Age and Body Size Influence Sperm Quantity in Male Aedes albopictus (Diptera: Culicidae) Mosquitoes.

Aedes albopictus (Skuse) (Diptera: Culicidae) is a vector of several arboviruses impacting human health, including dengue, chikungunya, and potentially Zika. Vector control strategies that deploy modified males into the field are in use or under development and require a solid understanding of male biology; unfortunately, there has been limited effort to understand male Ae. albopictus reproductive biology, including sperm production and capacity. We tested whether body size and age affect spermatogenesis in Ae. albopictus. In general, older and larger males produced more sperm than their younger or smaller counterparts. Large males continued spermatogenesis well after 10-d post-eclosion (dpe), augmenting their reserves by 39%. By contrast, small males stopped producing sperm at 10 dpe. These results contribute to a deeper understanding of Ae. albopictus reproductive physiology. We discuss the usefulness of these findings in the context of Ae. albopictus life history and their utility in optimizing male mosquito release strategies.

Differences in development, glycogen, and lipid content associated with cytochrome P450-mediated permethrin resistance in Culex pipiens quinquefasciatus (Diptera: Culicidae).

Insecticide resistance in populations of mosquitoes is an escalating problem that can directly affect disease prevalence. Determining the fitness associated with an insecticide resistance mechanism (allele) will provide for greater understanding of the evolution of resistance, and help inform effective vector management programs. Previously, a population cage experiment in which the alleles of two highly related strains of Culex pipiens quinquefasciatus (Say) SLAB (susceptible) and ISOP450 (permethrin resistant because of cytochrome P450-mediated detoxification) were placed in direct competition in the absence of insecticide revealed that the P450 resistance allele frequency decreased over time. In the current study, SLAB and ISOP450 development, mortality and energetic resources derived from glycogen, other sugars and lipids were measured to identify biological parameters that might explain the previously observed fitness cost. SLAB exhibited shorter egg-to-adult female development time and larger body size when reared in groups when compared with ISOP450. ISOP450 female adults provided 20% sugar water lived longer than 20% sugar water fed females of the SLAB strain. No significant differences in larval development time, larval mortality, pupal stage duration time, pupal mortality, longevity of male and female adults provided with distilled water and males provided sugar water were found between the strains. The caloric content from glycogen and lipids were significantly higher in SLAB relative to ISOP450 in adults. The slower female emergence time and smaller body size when reared in groups combined with lower energy reserves (glycogen and lipids) associated with the resistance allele (in ISOP450) are likely fitness costs associated with the resistance allele of P450-mediated detoxification.

Avian malaria parasites share congeneric mosquito vectors.

Assessing parasite specificity to vector is crucial to understanding the emergence of vector-borne diseases and the evolution of parasite diversity. Avian malaria parasites have a cosmopolitan distribution and broad avian host range, which together predict they are vector generalists, but little is known about parasite-vector associations in the wild. We tested this prediction by asking if 5 different mosquito species, known to feed on birds and abundant in the northeastern United States, were naturally infected in the field with identical avian Plasmodium spp. lineages. Mosquitoes were not pooled but rather analyzed individually, and, possibly as a result, lineage diversity was higher than reported in previous avian malaria vector studies. Plasmodium spp. lineages were rare in Aedes canadensis and absent in Aedes aurifer and Culiseta melanura. We sequenced a standard Plasmodium cytochrome b marker from Culex pipiens pipiens, Culex restuans, and Ae. canadensis. Most Plasmodium clades were shared by Cx. pipiens and Cx. restuans. In addition, 4 individual lineages were shared by both mosquito species, including the most common lineage. One Plasmodium clade, however, was only found in Cx. restuans. We therefore found limited support for our prediction that avian Plasmodium spp. vector breadth accompanies host breadth. The association of both Culex species with most Plasmodium clades, and the presence of a single parasite lineage in 3 mosquito species representing 2 genera, suggests that avian Plasmodium species are not tightly coevolved with vector species.

Flushing effect of rain on container-inhabiting mosquitoes Aedes aegypti and Culex pipiens (Diptera: Culicidae).

