available light traps and sticky traps baited with chemical light sticks for the collection of phlebotomine sand flies. Evaluations were conducted at Tallil Air Base, Iraq, in 2003. In an initial study, a Centers for Disease Control and Prevention (CDC)-style trap with UV bulb collected significantly more sand flies than did an up-draft CDC trap, a standard down-draft CDC trap (STD-CDC), or a sticky strap with a green chemical light stick. In a subsequent study, we found that the addition of chemical light sticks to sticky traps resulted in a significant increase in the number of sand flies collected compared with sticky traps without the light sticks. These data indicate that 1) the CDC light trap with an UV bulb is an effective alternative to the standard CDC light trap for collecting phlebotomine sand flies in Iraq, and 2) that the addition of a chemical light stick to a sticky trap can result in a field-expedient tool for the collection of sand flies.

Estimation of aerosol droplet sizes by using a modified DC-III portable droplet measurement system under laboratory and field conditions.

Modification of the DC-III portable droplet measurement system, permitting its use under field conditions, is described. Under laboratory conditions, the system effectively sampled water droplets from aerosols produced by a dry ice/water generator and high-pressure syringe. Seven droplet sizes, totaling 71,053 droplets within 22 tests (dry ice method), consisted of 1-, 2-, 6-, 11-, 18-, 25-, and 34-microm droplets with individual (rounded) percentages of 45.25, 37.22, 13.85, 3.17, 0.45, 0.02, and 0.005, respectively, for each size. Cumulatively, 1-microm droplets accounted for ca. 45.25% of the droplets sampled; combined with 2-microm (ca. 82.48% together), 6-microm (ca. 96.33% together), and 11-microm droplets, yielded ca. 99.51% of the droplets sampled. The syringe produced 12 droplet sizes, with 4,121 droplets sampled, consisting of 1, 2, 6, 11, 18, 25, 34, 45, 56, 69, 83, and 99 microm with individual percentages of 15.43, 21.91, 24.58, 17.30, 10.62, 4.65, 2.93, 1.33, 0.63, 0.33, 0.16, 0.07, respectively, for each size. The 6-microm droplets contributed the highest individual percentage, and cumulatively, these droplets combined with 1- and 2-microm droplets, yielding 61.93%, whereas 11- to 45-microm droplets contributed 36.83%, for a total of 98.76%. Droplets measuring 56-99 microm accounted for ca. 1.24% of droplets sampled. Hand-fogger oil aerosols produced 12 droplet sizes (1-38 microm) at test distances of 7.6 and 15.2 m, with 1,979 and 268 droplets sampled, respectively, during 10 tests at each distance. With analysis of variance of transformed individual percentages for each size at both distances, no significant differences were observed for 7.6 and 15.2 m. Cumulatively, 1-, 2-, 3-, and 5-microm droplets contributed 82.87 and 80.97%, whereas 8-, 11-, 14-, and 18-microm droplets added 14.55% to totals at both 7.6 and 15.2 m, respectively. Droplets measuring 22, 27, 32, and 38 microm contributed 2.57% and 4.47% to samples obtained at 7.6 and 15.2 m. The average mass median diameter (MMD) of mineral oil aerosols obtained at 7.6 and 15.2 m were 19.55 +/- 1.62 and 15.49 +/- 1.35, respectively. Positioned at 15 m (50 ft) within a field cage test plot during 12 separate tests, the MMD values obtained downrange were less than calibration MMD values in 5 tests (10-93% of calibration values), but 1.1 to 4.4 times greater in 7 remaining tests. With exception of 2 tests, susceptible Sebring strain Culex quinquefasciatus mortality at the 31-m (100-ft) stake, 15 m (50 ft) behind the DC-III, was consistently 100%. The average MMD values for malathion, permethrin, and resmethrin (at 0.003 and 0.007 rates) at this distance were 13.24 +/- 6.08, 7.48 +/- 5.69, 16.64 +/- 5.54, and 15.48 +/- 2.89, respectively.

A comparison of seven traps used for collection of Aedes albopictus and Aedes aegypti originating from a large tire repository in Harris County (Houston), Texas.

