An investigative study on the effects of black fly (diptera: simuliidae) sugar meals on reproductive success and parasite transmission /

Black flies are opportunistic sugar-feeders. They take sugar meals from Homopteran honeydew secretions or plant nectars, depending on availability. Homopteran honeydew secretions contain both simple and complex carbohydrates while plant nectars contain primarily simple carbohydrates. In order to determine whether honeydew secretions offer more energy than plant nectars to their insect visitors a study of wild-caught black flies was undertaken in Algonquin Provincial Park, Canada during the spring of 1 998 and 1 999. It was hypothesized that female black flies maintained on honeydew sugars will survive longer, produce more eggs and have a greater parasite vectoring potential than those maintained on artificial nectar or distilled water. Results demonstrated that: (1) host-seeking female Prosimulimfuscum/mixtum and Simulium venustum maintained on artificial honeydew did not survive longer than those maintained on artificial nectar when fed ad libitum; (2) fiiUy engorged S. venustum and Simulium rugglesi maintained on artificial honeydew did not produce more eggs than those maintained on artificial nectar when fed ad libitum; and (3) S. rugglesi did not have a greater vectoring potential of Leucocytozoon simondi when maintained on artificial honeydew as opposed to artificial nectar when fed ad libitum. However, all flies maintained on the two sugars (artificial honeydew and artificial nectar) survived longer, produce more eggs and had greater vectoring potential than those maintained on distilled water alone.

Behavioural characteristics in phylogenetics : a case study using black fly (Diptera: Simuliidae) cocoon spinning behaviour

1-1 is torically, the predominan t method of reconstructing phylogenies has been through the use of morphological characters. There are new techniques now gaining acceptance, including molecular techniques al1d chromosomal information. Altl10ugh the study of behaviour has been used in a comparative framework, these analyses have, historically, been based on intuition. Hennig (1966) devised a neV\' method of reconstructing phylogenies which provided a 110ncircular method for formulating, testing and refining phylogenies. Subsequent s)Tstematists had virtually abandoned ecological and beha\lioural data as primary indicators of phylogenetic relationships (Brooks and McLennan 1991). Therefore, in a modern cladistic framework (sensu Hennig) the analysis of behavioural traits remains underrepresented as a method of reconstructing phylogenies. This thesis will reconstruct the phylogeny for species of black flies (Diptera: Simuliidae), using two steps. The first step is to thoroughl)' understand and explain the cocoon spinning in black fly larvae. There have bee115 previous descriptions of cocoon spinning, but all were incomplete or erroneous. The advances in technology, including video recorders and VCRs, have allowed this behaviour to be analyzed in great detail in 20 different species. A complete description of the cocoon spinning of Simulium \littatum is given. This description will be used as a template for the other species observed. The description and understanding of cococ)n spinning was the first step in undertaking a phylogenetic analysis using this behaviour. The behaviour was then broken down and analyzed, revealing 23 characters, 3 either qualitative and quantitative in nature. These characters were assessed in a cladistic framework (sensu Hennig) and a phylogenetic tree was reconstructed with a e.I of 0.91 and an R.I. of 0.96. This phylogenetic tree closely resembles a previously established pllylogenetic tree produced from morphological and cytological information. The importance of this result is the indication that, contrary to some authors, behavioural characters, if used properly, can add very informative characters to a data set.

The effect of feeding regime on larval black fly (Diptera: simuliidae) primary head fan ray number

Identification of larval simuliids has always been difficult due to the morphological similarity many species bear to one another. For this reason all characters available have been drawn upon to aid in species identification, including head fan ray number. Even in light of an increasing body of anecdotal reports that head fan ray number is not fixed, it has continued to be used to aid species identification. In the current experiment simuliid larvae were reared under controlled laboratory conditions to last instar in one of three feeding regimes. Out of nine trials, the results of six showed a significant inverse relationship between feeding regime and head fan ray number. In addition to the laboratory experiments, larvae were also collected from the field over the course of the spring and summer, 1994. From these samples significant interspecific and intraspecific variations in head fan ray number were found both spatially and temporally within Algonquin Park. From these data it is concluded that head fan ray number for the species analysed is a developmentally plastic character, which varies in response to food availability. Furthermore, given the extreme variations in head fan ray number found in some species, I recommend that head fan ray number not be used as an aid to identification unless it can be shown to be a fixed character for the species in question.

