Rift Valley Fever Virus Primes Immune Responses in Aedes aegypti Cells.

The ongoing global emergence of arthropod-borne (arbo) viruses has accelerated research into the interactions of these viruses with the immune systems of their vectors. Only limited information exists on how bunyaviruses, such as Rift Valley fever virus (RVFV), are sensed by mosquito immunity or escape detection. RVFV is a zoonotic phlebovirus (Bunyavirales; Phenuiviridae) of veterinary and human public health and economic importance. We have shown that the infection of mosquitoes with RVFV triggers the activation of RNA interference pathways, which moderately restrict viral replication. Here, we aimed to better understand the interactions between RVFV and other vector immune signaling pathways that might influence RVFV replication and transmission. For this, we used the immunocompetent Aedes aegypti Aag2 cell line as a model. We found that bacteria-induced immune responses restricted RVFV replication. However, virus infection alone did not alter the gene expression levels of immune effectors. Instead, it resulted in the marked enhancement of immune responses to subsequent bacterial stimulation. The gene expression levels of several mosquito immune pattern recognition receptors were altered by RVFV infection, which may contribute to this immune priming. Our findings imply that there is a complex interplay between RVFV and mosquito immunity that could be targeted in disease prevention strategies.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

Effect of cage size on Aedes albopictus wing length, survival and egg production.

BACKGROUND: Aedes albopictus is currently the most widespread invasive mosquito species in the world. It has paramount medical importance since females are efficient vectors of important viruses affecting humans. The development of alternative control strategies to complement control measures has become an imperative and involves the Sterile Insect Technique (SIT). Research to improve the productivity of mass-rearing, as well as the quality of mass-reared males is of essential importance for the success of SIT. METHODS: This study compared the influence of three differently sized cages for Ae. albopictus mass-rearing on wing length, adult survival and egg production during 20 generations of colonization. Plexiglas cages of 40x40x40 cm (C1), 100 × 20 × 100 cm (C2) and 100 × 65 × 100 cm (C3) were loaded with equal adult density, and sex ratio of 1:1. An open source image processing and analysis programme (ImageJ) was used for the wing measurement and egg counting. RESULTS: In all tested cages, we identified two periods separated by the generation showing the minimum value of each considered parameter (wing length, adult survival and egg production). The wing length and adult survival passed through the phases of initial decrease to about intermediate colonization time, and increased afterwards. Fecundity was steady during the first period and increased in the second one. Cage C1 demonstrated not only the best values for all parameters but also the smallest decrease in the initial phase. Recovering of the caged mosquitoes in the second half of the study was higher in cages C1 and C2, than in C3. CONCLUSIONS: C1 provided the least negative selection pressure on wing length, adult survival and egg production for reared Ae. albopictus. Anyhow, since maximising mosquito density by exploiting the minimum space is a priority in mosquito mass-rearing, C2 might be a better choice for better fitting the space of mass-rearing rooms.

Comparative study on the effectiveness of different mosquito traps in arbovirus surveillance with a focus on WNV detection.

The selection of the ideal trap for arbovirus surveillance is an issue of primary importance to increase the sensitivity of virus detection and the cost-effectiveness of the entomological surveillance. During the summer 2011, the effectiveness of five types of mosquito traps (CDC gravid trap, CO2(-)baited trap, BG-Sentinel™ and two experimental prototypes) to attract females potentially infected with West Nile virus were assessed. The study was carried out in three natural wetland sites located in the Emilia-Romagna Region (Northern Italy), using a Latin square scheme. Single night collections of adult females were performed and determination of species and physiological state (gravid, nulliparous or parous) was made upon return to the laboratory. The species most frequently collected in the gravid trap was Culex pipiens sl. L., being gravid females the large majority of the individuals. Species diversity was much higher in CO2(-)baited traps, which may therefore enable a more comprehensive description of the vector species composition and their role in arboviruses circulation. Our findings indicate that gravid traps can be a valid tool and should be integrated in the West Nile virus surveillance system in the Emilia-Romagna region, mainly based on collections made with CO2-baited traps.

Reduced survival and reproductive success generates selection pressure for the dengue mosquito Aedes aegypti to evolve resistance against infection by the microsporidian parasite Vavraia culicis.

The success and sustainability of control measures aimed at reducing the transmission of mosquito-borne diseases will depend on how they influence the fitness of mosquitoes in targeted populations. We investigated the effects of the microsporidian parasite Vavraia culicis on the survival, blood-feeding behaviour and reproductive success of female Aedes aegypti mosquitoes, the main vector of dengue. Infection reduced survival to adulthood and increased adult female mosquito age-dependent mortality relative to uninfected individuals; this additional mortality was closely correlated with the number of parasite spores they harboured when they died. In the first gonotrophic cycle, infected females were less likely to blood-feed, took smaller meals when they did so, and developed fewer eggs than uninfected females. Even though the conditions of this laboratory study favoured minimal developmental times, the costs of infection were already being experienced by the time females reached an age at which they could first reproduce. These results suggest there will be selection pressure for mosquitoes to evolve resistance against this pathogen if it is used as an agent in a control program to reduce the transmission of mosquito-borne human diseases.