Among 7 traps tested, significantly higher (P < 0.01) mean numbers of Aedes albopictus (269) and Aedes aegypti (55) females were collected within the Mosquito Magnet Liberty trap compared with the remaining traps. The second highest mean captures for both species were obtained from omnidirectional Fay-Prince (77 Ae. albopictus) and Dragonfly (13 Ae. aegypti) traps, which were not significantly different (P > 0.01) from an experimental moving-target trap that produced mean captures of 40 Ae. albopictus and 6 Ae. aegypti (alpha = 0.01). In terms of Ae. albopictus capture, no significant differences (P > 0.01) existed between Dragonfly, CDC without light (CDC -), and CDC with light (CDC +) captures, which were significantly different (P < 0.01) from Mosquito Deleto. No statistical significance existed between moving-target, omnidirectional, CDC +, CDC -, and Mosquito Deleto traps in terms of Ae. aegypti capture (P > 0.01), individual trap positions, or number of Ae. albopictus and Ae. aegypti females collected throughout the 21-day test (P > 0.05). Mosquito Magnet Liberty collected 7,208 Ae. albopictus, 1467 Ae. aegypti, and 13 other species representing 5 genera, which comprised the largest total (9662) and percentage (62.5%) of mosquitoes collected by all traps combined. Omnidirectional and moving-target traps captured 1941 and 1050 Ae. albopictus, 138 and 220 Ae. aegypti, and 2171 (14.0%) and 1397 (9.0%) of the total mosquitoes captured by all traps, with 8 and 10 species representing 5 genera, respectively, included in these collections. The Dragonfly captured 476 Ae. albopictus, 376 Ae. aegypti, and 1008 total specimens (6.5%) representing 8 species and 4 genera in these collections. CDC + and CDC - traps collected nearly identical numbers of Ae. albopictus (431, 450) and Ae. aegypti (71, 71) with 537 (3.4%) and 551 (3.5%) total specimens, respectively. Eight species representing 5 genera were captured from CDC +, whereas CDC - captured 6 species representing 4 genera. Mosquito Deleto captured 118 mosquitoes, including 19 Ae. albopictus and 62 Ae. aegypti females (0.7%), with 6 species representing 4 genera. Battery-powered traps with contrasting color schemes and movement worked considerably better than stationary CDC miniatures without color or movement. Omnidirectional Fay-Prince and moving-target traps without octenol captured Ae. albopictus and Ae. aegypti females as frequently as some commercial traps. Additionally, costs incurred per mosquito trapped, future trap design, and important consumer-centered issues are briefly discussed.

Operational note effects of fipronil and lambda-cyhalothrin against larval Anopheles quadrimaculatus and nontarget aquatic mosquito predators in Arkansas small rice plots.

The effects of fipronil and lambda-cyhalothrin, applied at rates labeled for control of the rice water weevil, Lissorhoptrus oryzophilus, on 3 nontarget indigenous insect species in Arkansas rice are described. Three replicates of untreated control checks and fipronil- and lambda-cyhalothrin-treated plots containing 3 sentinel cages each were performed. Ten 4th-stage larvae of Anopheles quadrimaculatus, 10 adult Tropisternus lateralis, or 10 adult Notonecta indica were placed within individual cages in small rice plots treated with ICON 6.2 FS (fipronil) at 0.025 lb active ingredient (AI)/acre (0.028 kg/ha) or KARATEZ 2.08 CS (lambda-cyhalothrin) at 0.03 lb AI/acre (0.033 kg/ha) applied over vegetation and water with a single-boom sprayer. At 24 h after treatment in fipronil plots, significantly higher control of An. quadrimaculatus and T. lateralis (69 and 48% control, respectively) was achieved, compared to N. indica (18%). In lambda-cyhalothrin plots 24 h after treatment, 100% reductions of both T. lateralis and N. indica were highly significant (P < 0.05) from the lower level of control in An. quadrimaculatus (10%). At 48 h after treatment, no significant, differences existed between all species within fipronil plots, with An. quadrimaculatus, T. lateralis, and N. indica obtaining 41, 10, and 7% control, respectively. Significantly higher (P < 0.05) control was obtained in lambda-cyhalothrin plots 48 h after treatment, with 93 and 53% control of T. lateralis and N. indica, respectively, compared to 7% control of An. quadrimaculatus. A marked difference in susceptibility was found between selected nontarget organisms used in this study. When using lambda-cyhalothrin to control adult L. oryzophilus, populations of nontarget beneficial insects, such as T. lateralis and N. indica, could be adversely affected, whereas nontarget pestilent species, such as An. quadrimaculatus, could proliferate. Fipronil achieved higher percentages of control against An. quadrimaculatus, compared to lambda-cyhalothrin, and was less harmful to both nontarget predators.