Abiotic factors affecting the prevalence of Wolbachia (Rickettsiaceae) in immature Aedes albopictus (Skuse) (Culicidae).

Wolbachia is a genus of gram-negative endosymbiotic bacterium of maternal transmission, located mainly in the gonads of arthropods, including mosquitoes such as Aedes albopictus. The current distribution of Ae. albopictus in Argentina is restricted to the subtropical northeastern region of the country. Here, we studied the seasonal prevalence of Wolbachia detected in Ae. albopictus larvae and the relationship between the abiotic factors of the larval microhabitat and the infection status, in Eldorado city, Misiones province, subtropical region. The prevalence of Wolbachia infection found was 76.89% (n = 312). From the total samples examined, 52.80% (n = 214) showed double infection with the wAlbA/wAlbB strains, 23.84% (n = 97) infection only with wAlbB, and 0.25% (n = 1) only with wAlbA. The prevalence of double infection did not present statistically significant differences between the sites studied. For single infection, the lowest prevalence value of the wAlbB strain (13.33%) was found in the natural park, whereas the highest was found in the family dwellings and cemeteries. Tire repair shops showed an intermediate value. The wAlbA single infection was identified once. Our results also showed an association between temperature and slightly turbid waters with exposure to the sun in the larval habitats and the probability of infection by Wolbachia.

Redescription of immature stages of Culicoides paraensis (Goeldi) (Diptera: Ceratopogonidae), vector of the Oropouche virus.

Oropouche fever is an emerging zoonotic disease caused by Oropouche virus (OROV). It has two distinct transmission cycles, with the anthropophilic biting midge Culicoides paraensis (Goeldi) (Diptera: Ceratopogonidae) being the primary vector in the urban cycle. Species identification of Culicoides typically has been carried out on the basis of morphological characters, but molecular tools applied to taxonomy can provide rapid and efficient methods to the identification of vector species. The aim of this work was to obtain the first DNA barcode for C. paraensis collected in Argentina and redescribe the larvae and pupae of this species. Nested PCR amplification was applied in this study to increase the DNA amplification, because the material was preserved in alcohol 70% for a long period of time. The immature stages of C. paraensis are fully described from material collected in Misiones province, Argentina. Both stages are compared with their most similar congeners. This COI sequence complements the identification based on morphological characters and the values of genetic distance between the analysed species show that this sequence is useful to discriminate between species of the Culicoides genus.

Presence of Ascogregarina culicis and Ascogregarina sp. in natural sympatric populations of Aedes aegypti and Ae. albopictus (Diptera: Culicidae) in Argentina.

Aedes aegypti is the main vector of the four arboviruses in America which have the greatest impact on human health. The introduction of Aedes albopictus in South America and Argentina acquires importance given the possibility that this species may be a new vector of arboviruses in this region. For this reason, the studies of the biology of their parasites, such as Ascogregarina spp., should be important for the knowledge of the invasive behavior of these vectors. We reported the finding of Ascogregarina culicis in Aedes aegypti and Ascogregarina sp. in Ae. albopictus populations in subtropical Argentina. The prevalence of parasitism by A. culicis in Ae. aegypti and Ascogregrarina sp. in Ae. albopictus was 34.81% (n = 464) and 37.23% (n = 70), respectively, differing between the seasons and habitats. The infection intensity caused by A. culicis and Ascogregarina sp. varied between 1 to 250 and 1 to 327 trophozoites respectively. Ascogregarina culicis was found throughout the all sampling period of Ae. aegypti (June 2016-April 2018). However the presence of Ascogregarina sp. in the midgut of Ae. albopictus was not recorded throughout the whole sampling period despite the presence of the host.

Detection of Flaviviral-Like DNA Sequences in Aedes aegypti (Diptera: Culicidae) Collected From Argentina.

