Multiple symbiotes in larval black flies (Diptera: Simuliidae): associations of trichomycete fungi with ichthyosporeans, mermithid nematodes, and microsporidia

Studies of the interactions between two or more species of symbiotic microorganisms in a single host animal have lagged behind those involving interactions between hymenopteran or dipteran parasitoids. We screened the larvae of four species of black flies (Cnephia ornithophilia, Simulium furculatum, Simulium innoxium, and Simulium vandalicum) to determine if the colonization and prevalence of trichomycete fungi were influenced by ichthyosporeans, mermithid nematodes, microsporidia, or other species of trichomycetes. Differences in the layering of the peritrophic matrix of microsporidian-infected larvae, compared with that of uninfected larvae, were associated with greater levels of colonization by the midgut-inhabiting trichomycete Harpella melusinae. Colonization levels of different species of trichomycetes were significantly correlated with one another in only 2 of 11 cases. Aside from the consistent attachment of the trichomycete Simuliomyces microsporus to the ichthyosporean Paramoebidium chattoni, we found little evidence that prevalence and colonization levels of trichomycetes were influenced by other symbiotes, including other trichomycetes. This trend might reflect the physical separation of trichomycetes, which are gut specific, from other symbiotes. In addition, any combinations of symbiotes that were wholly incompatible would not have been observed in our field-collected material.     

Comparison of water ingestion between black flies (Simuliidae) and a mosquito (Aedes aegypti; Culicidae) using radiolabelled glucose

Ingestion of water by larvae of the black flies Simulium verecundum, Prosimulium mixtum/fuscum and Cnephnia dacotensis and by larvae of the mosquito Aedes aegypti was studied with radiolabelled glucose as a tracer. Extremely low ( 200 dpm/3 h) radiolabel uptake occurred in the black flies species, while Aedes aegypti, a species that is known to drink, had high radiolabel counts (> 6000 dpm/3 h). Radiolabel uptake by S. verecundum did not change for larvae fed in absence of current or with plugged mouthparts versus normal larvae. Radiolabel uptake by Aedes aegypti larvae with plugged mouthparts was < 3% of radiolabel uptake in normal larvae. Aedes aegypti was able to ingest the soluble glucose. Simulium verecundum, Prosimulium mixtum/fuscum and Cnephia dacotensis were unable to ingest the soluble sugar. The rationale for this difference is in the different filter-feeding mechanism of Culicidae and Simuliidae.     

Field and laboratory observations on a cytoplasmic polyhedrosis virus of blackflies (Diptera: Simuliidae)

Larval blackflies were sampled from streams in Newfoundland, Canada, from April 1974 through October 1975 for the presence of cytoplasmic polyhedrosis virus (CPV). CPV was found in Cnephia mutata, Prosimulium mixtum fuscum, Simulium tuberosum, S. venustum, and S. vittatum. Virus was most common in C. mutata occurring in seven of nine streams from which this host was sampled and with an incidence of infection up to 54%. Infections were rarer in other species, occurring in 10–30% of the streams sampled, depending on host species. Incidence of infection was usually less than 1% and never exceeded 5% for any species other than C. mutata. Results of preliminary infectivity studies are given, and the bionomics of the disease in nature are discussed.     

Seasonality of trichomycetes in larval black flies from South Carolina, USA

Trichomycete fungi are common endobionts of aquatic insect larvae, but little is known of their ecology. In this study, the seasonality of trichomycete colonization of larval black flies (Diptera: Simuliidae) was investigated in three streams in northwestern South Carolina. At least eight species of trichomycetes were found in two species of black flies, and 93.8% of 1819 larval black flies examined contained trichomycetes. Significant differences were found in the seasonal prevalence of Harpella melusinae, Simuliomyces microsporus, and Paramoebidium spp. at one of three sites. At this site, the lowest mean prevalence for H. melusinae occurred in winter (67%) versus the other seasons (96-100%), whereas mean prevalence was lowest in summer for Simuliomyces microsporus (1%) versus the other seasons (2-21%) and lowest in summer for P. spp. (9%) versus the other seasons (45-67%). Significant differences in levels of colonization among seasons were not detected. Conjugations of H. melusinae (representing early stages of sexual reproduction) occurred most frequently in the spring and winter (up to 14% of larvae). Sexual reproduction (represented by zygospores) of Legeriomycetaceae occurred most frequently in the spring and fall (up to 17% of larvae).     