An investigation into the effects of homopteran honeydew sugars versus floral nectar sugars on black fly longevity, flight performance and digestion /

Floral nectar is thought to be the primary carbohydrate source for most dipteran species. However, it has been shown that black flies (Burgin & Hunter 1997 a,b,c), mosquitoes (Foster 1995; Burkett et al. 1999; Russell & Hunter 2002), deer flies (Magnarelli & Burger 1984; Janzen & Hunter 1998; Ossowski & Hunter 2000), horse flies (Schutz & Gaugler 1989; Hunter & Ossowski 1999) and sand flies (MacVicker et al. 1990; Wallbanks et al. 1990; Cameron et al. 1992, 1995; Schlein & Jacobson 1994, 1999; Hamilton & EI Naiem 2000) feed on homopteran honeydew as well as floral nectar. Prior to 1997 floral nectar was thought to be the main source of carbohydrates for black flies. However, Burgin & Hunter (1 997a) demonstrated that up to 35% of black flies had recently consumed meals of homo pte ran honeydew. This information has necessitated a re-assessment of many life history aspects of black flies. Attempts are being made to examine the effects of nectar versus honeydew on black fly fecundity and parasite transmission (Hazzard 2003). Recently, Stanfield and Hunter (unpublished data) have shown that in female black flies, honeydew sugars produce flights of longer distance and duration than do nectar sugars. This thesis examines two aspects of black fly biology as it relates to sugar meal consumption. First, the effects of honeydew and nectar on black fly longevity are examined. Second, the proximate causation behind longer flight performances in honeydew-fed flies will be examined. The comparison between these two sources is important because nectar is composed of mainly simple sugars (monosaccharides and disaccharides) whereas honeydew is composed of both simple and complex sugars (including trisaccharides and tetrasaccharides ).

Characterization of black fly (Diptera: Simuliidae) silk proteins

Black fly (Simuliidae) silk is produced by the larvae and pharate pupae and is used for anchorage and cocoon production. There exists limited information on simuliid silks, including protein composition and genetic sequences encoding such proteins. The present study aimed to expand what is known about simuliid silks by examining the silks of several simuliid species and by making comparisons to the silk of non-biting midges (Chironomidae). Silk glands were dissected out of larval and pupal simuliids, and protein contents were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with silver stain. Protein contents were compared by mass in kilodaltons (kDa) between life stages and among species. Polymerase chain reaction (PCR) was used to expand upon known gene sequence information, and to determine the presence of genes homologous to chironomid silk. SDS-PAGE of cocoons revealed the presence of a 56 kDa and a 67 kDa protein. Silk gland contained as many as 28 different proteins ranging from 319 kDa to 8 kDa. Protein profiles vary among species, and group into large (>200), intermediate(>100), and small (<100) protein classes as is found in chironomids. It is likely that silk evolved in a common ancestor of simuliids and chironomids

An investigation of sugar feeding by black flies (Diptera: Simuliidae)