Diseases caused by flaviviruses are a major public health burden across the world. In the past decades, South America has suffered dengue epidemics, the re-emergence of yellow fever and St. Louis encephalitis viruses, and the introduction of West Nile and Zika viruses. Many insect-specific flaviviruses (ISFs) that cannot replicate in vertebrate cells have recently been described. In this study, we analyzed field-collected mosquito samples from six different ecoregions of Argentina to detect flaviviruses. We did not find any RNA belonging to pathogenic flaviviruses or ISFs in adults or immature stages. However, flaviviral-like DNA similar to flavivirus NS5 region was detected in 83-100% of Aedes aegypti (L.). Despite being previously described as an ancient element in the Ae. aegypti genome, the flaviviral-like DNA sequence was not detected in all Ae. aegypti samples and sequences obtained did not form a monophyletic group, possibly reflecting the genetic diversity of mosquito populations in Argentina.

Biological characterization of Aedes albopictus (Diptera: Culicidae) in Argentina: implications for arbovirus transmission.

Aedes albopictus (Diptera: Culicidae) is an invasive mosquito, native to Asia, that has expanded its range worldwide. It is considered to be a public health threat as it is a competent vector of viruses of medical importance, including dengue, chikungunya, and Zika. Despite its medical importance there is almost no information on biologically important traits of Ae. albopictus in Argentina. We studied life cycle traits, demographic parameters and analyzed the competence of this mosquito as a virus vector. In addition, we determined the prevalence of Wolbachia strains in Ae. albopictus as a first approach to investigate the potential role of this bacteria in modulating vector competence for arboviruses. We observed low hatch rates of eggs, which led to a negative growth rate. We found that Ae. albopictus individuals were infected with Wolbachia in the F1 but while standard superinfection with wAlbA and wAlbB types was found in 66.7% of the females, 16.7% of the females and 62.5% of the males were single-infected with the wAlbB strain. Finally, despite high levels of infection and dissemination, particularly for chikungunya virus, Ae. albopictus from subtropical Argentina were found to be relatively inefficient vectors for transmission of both chikungunya and dengue viruses.

Vector competence of Argentine mosquitoes (Diptera: Culicidae) for West Nile virus (Flaviviridae: Flavivirus).

We examined the ability of Culex pipiens L. complex mosquitoes from Argentina to vector West Nile virus (WNV) to assess their role in the transmission of WNV in South America. Several egg rafts of Culex spp. were collected from different breeding sites in the suburbs of the city of La Plata, Argentina, and a subset of each progeny was scored with morphological and genetic species indicators. Surprisingly, we did not find Cx. pipiens form pipiens, but found evidence of genetic hybrids of Culex quinquefasciatus and Cx. pipiens f. molestus. We then used morphological traits to create two colonies predominantly composed of one of these two taxa, although some hybrids are likely to have been included in both. These colonies were used in vector competence studies using NY99 and WN02 genotype strains of WNV obtained in New York State. As controls, we also tested colonies of U.S. Cx. quinquefasciatus and Cx. pipiens f. molestus. Additional Culex larvae from three drainage ditches near the cities of La Plata and Berisso, Argentina, were identified by morphological and high-resolution molecular markers (microsatellites) as Cx. quinquefasciatus Say, Cx. pipiens form molestus, and hybrids. Results indicate that Argentinian Culex are competent but only moderately efficient vectors of WNV and are less susceptible to this virus than comparable U.S. mosquito strains. Studies of vertical transmission of NY99 virus by Cx. pipiens f. molestus hybrids from Argentina yielded a minimal filial infection rate of 1.19 from females feeding during their second and later bloodmeals.

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.

Effect of temperature, photoperiod, flooding, and drying on the hatching pattern of the eggs of Strelkovimermis spiculatus (Nematoda: Mermithidae).