Molecular differentiation of two sibling species of the black fly Simulium vittatum (Diptera: Simuliidae) based on random amplified polymorphic DNA.

Larvae of the black fly morphospecies Simulium vittatum from Colorado, Montana, Nebraska, and New Hampshire were cytologically identified as either the IS-7 or the IIIL-1 cytospecies. DNA was PCR amplified from cytotyped larvae using eight 10-mer primers, labeled with 33P, and run on polyacrylamide gels. The entire data set of 96 amplicons produced incomplete separation of the two cytospecies when subjected to neighbor-joining and maximum parsimony analyses. However, when analyzed within geographical regions, separate species status was supported. Bootstrap support for distinctness of the two cytospecies was best in Colorado where they were collected in true sympatry. The IS-7 cytospecies was more polymorphic in the western states, where it differed most from IIIL-1, which was most variable in the eastern states. The frequencies of the 17 most common amplicons in the two cytospecies were inversely correlated. A model of speciation derived from the molecular evidence suggests that IS-7 evolved in the west and spread eastward, whereas IIIL-1 later originated in the east and spread westward.     

Parasitism of Hylodes phyllodes (Anura: Cycloramphidae) by Hannemania sp (Acari: Trombicuudae) in an area of Atlantic Forest, Ilha Grande, southeastern Brazil

We studied some parameters of the parasitism by the mite Hannemonia sp. on the endemic frog Hylodes phyllodes in the Atlantic Forest of Ilha Grande (Rio de Janeiro State, Brazil). Prevalence, mean abundance, mean intensity and total intensity of infestation, body regions infected, and host sexual differences in parasitism rate of the larvae of Hannemania sp. on individuals of H. phyllodes were assessed. Prevalence was 86.5% (N = 37; total of 1,745 larvae of Hannemania sp) for male hosts and 91.7% (N = 12; total of 330 larvae) for female hosts, with no significant difference between the sexes. Overall prevalence of Hannemania sp. on H. phyllodes was 87.7%. Mean intensity of infestation in males (54.5 +/- 42.5; range 1-173 larvae) was higher than in females (29.9 +/- 47.6; range 1-166). We conclude that the rates of intensity, abundance, and prevalence of Hannemania larvae parasitizing Hylodes phyllodes at Ilha Grande were considerably high, suggesting that this species of anuran constitutes a relevant host for this mite species to complete its life-cycle in the area. Differences between males and females in infestation rates probably reflect their differential use of space in the forest.     

Assessment of rDNA IGS as a molecular marker in the Simulium damnosum complex

For five cytospecies of the Simulium damnosum Theobald complex of blackflies (Diptera: Simuliidae) from West Africa, both ends of the intergenic spacer region (IGS) of the rDNA have been sequenced with the aim of developing specific molecular markers. No specific differences in these two regions were detected between Simulium sanctipauli V. & D., Simulium sirbanum V. & D., Simulium soubrense V. & D., Simulium squamosum Enderlein and Simulium yahense V. & D., except in the number of A subrepeats at the 5' end of the IGS (two in S. squamosum and four or five in the others) and in position 310 of the 3' end (a C in S. squamosum and a G in the others). However, genetic distances within and between species overlapped. These DNA sequences had no strong phylogenetic signal, and the trees obtained were mostly unresolved. Although most sequences from S. squamosum clustered together, a few of them were more similar to those in other cytospecies. These results could be explained either by hybridization with genetic introgression or by ancestral polymorphism and recent speciation.     

Wild birds as hosts of Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae).

We evaluated the prevalence, mean intensity and relative density of ticks in 467 wild birds of 67 species (12 families) from forest and cerrado habitats at two protected areas of Minas Gerais, between March and September 1997. Ticks collected (n=177) were identified as larvae and nymphs of Amblyomma cajennense and four other species of Amblyomma. We report for the first time 28 bird species as hosts of the immature stages of A. cajennense, demonstrating the lack of host specificity of the larvae and nymphs. A. cajennense had 15% prevalence on birds, with a mean infestation intensity of 0.37 ticks per host sampled, and 2.5 ticks per infested bird. Prevalence varied in relation to host species, diet and between birds from forests at two successional stages. There were no differences in relation to host forest dependence, participation in mixed flocks of birds, and nest type constructed. A. cajennense is a species of medical and veterinary importance, occurring on domestic animals but is known little of its occurrence on wildlife.     