Although much research has been conducted on blood-meal acquisition in adult female black flies (Diptera: Simuliidae), the same cannot be said for sugarmeals. Both sexes feed on sugar which provides energy for flight and it has been commonly held that nectar is the major carbohydrate source. This thesis addresses the question of whether a non-floral carbohydrate source, specifically homopteran honeydew, is ingested by male and female black flies. Black flies reared in the laboratory have been observed to readily ingest freshly excreted and older (dry) honeydew when presented with honeydew coated tamarack branches. Field work was conducted in Algonquin Park, Ontario in the spring and summer of 1993. Three separate studies were designed to test whether homopteran honeydew is an important carbohydrate source for black flies and whether flies from different habitats utilize different sugar sources. The sugars melezitose and / or stachyose are known to occur in a variety of homopteran honeydews and therefore were used as indicators of honeydew feeding by black flies. In the first study, black flies were collected with insect nets from a stand of Larix larcina heavily infested with honeydew - producing homopterans (Adelges lariciatus). Six black fly species were captured: Simulium venustum, S. rostra tum, S. vittatum, Stegopterna mutata, S. aureum and S. quebecense. Samples of honeydew and individual black flies were tested using thin layer chromatography (T. L. C.) with fructose, glucose, sucrose, turanose, melezitose, raffinose and stachyose as standards. All sugars except turanose and melezitose were found in the adelgid honeydew samples. Since the sugar melezitose was absent from ~ honeydew samples, stachyose was used to indicate that black flies were feeding from this particular honeydew source. Of the 201 black flies tested, 194 contained sugars which occurred in 16 combinations. Stachyose combinations excluding melezitose, present in 45.9 % of flies, were used to indicate that black flies had been feeding on the adelgid honeydew. In the second study, black flies were collected in the morning and evening on 8 collection dates, using a vehicle mounted insect net. The crops and midguts of 10 male and 10 female Simulium venustum were dissected on each sample date. In total the gut contents of 320 individual flies were analysed by T. L. C. The sugars identified from these flies were present in the following proportions: fructose (100.0%), glucose (100.0%), sucrose/turanose (50.4%), melezitose (30.3%), raffinose (18.8%) and stachyose (8.7%). These sugars occurred in fourteen different combinations. It is argued that the presence of melezitose and / or stachyose indicates that black flies had fed on homopteran honeydew. Significantly more female flies (40.0%) than male flies (27.5%) had fed on honeydew. In the third study, adult black flies were sampled by sweep netting vegetation in four habitats in the morning and evening on 8 collection dates. The habitats are as follows: (1) Davies Bog, (2) Abandoned Air Field (dominated by blueberries, Vaccinium spp.), (3) Deciduous Habitat and (4) Coniferous Habitat. Sugars in the crops and midguts of female flies were tested by T. L. C. and, for S. venustum, it was found that significantly fewer flies (18.8%) from the Air Field contained honeydew than from the other three sites (Davies Bog, 34.4%; Deciduous Habitat, 36.2%; Coniferous Habitat, 25.0%). Of the 1287 black flies tested individually by T. L. C. 441 (34.3%) contained melezitose and / or stachyose sugars indicating that this proportion of the population were feeding from Homopteran honeydew. It is therefore clear that floral (nectar) sugars are not the only source of carbohydrates available to black flies.

Gut-specific transcriptional regulatory elements of the carboxypeptidase gene are conserved between black flies and Drosophila.

Millions of people die every year in the tropical world from diseases transmitted by hematophagous insects. Failure of conventional containment measures emphasizes the need for additional approaches, such as transformation of vector insects with genes that restrict vectorial capacity. The availability of an efficient promoter to drive foreign genes in transgenic insects is a necessary tool to test the feasibility of such approach. Here we characterize the putative promoter region of a black fly midgut carboxypeptidase gene and show that these sequences correctly direct the expression of a beta-glucuronidase reporter in Drosophila melanogaster. By histochemical staining and mRNA analysis, we found that the gene is expressed strongly and gut-specifically in the transgenic Drosophila. This gut-specific black fly carboxypeptidase promoter provides a valuable tool for the study of disease vectors.

Ocorrência da Mosca-negra-dos-citros (Aleurocanthus woglumi ashby) (Hemiptera: aleyrodidae) em Pernâmbuco

This paper aimed to verify the presence of black-fly of citrus in the state of Pernambuco in Brazil and wordwide. The material with symptoms were collected in the metropolitan area of Recife/PE and subsequently led to the identification in the Insect Biology Laboratory of the Federal Rural University of Pernambuco - UFRPE on February 4, 2011. The samples collected from the branches of citrus and bilocular, found the presence of eggs, nymphs, "pupae" and adults of Aleurocanthus woglumi, confirming its occurrence in the state of Pernambuco. The presence of blackfly of citrus had been detected in the city in the state of Pernambuco as Timbauba, Bom Jardim, Macaparana, Axes, Orobo, Sao Vincent Farrer and Itambe in citrus plants. Before that had a suspect in the Valley Siriji/PE, now it is in the metropolitan area of Recife, therefore we can confirm that the material collected and identified in the metropolitan area of Pernambuco is A. woglumi.