We assessed the number of Strelkovimermis spiculatus preparasites obtained from a known initial number of nematode eggs and the effect of abiotic conditions (temperature, photoperiod, flooding-drying) on the number of emerged preparasites. Two egg groups were maintained: one continuously flooded, another with flooding-drying cycles (every 15, 30, 60 days). Each egg group was studied at 25°C and 14:10 (L:D) and 16°C and 12:12 (L:D). The flooded eggs contained a higher overall percentage of S. spiculatus preparasites compared to the wet-dry-cycle eggs. The conditions of continuous flooding at 16°C and 12:12 (L:D) produced the maximum percent of emerged J2s (30±15%). Preparasites were recorded by 7 (25°C) and 14 (16°C) days, suggesting this period as the minimum time for embryonic development. The preparasite-emergence time observed from the same flooded-egg batch (98 and 112 days at 25°C and 16°C, respectively) suggested a nonsynchronous hatching, possibly through nonuniform egg embryonation. The time of exposure to drought in the assays did not significantly affect the total average percentage of J2s obtained at 25°C and 14:10 (L:D), whereas at 16°C the number of emerged J2s diminished with a prolongation of the drying period. The oviposition period was also recorded only at 16°C and 12:12 (L:D): S. spiculatus eggs were detected at 12.6 days after postparasite emergence, and oviposition was complete at 51days under those conditions. We propose a flooding schedule to optimize the mass-rearing of S. spiculatus.

Native Argentinean cyclopoids (Crustacea: Copepoda) as predators of Aedes aegypti and Culex pipiens (Diptera: Culicidae) mosquitoes

Copepods from La Plata, Argentina were investigated to characterize the local community of larvivorous copepods inhabiting mosquito breeding sites and to identify new predator species of the mosquitoes which occur in artificial containers, Aedes aegypti and Culex pipiens. Diversity of larvivorous cyclopoids was highest in permanent pools. Predation by sex and age, selectivity on mosquito species, and daily predation rate during five days were studied for Acanthocyclops robustus, Diacyclops uruguayensis, Macrocyclops albidus and Mesocyclops longisetus. Female copepods presented the highest predatory capacity. No predatory preference for mosquito species was found. According to overall predation potential, copepods were ranked as follows: D. uruguayensis < A. robustus < M. albidus < M. longisetus. Copepod tolerance to desiccation and capacity to survive in water from artificial containers were also evaluated. D. uruguayensis and A. robustus survived under dry conditions, but D. uruguayensis showed lower survival in water from cemetery flower vases. M. albidus did not survive under dry conditions and did not tolerate water extracted from artificial containers. M. longisetus survival was not severely reduced after desiccation or breeding in water from flower vases. The Neotropical cyclopoids D. uruguayensis and A. robustus can be considered good candidates and merit further research as biological control agents for mosquitoes. Rev. Biol. Trop. 57 (4): 1059-1068. Epub 2009 December 01.

Se hizo una prospección de copépodos en La Plata, Argentina, con los objetivos de caracterizar la comunidad local de copépodos larvívoros en sitios de cría de mosquitos, e identificar nuevas especies depredadoras de los mosquitos de contenedores artificiales Aedes aegypti y Culex pipiens. La diversidad de ciclopoides larvívoros fue máxima en charcos permanentes. Se examinó la depredación por sexos y edad, la selectividad por especies de mosquito, y la tasa de depredación diaria durante cinco días en Acanthocyclops robustus, Diacyclops uruguayensis, Macrocyclops albidus y Mesocyclops longisetus. Los copépodos hembra presentaron la capacidad depredadora más alta. No se encontró preferencia por alguna especie de mosquito. De acuerdo al potencial de depredación en general, los copépodos se ordenan así: D. uruguayensis < A. robustus < M. albidus < M. longisetus. También se evaluó la tolerancia a la desecación del hábitat y la capacidad de resistir en agua de contenedores artificiales. D. uruguayensis y A. robustus sobrevivieron en condiciones de sequía, pero D. uruguayensis presentó menor supervivencia en agua de floreros de cementerio. M. albidus no sobrevivió condiciones de sequía y no toleró el agua extraída de contenedores artificiales. Los ciclopoides neotropicales D. uruguayensis and A. robustus son buenos candidatos y merecen investigación ulterior como agentes de control biológico de mosquitos.

