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Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae).

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

Animals↗

Cytotaxonomy of Simulium cauchense Floch & Abonnenc and Simulium quadrifidum Lutz (Diptera: Simuliidae) in Brazilian Amazonia.

Simulium cauchense Floch & Abonnenc and Simulium quadrifidum Lutz are widely distributed in the Amazon region and are morphologically similar at the larval and pupal stages. Chromosomally, these species are readily distinguished by the position of the nucleolar organizer, which is in the short arm of chromosome I in S. cauchense and in the long arm of chromosomes III in S. quadrifidum. They also differ by three fixed inversions. Sex chromosomes are undifferentiated in both species. Chromosomal resolution of the two species allowed us to evaluate four structural features previously used as diagnostic aids at the larval stage. Characters that distinguish larvae of the two species are the number of branches and branching patterns of the dorsal abdominal setae and the dark band on each primary fan. Branching patterns of the gill histoblasts were often diagnostic, with S. quadrifidum exhibiting more proximal branching and S. cauchense more distal branching. Sites where both species occurred sometimes had larvae with one petiole branching proximally and the other distally; in these cases examination of the chromosomes permitted assignment of the specimen to species. Pigmentation patterns of larvae, on the other hand, are highly variable. Color typically is sex linked in both species.

Animals↗

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae.

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinating B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.

Aedes↗

[Insecticide resistance in Simulium populations (Diptera, Simuliidae)].

Populations of Simulium (Chirostilbia) pertinax Kollar, 1832 from Southern and Southeastern States of Brazil were analyzed for temephos susceptibility considering control historical information and possible resistance. In situ bioassays were carried out for populations from the states of Paraná (Tibaji and Rolândia), Rio de Janeiro (Muriqui) and São Paulo (Barra do Una, Ilhabela and Morungaba). The populations were characterized as susceptible (S) or resistant (R) by submitting larvae in the last instars to an operational concentration (0.1ppm a.i./10min) of temephos (Abate 500E) as diagnostic. The possible mechanisms for the organophosphorus resistance development are discussed considering old and new control strategies.

Animals↗

[Adolpho Lutz's collection of black flies (Diptera - Simuliidae), its history and importance].

This is part of a master's thesis currently being written under the auspices of the Post-Graduate Program in Animal Biology of the Federal University of Rio de Janeiro, with support from CAPES. It presents the species of black flies in Adolpho Lutz's collection, held at the Laboratory of Black Flies and Oncocercosis of the Department of Entomology of the Instituto Oswaldo Cruz. A pioneer in the study of these dipterons, Lutz described about 25 species from different places in Brazil. A vector of round worm, the black fly's importance to public health was recognized at the end of the 1920s.

Animals↗

Spatial and temporal distribution of blackflies (Diptera: Simuliidae) in the Itatiaia National Park, Brazil.

The distribution of the blackfly larval taxocenoses of different altitudes in the Itatiaia National Park (located in the southeast of Rio de Janeiro state, Brazil 44 degrees 34' - 44 degrees 42' W, 22 degrees 16' - 22 degrees 28' S) was investigated on four campaigns, one in each season of 2003. On each campaign 60 samples were collected, using 30 x 30 cm wooden quadrats, distributed at four sites: Taquaral, Véu da Noiva, Três Picos and Brejo da Lapa. The winter and spring campaigns included two additional sampling sites: Alsene and Agulhas Negras. A total of 3578 larvae and 292 pupae were sampled, representative of six species: Simulium clavibranchium Lutz, S. subnigrum Lutz, S. rappae Py-Daniel, S. incrustatum Lutz, S. stellatum Gil-Azevedo, Figueiró & Maia-Herzog and Lutzsimulium pernigrum Lutz 1910. The highest larvae abundance and diversity were reported at the intermediate altitude site, Brejo da Lapa. Principal Components Analysis (PCA) revealed that L. pernigrum, S. stellatum and S. incrustatum were associated with the smaller breeding sites, the first two species being associated with sunny sites, while S. incrustatum occurred in sites with little sunlight.

Altitude↗

Larval description of Simulium (Notolepria) cuasiexiguum and Simulium (Chirostilbia) obesum and new records of black fly species (Diptera: Simuliidae) in the states of São Paulo and Minas Gerais, Brazil.

We describe the larval stage of Simulium cuasiexiguum Shelley, Luna Dias, Maia-Herzog & Lowry and Simulium obesum Vulcano and provide new distribution records of four black fly species in the State of São Paulo (Simulium cuasiexiguum, Simulium metallicum s.l. Bellardi, Simulium stellatum Gil Azevedo, Figuer6 & Maia-Herzog and Simulium oyapockense s.l. Floch & Abonnenc) and three species in the State of Minas Gerais (Simulium duodenicornium Pepinelli, Hamada & Trivinho-Strixino, Simulium obesum and Lutzsimulium pernigrum Lutz).