Estudo de Simuliidae (Diptera : Nematocera) e seus criadouros em igarapés do município de Santo Antônio do Tauá, estado do Pará, Brasil

Considerando a grande importância dos simulídeos (piuns e borrachudos) do ponto de vista bio-ecológico, médico e veterinário, desenvolveu-se este estudo sobre as espécies do gênero Simulium que se criam em igarapés do município de Santo Antônio do Tauá, Estado do Pará, Brasil. Os estudos foram desenvolvidos pelas seguintes metodologias: observações diretas dos criadouros nos igarapés; instalação de criadouros artificiais para a colonização por simulídeos; análise das freqüências de picadas em pessoas durante o dia; coletas e amostragens da entomofauna associada aos habitats aquáticos; amostragens qualitativas e quantitativas da vegetação nos criadouros; análises físico-químicas da água dos igarapés e criadouros; análises de fatores climáticos locais; e morfometria das larvas para a determinação do número de estádios larvais de Simulium quadrifidum. Estudou-se a entomofauna aquática e a respectiva abundância relativa de Simuliidae em dois igarapés. As espécies Simulium perflavum,Simulium quadrifidum, Simulium incrustaium e Simulium goeldii foram coletadas. As formas imaturas de Simulium perflavum foram as predominantes em ambientes perturbados com águas claras. Simulium quadrifidum foi predominante em ambientes com pouca alteração, de águas pretas, com baixos teores de nutrientes. Simulium incrustatum foi mais abundante em ambientes arbustivos e somente em águas claras. Simulium goeldii foi registrada apenas em áreas de matas primárias (com pouca alteração) e mais abundante em águas pretas. Os fatores que mais interferiram na colonização natural dos substratos artificiais por Simuliidae foram as constantes oscilações no nível d'água durante o período quando em que substratos ficaram expostos nos igarapés. A entomofauna aquática apresentou-se abundante no período seco. Constatou-se pela primeira vez a antropofilia da espécie Simulium incrustatum no Pará. Esta espécie mostrou dois picos diários de freqüência de picadas em voluntários, um pela manhã e outro à tarde. Houve diferenças significativas na freqüência de picadas entre áreas com tipos diferentes de vegetação nos mesmos períodos, e alta correlação negativa com a temperatura do ar, apenas na área de capoeira e no período seco. Determinou-se, pela primeira vez, o número de estádios larvais de Simulium quadrifidum e registrou-se a ovipostura de suas fêmeas somente pela parte da tarde, às 16:30h. Novos registros de predadores de adultos de simulídeos foram acrescentados.

Simulídeos (Diptera: Nematocera) das bacias do leste do estado do Pará, com ênfase na Serra dos Martírios-Andorinhas

Os estudos neste trabalho acrescentam diversas informações sobre simulídeos (Diptera: Simuliidae) do leste do estado do Pará, Brasil. Tem como objetivo avaliar a distribuição geográfica das espécies; elaborar uma chave para a identificação de pupas de simulídeos do leste paraense; estimar a riqueza de espécies; avaliar a atividade hematofágica diurna; avaliar a relação dos fatores limnológicos e meteorológicos com as populações de simulídeos e outros insetos aquáticos associados; registrar o estado de conservação e similaridade de três áreas (Atlântico-Nordeste, Serras das Andorinhas e Carajás), baseadas em um protocolo ambiental e nas espécies de piuns. Foram registradas 14 espécies: S. nigrimanum, S. incrustatum, S. minusculum, S. quadrifidum, S. limbatum, S. perflavum, S. iracouboense, S. rorotaense, S. spinibranchium, S. subpallidum, S. pertinax, S. subnigrum, S. brachycladum e S. goeldii. Chave de identificação baseada nas pupas dessas espécies foi elaborada. Estes dados são inéditos e as espécies de interesse em saúde pública (S. nigrimanum, S. crustatum, S. rorotaense, S. minusculum, S. subnigrum e S. pertinax) foram encontradas em diversos ambientes, com ampla distribuição e registros de novas ocorrências no Pará e Amazônia Oriental. Estudou-se a hematofagia de S. rorotaense, S. minusculum e S. pertinax nos meses de janeiro, abril, agosto e dezembro de 2006, na Serra das Andorinhas. Estas atividades de ataque foram correlacionadas principalmente à temperatura e umidade relativa do ar, exibindo dois picos de atividades, um pela manhã e outro pela tarde. A preferência por regiões do corpo de humanos também foi estudada. Os simulídeos e entomofauna aquática associada dos sistemas aquáticos foram ordenados em dois grupos e correlacionaram em maior ou menor grau à vazão, velocidade, profundidade, largura, alcalinidade e ferro, em ambas as regiões estudadas (Costa Atlântica-Nordeste e Tocantins-Araguaia). Registrou-se ainda que o meio ambiente apresenta-se bem conservado na Serra das Andorinhas, mas bastante alterado na Serra dos Carajás e em localidades da Costa Atlântica-Nordeste. A maior similaridade na composição das espécies de simulídeos foi observada entre as Serras das Andorinhas e Carajás, seguida pela similaridade com Costa Atlântica-Nordeste.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Biological control of the citrus blackfly in Mexico /