Factors affecting horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host Mesocyclops annulatus.

Factors that directly impact horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host, Mesocyclops annulatus were examined in laboratory bioassays. Results were evaluated in relation to life history strategies that facilitate persistence of the parasite in natural populations of its definitive mosquito host, Ochlerotatus albifasciatus. A moderately high quantity of meiospores from mosquito larvae was required to infect adult female copepods; the IC50 was estimated at 3.6 x 10(4) meiospores/ml. Meiospore infectivity following storage at 25 degrees C was detected up to 30 days, while meiospores stored at 4 degrees C remained infectious to copepods for 17 months with virtually no decline in infectivity. Uninfected female M. annulatus are long-lived; no appreciable mortality was observed in field-collected individuals for 26 days, with a few individuals surviving up to 70 days. The pathological impact of A. albifasciati infection on M. annulatus resulted in a 30% reduction in survivorship after 7 days followed by gradual progressive mortality with no infected individuals surviving more than 40 days. This moderate level of pathogenicity allows for a steady continual release of spores into the environment where they may be ingested by mosquito larvae. Infected female copepods survived in sediment under conditions of desiccation up to 30 days, thus demonstrating their capacity to function as a link for maintaining A. albifasciati between mosquito generations following periods of desiccation. The susceptibility of late stage copepodid M. annulatus to meiospores of A. albifasciati and subsequent transstadial transmission of infection to adult females was established.

Native Argentinean cyclopoids (Crustacea: Copepoda) as predators of Aedes aegypti and Culex pipiens (Diptera: Culicidae) mosquitoes.

Copepods from La Plata, Argentina were investigated to characterize the local community of larvivorous copepods inhabiting mosquito breeding sites and to identify new predator species of the mosquitoes which occur in artificial containers, Aedes aegypti and Culex pipiens. Diversity of larvivorous cyclopoids was highest in permanent pools. Predation by sex and age, selectivity on mosquito species, and daily predation rate during five days were studied for Acanthocyclops robustus, Diacyclops uruguayensis, Macrocyclops albidus and Mesocyclops longisetus. Female copepods presented the highest predatory capacity. No predatory preference for mosquito species was found. According to overall predation potential, copepods were ranked as follows: D. uruguayensis < A. robustus < M. albidus < M. longisetus. Copepod tolerance to desiccation and capacity to survive in water from artificial containers were also evaluated. D. uruguayensis and A. robustus survived under dry conditions, but D. uruguayensis showed lower survival in water from cemetery flower vases. M. albidus did not survive under dry conditions and did not tolerate water extracted from artificial containers. M. longisetus survival was not severely reduced after desiccation or breeding in water from flower vases. The Neotropical cyclopoids D. uruguayensis and A. robustus can be considered good candidates and merit further research as biological control agents for mosquitoes.

Seasonality and abundance of the mosquito Isostomyia paranensis from phytotelmata in temperate Argentina.

Sampling was conducted for 1 year in a marsh near Buenos Aires from axils of Scirpus giganteus, a larval habitat of the poorly known sabethine mosquito, Isostomyia paranensis. Immatures of this species were recovered on every sampling date, averaging 3-4/plant in April and decreasing to 0-1/plant in October-December. The spatial distribution of Is. paranensis immatures was clumped, and larval age skewed toward 1st instars. The percentage of mosquito-positive S. giganteus was negatively correlated with accumulated rainfall 1 wk before collection. Microcrustacea were the only other invertebrates common in this phytotelmata, and no parasites or pathogens were detected in Is. paranensis. Fourth instars of this species attacked and killed one another in the laboratory, but only algae were recovered from dissected digestive tracts of field-collected larvae. Adult females of this species emerged from independent collections of pupae refused blood, but females captured at human bait readily consumed human blood. Mean (+/- SD) number of eggs developed by females collected at human bait and fed with blood (77.4 +/- 22.8) was not significantly different from the mean number of eggs developed by females collected as pupae and fed on sugar (72.0 +/-23.0).