We investigated the role of heavy rain on container-inhabiting mosquito (Diptera: Culicidae) populations, and how different species may have adapted to such conditions. Rains were created with a rain simulator calibrated to natural rain intensities in the habitats of two important vector species: Aedes aegypti (L.) from northern Thailand and Culex pipiens L. from New York state, USA. Immature stages of Ae. aegypti were able to resist the flushing effect of rain better than Cx. pipiens. This difference was most dramatic during the pupal stage. Fourth instars of Ae. aegypti were not affected by flushing when exposed for longer rain intervals (30 versus 60 min) or at a colder water temperature (24 versus 16 degrees C). In contrast, significantly more Cx. pipiens larvae flushed out with longer rain exposure. Warmer water temperatures also increased the proportion of Cx. pipiens flushed out, but mostly at the longest exposure time. Container position (tilted at a 7 degrees angle or level) did not affect proportions of fourth instars flushed out for both species. More accurate models of vector-borne diseases can be developed by incorporating the described effects of rain on container-breeding mosquito populations. Such models may provide more realistic assessments of disease risk and ensure optimal use of limited financial resources of mosquito control agencies.

Influence of container size, location, and time of day on oviposition patterns of the dengue vector, Aedes aegypti, in Thailand.

We conducted a study to determine the effect of container size and location on oviposition site selection by Ae. aegypti in large outdoor field enclosures (10 x 10 x 4 m high). There was a strong positive relationship between increasing container diameter, container volume, and water surface area with egg numbers over both high (rainy, July) and low (cool-dry, January) dengue transmission seasons. Location of containers (indoors versus immediately outdoors and underneath houses) did not influence the number of eggs deposited for containers 5-32 cm in diameter in either season. No trends based on container color (black, brown, or grey) were observed. A slight trend with a greater numbers of eggs laid outdoors in the largest containers (42 cm diameter) during the dry season was observed. Three separate models were run using the mixed model procedure in SAS for each container attribute. Controlling for season, time, and date, the most important container attribute predicting total egg numbers was container volume (total capacity) explaining 88% of the variation, followed by water surface area (85%), and container diameter opening (83%). Oviposition peaked in the afternoon at 1600 hrs and 2000 hrs in the dry and rainy seasons, respectively. Few eggs were laid overnight (2000 hrs-0600 hrs). Our results indicate that physical attributes of oviposition sites, such as size, light-dark contrasts, and specular reflectance from water surfaces, play a significant role in oviposition site selection.

Field validation of Aedes aegypti (Diptera: Culicidae) age estimation by analysis of cuticular hydrocarbons.

In previous studies, we developed linear regression models to age-grade female Aedes aegypti L. reared and maintained under controlled laboratory conditions. The models were based on temporal differences between two cuticular hydrocarbons, pentacosane (C25H52) and nonacosane (C29H60), which were extracted from Ae. aegypti legs and analyzed by gas-liquid chromatography. These initial models predicted adult female age up to 165 DD (12-15 calendar d at 28 degrees C). The age of older mosquitoes, however, could not be accurately predicted. In this study, our original regression models were tested using age data obtained from mosquitoes maintained in a field laboratory and those that were marked, released, and recaptured in northwestern Thailand. Our field data led to the development of two new regression models: one for the cool-dry season (February-March) and one for the rainy season (July-August). Both models resulted in better estimates of age than the original model and thus improved our ability to predict the age of Ae. aegypti to 15 calendar d. Females older than 15 d can be identified as such, but their exact age cannot yet be estimated. The new models will be useful for epidemiological studies and evaluating the impact of Ae. aegypti control interventions for disease prevention.

Indirect evidence against delayed "skip-oviposition" behavior by Aedes aegypti (Diptera: Culicidae) in Thailand.