Effect of diet composition on the development of the floodwater Mosquito Ochlerotatus (Ochletotatus) albifasciatus (Macquart) (Diptera: Culicidae) / Efecto de la composición de la dieta sobre el desarrollo del mosquito de inundación Ochlerotatus (Ochletotatus) albifasciatus (Macquart) (Diptera: Culicidae)

One important step for the colonization of Ochlerotatus albifasciatus (Macquart) is to determine the optimal conditions for larval rearing, which makes possible the development of experiments comprising larval rearing in the laboratory. In this research the effect of diet composition on the development of O.albifasciatus was studied. For this purpose, cohorts of 20 first instars were reared using five diets: 1:1 mix of yeast and TetraMin®, 1:1 mix of fine and coarse organic matter, grass cuts soaked in water for 1h or 24h before larvae incorporation, and 1:1:1 mix of grass cuts, fine organic matter and coarse organic matter. Survival was recorded for each cohort, while development time from first instar to pupa, and adult wing length were recorded for each individual. The effects of the diet on the observed traits were analyzed by one way ANOVA. Both survival and wing length were affected by diet, being significantly higher (79-100% and ~ 4.46 mm) on diets consisting of organic matter or organic matter plus grass than on those consisting of yeast plus TetraMin® or grass. Development time was not affected by the diet, although the shortest time (8.1 to 8.3 days) and lower variation between replicates were recorded when larvae were reared using organic matter or organic matter plus grass. Thus, it is concluded that the diets consisting of organic matter or a mix of it and grass are the most effective for O.albifasciatus rearing.

Un paso importante para la colonización de Ochlerotatus albifasciatus (Macquart) es conocer las condiciones óptimas para la cría de las larvas, lo cual además posibilita el desarrollo de experimentos en el laboratorio. En este trabajo se estudió el efecto de la dieta sobre el desarrollo de O. albifasciatus. Para ello se criaron cohortes de 20 larvas I utilizando cinco tipos de dietas: una mezcla 1:1 de levadura y TetraMin®, una mezcla 1:1 de materia orgánica fina y gruesa, gramíneas cortadas y sumergidas en agua 1h o 24h antes de incorporar las larvas, y una mezcla 1:1:1 de gramíneas cortadas, materia orgánica fina y materia orgánica gruesa. Para cada cohorte se registró la supervivencia, y para cada individuo, el tiempo de desarrollo desde larva I hasta pupa y la longitud del ala de los adultos. El efecto de la dieta sobre estas características se analizó mediante un ANOVA de una vía. Los resultados mostraron que la dieta afecta la supervivencia y la longitud del ala, obteniéndose los mayores valores de estas variables (79-100% y ~ 4,46 mm) con las dietas consistentes en materia orgánica o materia orgánica más gramíneas. El tiempo de desarrollo no estuvo afectado por la dieta, aunque el tiempo más corto (8,1-8,3 dias) y la menor variación entre réplicas se registraron al criar las larvas con materia orgánica o materia orgánica más gramíneas. Se concluye que las dietas consistentes en materia orgánica o una mezcla de ésta y gramíneas, son las más efectivas para la cría de O. albifasciatus.

Effect of diet composition on the development of the floodwater Mosquito Ochlerotatus (Ochletotatus) albifasciatus (Macquart) (Diptera: Culicidae).

One important step for the colonization of Ochlerotatus albifasciatus (Macquart) is to determine the optimal conditions for larval rearing, which makes possible the development of experiments comprising larval rearing in the laboratory. In this research the effect of diet composition on the development of O.albifasciatus was studied. For this purpose, cohorts of 20 first instars were reared using five diets: 1:1 mix of yeast and TetraMin(R), 1:1 mix of fine and coarse organic matter, grass cuts soaked in water for 1h or 24h before larvae incorporation, and 1:1:1 mix of grass cuts, fine organic matter and coarse organic matter. Survival was recorded for each cohort, while development time from first instar to pupa, and adult wing length were recorded for each individual. The effects of the diet on the observed traits were analyzed by one way ANOVA. Both survival and wing length were affected by diet, being significantly higher (79-100% and ~ 4.46 mm) on diets consisting of organic matter or organic matter plus grass than on those consisting of yeast plus TetraMin(R) or grass. Development time was not affected by the diet, although the shortest time (8.1 to 8.3 days) and lower variation between replicates were recorded when larvae were reared using organic matter or organic matter plus grass. Thus, it is concluded that the diets consisting of organic matter or a mix of it and grass are the most effective for O.albifasciatus rearing.

Variation in the hatching response of Ochlerotatus albifasciatus egg batches (Diptera: Culicidae) in temperate Argentina.