Spatial and temporal abundance,substrate partitioning and species co-occurrence in a guild of Neotropical blackflies (Diptera: Simuliidae)

Blackfly larvae were sampled monthly from two smallNeotropical mountain streams 500 m apart from each other in northernVenezuela during a 15-month period, and ten habitat variables weremeasured at two altitudes (800 and 900 m) to determine local spatialand temporal variations in abundance, stream variables, speciesco-occurrence, and substrate preference within the blackflyguild. The blackfly species composition of each stream was the same,with five species (in order of abundance): Simulium ochraceumWalker s.l., S. paynei Vargas, S. metallicum Bellardicytospecies E, Simulium sp., and S. horacioi Okazawa &Onishi. There were clear differences in community structurebetween streams (not among altitudes), although S. ochraceums.l. was the most abundant species in both habitats. Principal component andcorrelation analyses revealed that structural (great depth, width,flow, and discharge) and chemical (low conductivity) factors werethe stream variables best associated to the spatial distribution ofmost blackfly species. Blackfly abundance was greater in the largerstream. Larvae were most abundant on submerged rocks and fallenleaves, although the patterns of substrate use within the guildrevealed substrate partitioning among most blackfly species.There was one general annual peak of abundance at the end of therainy season. We suggest that blackfly community structure in theseNeotropical headwaters streams, over the small spatial scaleexamined here, is largely influenced by interactions among streamsize and velocity-related factors (width, streamflow, discharge andsubstrate availability-stability) and the rainfall regime.     

Laboratory infection of blackflies (Simuliidae) and midges (Chironomidae) by the mosquito mermithid, Romanomermis culicivorax

Penetrating infective juveniles of Romanomermis culicivorax usually killed first-stage larvae of Chironomus maturus Johann., Chironomus sp., Simulium damnosum Theo., and S. venustum Say. Nematodes were melanized and died after they entered fourth stage larvae of 2 chironomid species, but no host reaction was evident after entry into fourth-stage blackfly larvae. In contrast, the nematodes initiated development in the latter hosts, which died before the nematodes completed their development.     

Estimation of preferred water flow parameters for four species of Simulium (Diptera: Simuliidae) in small clear streams in South Africa

Blackfly larvae typically occur in fast-flowing riffle sections of rivers, with different blackfly species showing preferences for different hydraulic conditions. Very little quantitative data exist on hydraulic conditions linked to the blackfly species occurring in South African streams. Stones-in-current biotopes (i.e. fast riffle flows over cobbles) were sampled from four sites in three small clear streams in the Eastern and Western Cape provinces of South Africa. Mean water column velocities at each sampled stone were measured using a mini current meter, while flow velocities closer to the boundary layer where blackfly larvae occurred were estimated using indirect techniques (standard hemispheres and aerating tablets). Standard hemispheres were also used to calculate more complex hydraulic parameters such as Froude and Reynolds numbers. Four species of Simuliid were sampled in sufficient numbers to show trends in flow velocity preferences. Simulium impukane and S. rutherfoordi both occurred at their highest densities at velocities of 0.3m s^?1, while S. merops preferred velocities of 0.7m s^?1. Simulium nigritarse SL attained the highest densities of all the blackfly species sampled, and its relative abundances were greatest at velocities of 0.8–0.9m s^?1. Within the streams surveyed, all blackfly species occurred in subcritical-turbulent flows — based on a classification using Froude and Reynolds numbers — although two of the species were also found in high densities in supercritical flows where these existed at the sites. Local hydraulics within the stones-in-current biotope are complex, but in the absence of fine-scale equipment for measuring micro-velocities, standard hemispheres are a useful, cost-effective technique for the initial quantification of hydraulic parameters in small, clear streams. Such an approach facilitates further understanding of links between hydraulics and aquatic invertebrates in South African streams.     