Mosquitoes that do not oviposit their entire batch of eggs in one location, but choose instead to oviposit a few eggs in several different sites exhibit skip oviposition. Although skip oviposition may ensure the greater distribution of progeny from an individual female and reduce sibling competition, it also uses greater maternal energy reserves, may increase the risk of adult female mortality and does not reduce other competition. To test the hypothesis that female Aedes aegypti (L.) do not oviposit all of their eggs at one time and place, we examined females collected with an aspirator resting inside houses in Thailand. Females (n = 384) were dissected and the stage and number of developing oocytes were enumerated. If skip oviposition is a common behavior among female Ae. aegypti, one would expect to find significantly fewer mature eggs (Christophers' stage V) in gravid females than developing oocytes in half-gravid females (Christophers' stage IIIa-IVb). No significant differences were found between ovaries containing immature or mature oocytes, even when the data were stratified by female body size and when immature oocytes that potentially could undergo resorption (Christophers' stages IIIa-IIIb) were excluded from the analysis. We found no evidence that skip oviposition over several days is a common behavior among domestic populations of Ae. aegypti at the end of the rainy season in this region of Thailand.

Analysis of survival of young and old Aedes aegypti (Diptera: Culicidac) from Puerto Rico and Thailand.

It generally is assumed that the daily probability of survival of wild adult mosquitoes is independent of age. To test this assumption we conducted mark-release-recapture studies in Puerto Rico and Thailand to determine if estimated daily survival rates between two different age cohorts of the dengue vector Aedes aegypti (L.) were the same. Survivorship was estimated with nonlinear regression analysis using bootstrapping to obtain estimates of errors. Initial recapture success of the younger cohort was greater than the older cohort at both locations. Our analysis revealed a significantly greater survival rate for the younger cohort of females in Puerto Rico, and no significant differences between age cohorts in Thailand. For comparison, a traditional approach for analyzing these type of data, linear regression of log-transformed captures over time (exponential model), was used to calculate the probability of daily survival based on slopes of linear regression lines for recaptured mosquitoes. With this method, the estimated daily survival rate of older females (13-23 d old) was significantly greater than survival of younger ones (3-13 d old) in Puerto Rico and Thailand. In addition, short-range movement of mosquitoes was observed in Puerto Rico; maximum dispersal distance detected was 79 m. Survival rates of adult Ae. aegypti may be age-dependent and nonlinear regression analysis is a sensitive approach for comparing patterns of mosquito survival based on mark, single release, multiple recapture data.

Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood?

Adult female Aedes aegypti (L.), the vector of dengue and yellow fever viruses, have an affinity for feeding on human blood and a tendency to forego feeding on sugar. This observation challenges two tenets of mosquito biology: (1) mosquitoes imbibe plant carbohydrates for synthesis of energy reserves and blood for reproduction and (2) egg production is reduced when mosquitoes feed on human blood compared with blood from other species. Sub-optimal amounts of the amino acid isoleucine in human blood (particularly free isoleucine in plasma) are thought to be responsible for lowered egg production when human blood is ingested. We tested the hypothesis that feeding on human blood is associated with a selective advantage for Ae. aegypti and is an underlying reason for this mosquito's intimate and epidemiologically important relationship with human beings. Our five experiments examined the effects of different isoleucine concentrations on accumulated energy reserves, frequency of host contact, survival, and egg production. When mosquitoes imbibed blood meals over a 7- to 10-d period and were not fed sugar, increased isoleucine concentration decreased energy reserves and did not increase egg production. Aedes aegypti took smaller but more frequent blood meals when feeding on a low-isoleucine human host daily compared with a high-isoleucine mouse host. Previous reports that isoleucine enhances egg production were confirmed only when females were fed sugar, an unusual behavior for most domestic Ae. aegypti populations. Females fed human blood and water had greater age-specific survival (l(x)), reproductive output (m(x)), and cumulative net replacement (R0) than cohorts fed human blood plus sugar or isoleucine-rich mouse blood with or without access to sugar. The unique isoleucine concentration of human blood is associated with Ae. aegypti's unusual propensity to feed preferentially and frequently on humans--a behavior that increases this mosquito's fitness, synthesis of energy reserves, and contact with human hosts, making it an especially effective disseminator of human pathogens.

Aedes aegypti (Diptera: Culicidae) movement influenced by availability of oviposition sites.