Animals↗

Cytotaxonomic and molecular analysis of Simulium (Edwardsellum) damnosum sensu lato (Diptera: Simuliidae) from Abu Hamed, Sudan.

The northernmost focus for Onchocerca volvulus Leuckhart (Nematoda: Onchocercidae), the causative agent of human onchocerciasis, is found along the Nile near the town of Abu Hamed in Sudan. The vector for O. volvulus at this focus is a single monomorphic population of Simulium (Edwardsellum) damnosum Theobald. This black fly population is limited to a small area between the fourth and fifth cataracts of the Nile River that is isolated geographically from all other populations of S. damnosum sensu lato. Phylogenies produced from cytological analyses and sequence data derived from the NADH dehydrogenase subunit 4 and 16S rRNA genes indicate that Abu Hamed black flies are similar to, but distinct from, the savanna-dwelling sibling species of S. damnosum s.l., Simulium (Edwardsellum) damnosum sensu strictu Theobald, and S. (Edwardsellum) sirbanum Vajime & Dunbar. The DNA sequence and the cytological data support the hypothesis that the black fly population present in Abu Hamed may represent a new sibling species of S. damnosum s.l. We propose that this population be informally designated as the hamedense form of the Simulium damnosum complex.

Animals↗

Simulium vittatum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psychodidae) salivary gland hyaluronidase activity.

Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum (Zetterstedt) is described, and its optimal pH determined. Salivary activity was reduced significantly after a blood meal, indicating that it was secreted after blood feeding. Phlebotomus papatasi (Scopoli) also exhibited salivary hyaluronidase activity. These results indicate that hematophagous pool-feeding insects may secrete this enzyme to help the spread of salivary antihemostatic agents in the vicinity of the feeding lesion, and perhaps to increase the size of the feeding lesion itself. Additionally, this enzyme may affect local host immune reactions and promote arboviral transmission.

Aedes↗

Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York.

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.

Aedes↗

Human onchocerciasis in the Amazonian area of southern Venezuela: spatial and temporal variations in biting and parity rates of black fly (Diptera: Simuliidae) vectors.

We investigated some entomological factors underlying altitudinal prevalence variation in the Venezuelan Amazonia human onchocerciasis focus. Spatial and temporal variation in relative abundance, daily biting rate, proportion of parous flies, and monthly parous biting rate were studied for the three main simuliid vectors (based on their vectorial competence: Simulium oyapockense s.l. Floch & Abonnenc approximately = S. incrustatum Lutz << S. guianense s.l. Wise). Yanomami villages were selected among sentinel communities of the ivermectin control program, representing hypo- to hyperendemicity conditions of infection. Spatial variation was explored via increasing village altitude on two river systems (A: Ocamo-Putaco and B: Orinoco-Orinoquito). Temporal variation was studied between 1995 and 1999 by sampling the biting population during dry and rainy mouths. Environmental variables included monthly rainfall and maximum river height. Simuliid species composition itself varied along the altitudinal and prevalence gradient. S. oyapockense s.l. prevailed below 150 m. Above this altitude and up to 240 m, S. incrustatum and S. guianense s.l. became more frequently and evenly collected along A but not along B, where S. incrustatum remained absent. The daily biting rate of S. oyapockense s.l. was higher during the dry season along A, whereas the converse took place along B. Daily biting rate of S. incrustatum was lowest during early rains. By contrast, the daily biting rate of S. guianense s.l. was highest during this period. There was a significant negative cross-correlation between proportion of parous of S. oyapockense s.l. and river height (2 and 3 mo lagged), whereas this variable (1 and 2 mo lagged) was positively correlated with the proportion of parous flies for S. incrustatum. Monthly parous biting rate values suggest that the months contributing most to onchocerciasis transmission in the area are likely to be the dry season and the transition periods between seasons.

Animals↗

Suppression of mosquito (Diptera: Culicidae) and black fly (Diptera: Simuliidae) blood feeding from Hereford cattle and ponies treated with permethrin.