Context-dependent symbiosis between black flies (Diptera: Simuliidae) and trichomycete fungi (Harpellales: Legeriomycetaceae)

The context-dependent nature of a symbiotic relationship between a trichomycete fungus ( Smittium culisetae ) and a larval black fly ( Simulium vittatum ) is demonstrated in the present study. No significant difference was found between the size of larvae colonized by trichomycetes and those free of trichomycetes, regardless of the trichospore dosage or initial age of the larvae. This trend suggests that the trichomycete has no detectable effect on host fitness, indicating a commensalistic relationship. However, in half of the experiments, stressed (i.e. starved) larvae exposed to trichospores at a dosage of 20 000 spores ml⁻¹ had significantly higher survival than did trichomycete-free larvae, indicating a mutualistic relationship. Trichomycetes in adult female black flies can replace the ovaries. The symbiotic association between trichomycetes and simuliids, therefore, is dynamic: commensalistic when larvae are well fed, mutualistic when larvae are starved, and parasitic in adults. The trichomycete-black fly relationship represents a rare case of symbiosis shifting among three states.     

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.     

B chromosome polymorphism of blackflies (Diptera, Simuliidae) from the north-western region of Russia

We have studied karyofonds of natural populations and B-chromosome morphology of 8 species of blackflies from the North-Western region of Russia: Odagmia ornata Mg., Hellichiella crassa Rubz., Simulium morsitans Edw., Simulium argyreatum Mg., Shoenbaueria pusilla Fries., Cnetha fontinalis Radzv., Stegopterna duo-decimata Rubz., and Archesimulium tuberosum Lundstr. For this purpose we made slides of squashed blackflies larvae with salivary gland polytene chromosomes stained by aceto-orcein, in addition to similarly stained slides with mitotic chromosomes from gonads and ganglia. Morphology of polytene B-chromosomes of Shoenbaueria pusilla Fries., Cnetha fontinalis Radzv., Stegopterna duodecimata Rubz., and Archesimulium tuberosum Lundstr. has been first described. B-chromosome polymorphism was found in all species, but the number of B chromosomes was conserved within each differences in polytene individual. Stable and distinct interspecific differences in the morphology of polytene B-chromosomes were demonstrated, and these characters are advisable to use to distinguish the species. We have investigated for the first time karyofonds of Od. ornata populations from Arkhangelsk Region (Solovetskie Islands) and Leningrad Region (railway station Sablino), and those of S. argyreatum populations from Murmansk Region (Kandalaksha environs) and Karelia (railway station Chupa). A long term study of Od. ornata and S. argyrestum population from North-Western Russia revealed interspecific and interpopulation dynamics of the occurrence of specimens with B-chromosomes. Some populations showed an increased percentage of individuals with B-chromosomes. It is suggested that B-chromosomes may play a role in adaptation of polulations to severe environmental conditions.     

Molecular characterization and taxonomy of a new species of Caudosporidae (Microsporidia) from black flies (diptera: simuliidae) with host-derived relationships of the North American caudosporids

A new species of microsporidium, Caudospora palustris (Microsporidia: Caudosporidae), is described from 3 species of black flies (Cnephia ornithophilia and diploid and triploid cytospecies of Stegopterna mutata), bringing to 7 the total species of caudosporids recorded from North America. This new species of caudosporid is recorded from swamp streams of the Coastal Plain from New Jersey to Georgia, with single records from the New Jersey mountains and the Upper Peninsula of Michigan. Densities of patently infected larvae (up to 10,600/m2) and spore production (nearly 8x10(11)/m2) are the greatest recorded for any microsporidium of black flies. The ultrastructure of this new species is presented, along with the first molecular characterization for a microsporidium of black flies. The phylogenetic position of black fly microsporidia within the phylum Microsporidia is presented; however, the analysis does not support the inclusion of C. palustris in any clade. Key features of all North American caudosporids are provided, and possible evolutionary trajectories are proposed based on optimization of caudosporid species on the phylogeny of their 22 known host species, including 16 that represent new host species records.     