Egg hatching of winter-collected Ochlerotatus albifasciatus was studied for six months. Batches of eggs were divided into two groups, one of them was stored in the laboratory at 23 degrees C and 12:12 photoperiod, and the other in the field under dead leaves. Every month, from July to December, eggs from the two groups were flooded under both laboratory and field conditions. Unhatched eggs were returned to the original condition and flooded two more times separated by ten-day intervals. Results show that egg diapause is expressed in different intensities, not only on eggs exposed to different conditions but also in those exposed to the same condition, even when they were laid by the same female. Successive inundations yielded incomplete hatches of eggs, and favored the hatching response in the next flooding. Low environmental temperatures before and during the flooding depressed hatching response. This shows that eggs need a warm period before flooding as well as warm temperatures during flooding, to hatch. As drought period was longer hatching response increased, but this was also accompanied by warmer environmental conditions. The experiment performed in laboratory did not show that increment. Field studies showed that a layer of dead leaves protected eggs from extreme temperatures.

Variation in the hatching response of Ochlerotatus albifasciatus egg batches (Diptera: Culicidae) in temperate Argentina

Egg hatching of winter-collected Ochlerotatus albifasciatus was studied for six months. Batches of eggs were divided into two groups, one of them was stored in the laboratory at 23ºC and 12:12 photoperiod, and the other in the field under dead leaves. Every month, from July to December, eggs from the two groups were flooded under both laboratory and field conditions. Unhatched eggs were returned to the original condition and flooded two more times separated by ten-day intervals. Results show that egg diapause is expressed in different intensities, not only on eggs exposed to different conditions but also in those exposed to the same condition, even when they were laid by the same female. Successive inundations yielded incomplete hatches of eggs, and favored the hatching response in the next flooding. Low environmental temperatures before and during the flooding depressed hatching response. This shows that eggs need a warm period before flooding as well as warm temperatures during flooding, to hatch. As drought period was longer hatching response increased, but this was also accompanied by warmer environmental conditions. The experiment performed in laboratory did not show that increment. Field studies showed that a layer of dead leaves protected eggs from extreme temperatures.

Mortality in immatures of the floodwater mosquito Ochlerotatus albifasciatus (Diptera: Culicidae) and effects of parasitism by Strelkovimermis spiculatus (Nematoda: Mermithidae) in Buenos Aires Province, Argentina.

Life tables were constructed for six cohorts of immature stages of the floodwater mosquito Ochlerotatus albifasciatus (Macquart) in a park in Buenos Aires, highlighting the mortality attributable to the parasitic nematode, Strelkovimermis spiculatus Poinar & Camino. Two cohorts were selected to compare parasite incidence in all mosquito stages when low and high parasitism occurred. Development time of Oc. albifasciatus from first instar to adult was 7.7-10 days in the spring, 6 days in the summer, and 10.9-21.9 days in the fall. Survival was estimated as 0-1.4% in the spring, 2% in the summer and 0.2-4.4% in the fall. The highest "K" value (Killing power) occurred during a fall cohort when prevalence of the parasite was 86.9%, and the lowest in a spring cohort. Parasitism occurred during all seasons, but S. spiculatus persisted to adult only in the summer and fall, when adult mosquitoes developed from parasitized third and fourth instars larvae. The abundance of S. spiculatus differed between old and young larvae only when parasite prevalence was the highest. Although pupae and adults of Oc. albifasciatus were parasitized, no pupal mortality attributable to parasitism was recorded. The proportion of parasitized adults ranged from 14.2% and 5.7% in the two cohorts compared. Pupal wet weight and adult wing lengths did not differ between parasitized and unparasitized individuals.

Mortality in immatures of the floodwater mosquito Ochlerotatus albifasciatus (Diptera: Culicidae) and effects of parasitism by Strelkovimermis spiculatus (Nematoda: Mermithidae) in Buenos Aires Province, Argentina

Life tables were constructed for six cohorts of immature stages of the floodwater mosquito Ochlerotatus albifasciatus (Macquart) in a park in Buenos Aires, highlighting the mortality attributable to the parasitic nematode, Strelkovimermis spiculatus Poinar & Camino. Two cohorts were selected to compare parasite incidence in all mosquito stages when low and high parasitism occurred. Development time of Oc. albifasciatus from first instar to adult was 7.7-10 days in the spring, 6 days in the summer, and 10.9-21.9 days in the fall. Survival was estimated as 0-1.4 percent in the spring, 2 percent in the summer and 0.2-4.4 percent in the fall. The highest "K" value (Killing power) occurred during a fall cohort when prevalence of the parasite was 86.9 percent, and the lowest in a spring cohort. Parasitism occurred during all seasons, but S. spiculatus persisted to adult only in the summer and fall, when adult mosquitoes developed from parasitized third and fourth instars larvae. The abundance of S. spiculatus differed between old and young larvae only when parasite prevalence was the highest. Although pupae and adults of Oc. albifasciatus were parasitized, no pupal mortality attributable to parasitism was recorded. The proportion of parasitized adults ranged from 14.2 percent and 5.7 percent in the two cohorts compared. Pupal wet weight and adult wing lengths did not differ between parasitized and unparasitized individuals