The blood feeding of mosquitoes and black flies from Hereford cattle and ponies treated with commercial formulations of permethrin was evaluated using an animal enclosure trap sample system that allowed comparison of insect blood-feeding levels between treated and nontreated animals. Blood feeding of both Aedes dorsalis Meigen and A. melanimon Dyar from heifers treated with pour-on concentrate and whole body spray treatments was reduced significantly by 79-88% at 4 d posttreatment, with apparent but not significant reductions of 61-68% at 11 d posttreatment. Simulium bivittatum Malloch and S. griseum Coquillett blood feeding was reduced significantly by 96% to >99% at 4 d posttreatment, but apparent reductions of 30-87% at 11 d posttreatment were not significant. Blood feeding of S. bivittatum from ponies treated with a permethrin fly wipe was reduced significantly by 98 and 87% at 1 and 7 d posttreatment, respectively. No evidence of treatment-induced mortality was observed for recently blood-fed female mosquitoes or black flies captured from treated animals and held for 24 h. The potential benefit of using permethrin to protect livestock from insect-transmitted pathogens was estimated with a model based on level of host attack, pathogen infection rate in the vector, and suppression of blood feeding. Suppression of blood feeding by 90% is predicted to prevent the exposure of a host to a pathogen for up tolO d at 1,000 insect feedings per d when the vector population infection rate is one insect per 1,000. If insect feedings are lower (100/d) and the insect infection rate remains at one per 1,000, protection is predicted for 100 d. In contrast, a 90% suppression of blood feeding is predicted to provide protection for less than 1 d at 1,000 feeding per day and a vector infection rate of one insect per 100.

Aedes↗

Cytogenetics of laboratory colonies of simulium vittatum cytospecies IS-7 (Diptera: Simuliidae).

Laboratory colonies of the black fly Simulium vittatum Zetterstedt cytospecies IS-7 were analyzed cytogenetically and compared with the original New York population from which source material was collected 18 yr earlier. All sex chromosomes and major autosomal polymorphisms that were present in the source population were still represented in the laboratory colonies. However, the extent of sex linkage and the frequencies of four of the five major autosomal inversions changed significantly in at least one colony, perhaps because of bottlenecks experienced by the colony. A complete absence of males homozygous for the IS-7 inversion in both field and colony material is explained by postulating that the Y2 chromosome, representing the inverted condition for the IS-7 sequence, is absent from the population or acts as a rare reproductive lethal. This example possibly represents pseudo-partial sex linkage involving the X-linked sequences.

Animals↗

Biological transmission of vesicular stomatitis virus (New Jersey serotype) by Simulium vittatum (Diptera: Simuliidae) to domestic swine (Sus scrofa).

The role of hematophagous arthropods in vesicular stomatitis virus (New Jersey serotype; VSV-NJ) transmission during epizootics has remained unclear for decades in part because it has never been shown that clinical or subclinical disease in a livestock host results from the bite of an infected insect. In this study, we investigated the ability of VSV-NJ-infected black flies (Simulium vittatum Zetterstedt) to transmit the virus to domestic swine, Sus scrofa L. Experimental evidence presented here clearly demonstrates that VSV-NJ was transmitted from black flies to the swine. Transmission was confirmed by seroconversion or by the presence of clinical vesicular stomatitis followed by seroconversion. Our results represent the first report of clinical vesicular stomatitis in a livestock host after virus transmission by an insect.

Animals↗

Uptake of Onchocerca volvulus (Nematoda: Onchocercidae) by Simulium (Diptera: Simuliidae) is not strongly dependent on the density of skin microfilariae in the human host.

The relation between the number of microfilariae (mf) ingested by host-seeking vectors of human onchocerciasis and skin mf load is an important component of the population biology of Onchocerca volvulus, with implications for disease control and evaluation of the risk of transmission recrudescence. The microsimulation model ONCHOSIM has been used to assess such risk in the area of the Onchocerciasis Control Program (OCP) in West Africa, based on a strongly nonlinear relation between vector mf uptake and human mf skin density previously published. However, observed levels of recrudescence have exceeded predictions, warranting a recalibration of the model. To this end, we present the results of a series of fly-feeding experiments carried out in savanna and forest localities of West Africa. Flies belonging to Simulium damnosum s.s., S. sirbanum, S. soubrense, and S. leonense were fed on mf carriers and dissected to assess the number of ingested mf escaping imprisonment by the peritrophic matrix (the number of exo-peritrophic mf), a predictor of infective larval output. The method of instrumental variables was used to obtain (nearly) unbiased estimates of the parameters of interest, taking into account error in the measurement of skin mf density. This error is often neglected in these types of studies, making it difficult to ascertain the degree of density-dependence truly present in the relation between mf uptake and skin load. We conclude that this relation is weakly (yet significantly) nonlinear in savanna settings but indistinguishable from linearity in forest vectors. Exo-peritrophic mf uptake does not account for most of the density dependence in the transmission dynamics of the parasite as previously thought. The number of exo-mf in forest simuliids is at least five times higher than in the savanna vectors. Parasite abundance in human onchocerciasis is regulated by poorly known mechanisms operating mainly on other stages of the lifecycle.

Africa, Western↗

Development of Onchocerca volvulus (Filarioidea: Onchocercidae) in the West African black fly Simulium yahense (Diptera: Simuliidae) in Liberia.