Factors influencing nuisance blackfly (Diptera: Simuliidae) activity in the Scottish Highlands

Abstract. The nuisance activity of blackflies (Diptera: Simuliidae) was investigated in several habitats on Speyside, near Kincraig (57^o08'N, 3^o56'W), Invernesshire, in central Scotland during May-October 1987-89.
The main blackfly species caught landing/biting on humans were Simulium reptans, S. argyreatum, S. variegetum and the S. tuberosum complex, in order of prevalence. Blackfly biting activity occurred from mid-May to mid-September. Numbers of female blackflies attracted to volunteers were correlated with their body posture, habitat and the season. Overall, Simulium activity was greatest in mixed birch/juniper forest, least in spruce plantations and at intermediate levels on pasture, moorland and in Scots Pine forest.
Compared with an adult, a child experienced twice as many Simulium bites per hour (12.2 v 6.3) in the birch forest. On the child's body, 69% of blackfiies landed on the head, neck and back, whereas the majority landed on the legs (48%) and arms (28%) of adults. Bending over, especially during the exertion of gardening, was more attractive to anthropophilic blackfiies than standing or kneeling.     

Life cycle of Simulium jenningsi (Diptera: Simuliidae) in southern West Virginia.

The life cycle of Simulium jenningsi Malloch was compared at two study sites representing the largest and smallest streams (New River and Indian Creek, respectively) where this species is known to breed in southern West Virginia. Larvae first appeared in March, and the first generation emerged in April, followed by two to four more generations by September. A few larvae and adults persisted in autumn, then the population overwintered in the egg stage. There were considerable differences in life cycle at the two study sites. In the New River, emergence of the first generation was 2-3 wk earlier, there were five generations rather than three, and the last larvae of the season persisted 1 mo later as compared with Indian Creek. Warmer temperature and higher quality food in the New River are probable explanations for differences in life cycle. Effective pest management of this species will require larviciding at frequent intervals (1-2 wk) from April through September in many streams over a broad geographic area because of the species' nonsynchronous life cycle, occurrence in different size streams, and strong dispersal ability.     

The water velocity preferences of Austrosimulium furiosum and Simulium ornatipes (Diptera: Simuliidae) larvae,and the implications for micro-habitat partitioning

The aquatic larvae of two simuliid species, Austrosimulium furiosum (Skuse) and Simulium ornatipes Skuse, which often occur together in Victorian streams, were shown in laboratory experiments to have preferences for different water velocities: larvae of A. furiosum preferred water velocities of 0.2–0.3 m s^–1, and S. ornatipes preferred water velocities of 0.9–1.3 m s ^–1 . Final instar larvae of both species selected slow water speeds of less than 0.25 m s ^–1 prior to pupation. Flow patterns around a cylinder in a laboratory stream were mapped, and the distribution of A. furiosum larvae within the wake, paired vortices and horseshoe vortex was recorded. Larvae selected areas with suitable water velocities and aligned with the flow, providing flow visualization of micro-current speed and direction. The potential for micro-habitat partitioning is discussed in relation to benthic sampling strategies.     

Seasonal variation of black fly (Diptera: Simuliidae) species diversity and community structure in tropical streams of Thailand

Seasonal variation of the physicochemical conditions of streams plays an important role in aquatic insect diversity and community structure. Asian tropical streams profoundly change between seasons due to the effects of monsoons. However, little is known about how these changes affect aquatic insect diversity and community structure. The objectives of this study are to examine seasonal variations of the physicochemical conditions in tropical streams in Thailand and to assess the effects of these changes on black fly community structure and diversity. Black flies were sampled and physicochemical conditions recorded at eight sites between December 2007 and December 2008. A total of 10 black fly species were found. Comparisons of the streams between seasons revealed that physical conditions related to rainfall rate were significantly different. Canonical correspondence analysis differentiated sampling sites from each season. Streams in the rainy season were faster and deeper, with higher discharge and conductivity than those of the cold and dry seasons. Species richness was significantly higher in the rainy season than in the cold and dry seasons ( F  = 6.23, P  = 0.004). Community structure profoundly changed between the low-flow season (cold and dry) and high-flow season. Black fly species found predominantly in the low-flow season ( Simulium siamense "cytoform A", S. aureohirtum ) decreased dramatically during the high-flow season. In contrast, species found at high frequency during the high-flow season ( S. nakhonense , S. angulistylum ) disappeared in the low-flow season. The study demonstrates the importance of seasonal variation of stream conditions on black fly community structure and diversity.