[Population dynamics of the immature stages of Aedes aegypti (Diptera: Culicidae), vector of dengue: a longitudinal study (1996-2000)]. / Dinámica poblacional de los estadios inmaduros del vector del dengue Aedes aegypti (Diptera: Culicidae): un estudio longitudinal (1996-2000).

A four year study was conducted on a natural population of immature stages of Aedes aegypti after the re-invasion of Argentina by this vector in 1987. Thirty six plastic containers with 700 ml of dechlorinated water were placed in the La Plata Zoological Garden, La Plata, Argentina. A strip of filter paper around each container was added to facilitate egg counting. Eggs, larvae and pupae were counted weekly in each container from September, 1996 to August, 2000. After egg counting, papers were submerged to facilitate egg hatching and a new paper was placed in each container. Presence of A. aegypti immature stages was recorded from December-January to June during each of the four years of this study. In 1997, 13,105 eggs, 7,978 larvae and 1,476 pupae were registered with 54.7 % positive containers; during 1998, 8,194 eggs, 668 larvae and 142 pupae were recorded with 28.3 % positive containers; 13,510 eggs, 3,690 larvae and 743 pupae were registered during 1999 with 56.7 % positive containers; and 16,327 eggs, 4,669 larvae and 715 pupae during 2000 with 59.3 % of containers with presence of A. aegypti. Egg number and hatching rate were drastically reduced in 1998 when temperatures from December to May were 1 to 2.5 degrees C lower than the other years of this study. These colder than usual temperatures in the summer of 1998 were a consequence of the El Niño event.

Evaluation of Mesocyclops annulatus (Copepoda: Cyclopoidea) as a control agent of Aedes aegypti (Diptera: Culicidae) in Argentina.

We evaluated the potential of Mesocyclops annulatus as a control agent of Aedes aegypti in La Plata city (Argentina). Mosquito larval survivorship due to predation by these copepods was estimated at weekly intervals during the oviposition period of A. aegypti. Mean weekly A. aegypti larval survivorship in cylindrical plastic containers (12 cm height and 11 cm diameter) with copepods was significantly lower than in control containers. Furthermore, weekly larval survival was negatively correlated with M. annulatus adult density, and approximately 23 adult copepods/container would be a threshold density over which the weekly mosquito larval survivorship approached zero. The copepods were able to persist in all containers during approximately 100 days (in three of them until the end of the experiment: 155 days) without the resource represented by A. aegypti larvae. The predation and persistence observed suggest that M. annulatus is a potential control agent to be considered in biological control programs.

Evaluation of Mesocyclops annulatus (Copepoda: Cyclopoidea) as a control agent of Aedes aegypti (Diptera: Culicidae) in Argentina

We evaluated the potential of Mesocyclops annulatus as a control agent of Aedes aegypti in La Plata city (Argentina). Mosquito larval survivorship due to predation by these copepods was estimated at weekly intervals during the oviposition period of A. aegypti. Mean weekly A. aegypti larval survivorship in cylindrical plastic containers (12 cm height and 11 cm diameter) with copepods was significantly lower than in control containers. Furthermore, weekly larval survival was negatively correlated with M. annulatus adult density, and approximately 23 adult copepods/container would be a threshold density over which the weekly mosquito larval survivorship approached zero. The copepods were able to persist in all containers during approximately 100 days (in three of them until the end of the experiment: 155 days) without the resource represented by A. aegypti larvae. The predation and persistence observed suggest that M. annulatus is a potential control agent to be considered in biological control programs.

Life cycle and epizootiology of Amblyospora ferocis (Microspora: Amblyosporidae) in the mosquito Psorophora ferox (Diptera: Culicidae).