Simulium yahense black flies infected with microfilaria of Onchocerca volvulus were kept in a defined insectary environment in Liberia, West Africa. A daily sample of infected flies was dissected for larvae developing in the thoracic muscles and examined for growth in stadial development. Microfilariae ingested by black flies transformed to the L1 larval stage without molting. Successive larval development included molting to the L2 stage and, finally, to the L3 stage, which was infective in humans. The cephalic cap, consisting of a laterally located hook and central stoma, occurs in the first larval stage. The caudal appendix and the laterally located anal opening are apparent in the L1 larva. In the L2 stage, the cephalic cap is lost and the large circular stoma becomes surrounded with elevated flaps. The caudal appendix was lost after larvae molted to the L3 stage, and in its place, 3 terminal papillae developed. Sense organs, such as 2 opposing phasmids and 8 papillae that were arranged into 2 circles, developed in the cephalic region of the L3 larva. The evidence of pathological consequences due to the presence of the L3 larva in the fly host are illustrated and discussed.

Animals↗

Influence of developmental stage on susceptibilities and sensitivities of Simulium vittatum IS-7 and Simulium vittatum IIIL-1 (Diptera: Simuliidae) to chlorpyrifos.

Determination of the most sensitive and susceptible organismal life stage is important for use of the organism in toxicity assessments of environmental pollutants. In the present study, the sensitivities and susceptibilities of larval developmental stages of two black fly sibling species, Simulium vittatum Zetterstedt cytospecies IS-7 and S. vittatum Zetterstedt cytospecies IIIL-1, were determined using the organophosphate-insecticide chlorpyrifos. Differences in sensitivity and susceptibility were determined through analysis of slopes and median lethal concentration values produced from 24-h orbital shaker toxicity tests, respectively. The results showed no difference in sensitivity or susceptibility between sibling species. However, early instar (second and third) as well as mid-instar (fourth and fifth) groupings of S. vittatum IS-7 were significantly more susceptible than late-instar (sixth and seventh) larval groupings. Likewise, mid-instar groupings of S. vittatum IIIL-1 were more susceptible than late-instar larval groupings. However, neither species showed significant differences in sensitivity among instar groupings. The results of the present study indicate that the mid-instar groupings of black fly larvae are the best choice for use in toxicity tests, both because of their increased susceptibility compared to late-instar groupings and because of their ease in handling and manipulating in the laboratory compared to early instar groupings.

Animals↗

Nematocera (Ceratopogonidae, Psychodidae, Simuliidae and Culicidae) and control methods.

The biology, veterinary importance and control of certain Nematocera are described and discussed. Culicoides spp. (family Ceratopogonidae) transmit the arboviruses of bluetongue (BT), African horse sickness (AHS), bovine ephemeral fever (BEF) and Akabane. Some other arboviruses have been isolated from these species, while fowl pox has been transmitted experimentally by Culicoides. These insects are vectors of the parasitic protozoans Leucocytozoon caulleryi and Haemoproteus nettionis, and the parasitic nematodes Onchocerca gutturosa, O. gibsoni and O. cervicalis. They also cause recurrent summer hypersensitivity in horses, ponies, donkeys, cattle and sheep. Farm animals can die as a result of mass attack by Simulium spp., which are also vectors of Leucocytozoon simondi, L. smithi and the filariae O. gutturosa, O. linealis and O. ochengi. Venezuelan equine encephalomyelitis (VEE) and Rift Valley fever (RVF) have been isolated from simuliids, and vesicular stomatitis virus New Jersey strain has been replicated in Simulium vittatum. Simuliids are well known as vectors of O. volvulus, the cause of human onchocercosis (river blindness). The family Psychodidae includes the genera Phlebotomus and Lutzomyia (subfamily Phlebotominae), vectors of Leishmania spp. in humans, dogs and other mammals. Vesicular stomatitis virus Indiana strain has been regularly isolated from phlebotomine sandflies. Mass attack by mosquitoes can also prove fatal to farm animals. Mosquitoes are vectors of the viruses of Akabane, BEF, RVF, Japanese encephalitis, VEE, western equine encephalomyelitis, eastern equine encephalomyelitis and west Nile meningoencephalitis, secondary vectors of AHS and suspected vectors of Israel turkey meningoencephalitis. The viruses of hog cholera, fowl pox and reticuloendotheliosis, the rickettsiae Eperythrozoon ovis and E. suis, and the bacterium Borrelia anserina are mechanically transmitted by mosquitoes. These insects also induce allergic dermatitis in horses. They transmit several filarial worms of both animals and humans, and are of great medical importance as vectors of major human diseases, including malaria, yellow fever, dengue fever and many more diseases caused by arboviruses.

Animals↗