Marked Aedes aegypti (L.) (5-6 d old) were released inside 2 groups of 5 houses (100 females per house) in a residential community in Florida, PR, to compare behavior of gravid females at sites where oviposition containers were absent to sites where containers were abundant (i.e., 2 tires and 10 ovipots were added to each yard). Two sequential releases were made so that both groups of houses were evaluated with oviposition containers removed and added. Mosquitoes resting inside the 10 release houses plus 20 additional neighboring houses were collected with backpack aspirators for 4 consecutive days, beginning 2 d after release. Because 172 of the 185 recaptured females (93%) were collected in the same houses in which they had been released, dispersal patterns were not directly comparable. However, the recapture rate in houses with containers added (13%) was significantly higher (P < 0.0001) than in houses with containers removed (6%). No difference was observed in the mean number of potential oviposition containers among the nonrelease houses at the 2 sites (3.9 versus 3.8 aquatic containers per house in the prerelease survey). Our results are consistent with the hypothesis that availability of oviposition sites is inversely correlated with the potential for female Ae. aegypti to disperse. These results have important implications because campaigns to reduce Ae. aegypti larval sites during dengue epidemics could have the undesirable effect of inducing the dispersal of infected adult female mosquitoes.

Vaginal birth after cesarean in a hospital-based birth center staffed by certified nurse-midwives.

The efficacy and safety of a trial of labor after previous cesarean were evaluated in selected, low-risk women in a hospital-based birthing center staffed by certified nurse-midwives. A total of 303 low-risk women with one previous cesarean delivery underwent a trial of labor in the birthing center. A matched control, without a previous uterine incision, was selected for each study patient. Hospital charts of 298 matched pairs were available for analysis. Outcome measures included the requirement for intrapartum transfer to medical management, use of oxytocin, method of delivery, uterine scar separation, Apgar scores, birth weights, maternal febrile morbidity, and length of hospital stay. Intrapartum transfer to medical management was necessary in 26 (8.7%) study patients and 31 (10.4%) control subjects. The overall rate (98.3%) of vaginal birth after cesarean among study patients was not statistically different from the vaginal birth rate (99.3%) among control subjects. There were no differences in maternal or neonatal morbidity. The high percentage (84%) of study patients having had a previous uncomplicated vaginal birth after cesarean must be considered a potential limitation of the outcome data; however, the overall vaginal birth rate between study patients with and study patients without previous vaginal birth after cesarean was not statistically different. The latter group was more likely to require transfer to medical management and/or oxytocin augmentation. On the basis of these results, we concluded that for selected, low-risk patients, a trial of labor after one previous cesarean may be managed safely and effectively by certified nurse-midwives in a hospital setting.

Comparisons of sampling methods and seasonal abundance of Drosophila repleta in caged-layer poultry houses.

Four sampling methods were compared to determine their practicality and suitability for detecting population fluctuations of adult Drosophila repleta, a pest in caged-layer poultry houses. Five caged-layer poultry houses with gutter-flush manure removal systems in Franklin County, North Carolina, U.S.A., were sampled once every 2 weeks over 15 months, from June 1991 to August 1992. The flies were most abundant during the spring and early summer. Visual counts of adult flies resting on the feed troughs in front of cages in the aisles and counts of flies caught on a sticky ribbon lowered into the gutter used for manure removal by flushing were more satisfactory sampling methods than using counts of flies caught on sticky ribbons carried along the aisles or aspirator collections from the gutters. For the visual count method, the effects of location in the house, within each aisle, and feed trough height were determined; two counts on feed troughs in each of two inside rows of cages were sufficient to measure population changes. For routine monitoring of D. repleta populations, visual counts of flies resting on the feed troughs in the front area of two aisles and catching flies on sticky ribbons inserted into the rear gutter area are recommended.

The F pilus of Escherichia coli appears to support stable DNA transfer in the absence of wall-to-wall contact between cells.

Separation of HfrC-F- mating pairs of Escherichia coli by a filter 6 microns thick with straight-through pores 0.01 to 0.1 micron in diameter did not prevent DNA transfer. We conclude that the F pilus alone is capable of acting as a stable conduit for cell-to-cell DNA transfer.