A natural population of Psorophora ferox (Humbold, 1820) infected with the microsporidium Amblyospora ferocis Garcia et Becnel, 1994 was sampled weekly during a seven-month survey in Punta Lara, Buenos Aires Province, Argentina. The sequence of development of A. ferocis in larvae of P. ferox leading to the formation of meiospores followed the developmental pathway previously reported for various species of Amblyospora. The natural prevalence of A. ferocis in the larval population of P. ferox ranged from 0.4% to 13.8%. Spores were detected in the ovaries of field-collected females of P. ferox and were shown to be responsible for transovarial transmission of A. ferocis to the next generation of mosquito larvae in laboratory tests. These spores were binucleate and slightly pyriform in shape. The prevalence of A. ferocis in the adult population ranged from 2.7% to 13.9%. Data on effects of the infection on female fecundity showed that infected field-collected adults of P. ferox laid an average of 47.6 +/- 6.5 eggs of which 35.8% +/- 4.1% hatched. Uninfected field-collected adults of P. ferox laid 82.8 +/- 6.8 eggs of which 64.1% +/- 5.5% hatched. Six species of copepods living together with P. ferox were fed meiospores from field-infected larvae but none became infected. Horizontal transmission of A. ferocis to P. ferox larvae remains unknown.

Laboratory evaluation of Mesocyclops annulatus (Wierzejski, 1892) (Copepoda: Cyclopidea) as a predator of container-breeding mosquitoes in Argentina.

In laboratory bioassays we tested the predatory capacity of the copepod Mesocyclops annulatus on Aedes aegypti and Culex pipiens larvae. A single adult female of M. annulatus caused 51.6% and 52.3% mortality of 50 first instar larvae of Ae. aegypti and Cx. pipiens respectively, in a 72 h test period. When alternative food was added to the containers, mortality rates declined to 16% and 10.3% for Ae. aegypti and Cx. pipiens respectively. When 50 first instar larvae of each of the two mosquito species tested were placed together with a single adult female of M. annulatus, mortality rates were 75.5% for Ae. aegypti larvae and 23.5% for Cx. pipiens larvae in a three day test period. Different density of adult females of M. annulatus ranged from 5 to 25 females produced mortality rates of Ae. aegypti first instar larvae from 50% to 100% respectively. When a single adult female of M. annulatus was exposed to an increasing number of first-instar Ae. aegypti larvae ranging from 10 to 100, 100% mortality was recorded from 1 to 25 larvae, then mortality declined to 30% with 100 larvae. The average larvae killed per 24 h period by a single copepod were 29.

Laboratory evaluation of Mesocyclops annulatus (Wierzejski, 1892) (Copepoda: Cyclopidea) as a predator of container-breeding mosquitoes in Argentina

In laboratory bioassays we tested the predatory capacity of the copepod Mesocyclops annulatus on Aedes aegypti and Culex pipiens larvae. A single adult female of M. annulatus caused 51.6 percent and 52.3 percent mortality of 50 first instar larvae of Ae. aegypti and Cx. pipiens respectively, in a 72 h test period. When alternative food was added to the containers, mortality rates declined to 16 percent and 10.3 percent for Ae. aegypti and Cx. pipiens respectively. When 50 first instar larvae of each of the two mosquito species tested were placed together with a single adult female of M. annulatus, mortality rates were 75.5 percent for Ae. aegypti larvae and 23.5 percent for Cx. pipiens larvae in a three day test period. Different density of adult females of M. annulatus ranged from 5 to 25 females produced mortality rates of Ae. aegypti first instar larvae from 50 percent to 100 percent respectively. When a single adult female of M. annulatus was exposed to an increasing number of first-instar Ae. aegypti larvae ranging from 10 to 100, 100 percent mortality was recorded from 1 to 25 larvae, then mortality declined to 30 percent with 100 larvae. The average larvae killed per 24 h period by a single copepod were 29