Cover title.

One and the same: Integrative taxonomic evidence that Bactrocera invadens (Diptera: Tephritidae) is the same species as the Oriental fruit fly Bactrocera dorsalis

The invasive fruit fly Bactrocera invadens Drew, Tsuruta & White, and the Oriental fruit fly Bactrocera dorsalis (Hendel) are highly destructive horticultural pests of global significance. Bactrocera invadens originates from the Indian subcontinent and has recently invaded all of sub-Saharan Africa, while B. dorsalis principally occurs from the Indian subcontinent towards southern China and South-east Asia. High morphological and genetic similarity has cast doubt over whether B. invadens is a distinct species from B. dorsalis. Addressing this issue within an integrative taxonomic framework, we sampled from across the geographic distribution of both taxa and: (i) analysed morphological variation, including those characters considered diagnostic (scutum colour, length of aedeagus, width of postsutural lateral vittae, wing size, and wing shape); (ii) sequenced four loci (ITS1, ITS2, cox1 and nad4) for phylogenetic inference, and; (iii) generated a cox1 haplotype network to examine population structure. Molecular analyses included the closely related species, Bactrocera kandiensis Drew & Hancock. Scutum colour varies from red-brown to fully black for individuals from Africa and the Indian subcontinent. All individuals east of the Indian subcontinent are black except for a few red-brown individuals from China. The postsutural lateral vittae width of B. invadens is narrower than B. dorsalis from eastern Asia, but the variation is clinal, with subcontinent B. dorsalis populations intermediate in size. Aedeagus length, wing shape and wing size cannot discriminate between the two taxa. Phylogenetic analyses failed to resolve B. invadens from B. dorsalis, but did resolve B. kandiensis. Bactrocera dorsalis and B. invadens shared cox1 haplotypes, yet the haplotype network pattern does not reflect current taxonomy or patterns in thoracic colour. Some individuals of B. dorsalis/B. invadens possessed haplotypes more closely related to B. kandiensis than to conspecifics, suggestive of mitochondrial introgression between these species. The combined evidence fails to support the delimitation of B. dorsalis and B. invadens as separate biological species. Consequently, existing biological data for B. dorsalis may be applied to the invasive population in Africa. Our recommendation, in line with other recent publications, is that B. invadens be synonymized with B. dorsalis.

'Ladd traps' as a visual trap for male and female Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae)

Bactrocera tryoni (Froggatt) is Australia's major horticultural insect pest, yet monitoring females remains logistically difficult. We trialled the ‘Ladd trap’ as a potential female surveillance or monitoring tool. This trap design is used to trap and monitor fruit flies in countries other (e.g. USA) than Australia. The Ladd trap consists of a flat yellow panel (a traditional ‘sticky trap’), with a three dimensional red sphere (= a fruit mimic) attached in the middle. We confirmed, in field-cage trials, that the combination of yellow panel and red sphere was more attractive to B. tryoni than the two components in isolation. In a second set of field-cage trials, we showed that it was the red-yellow contrast, rather than the three dimensional effect, which was responsible for the trap's effectiveness, with B. tryoni equally attracted to a Ladd trap as to a two-dimensional yellow panel with a circular red centre. The sex ratio of catches was approximately even in the field-cage trials. In field trials, we tested the traditional red-sphere Ladd trap against traps for which the sphere was painted blue, black or yellow. The colour of sphere did not significantly influence trap efficiency in these trials, despite the fact the yellow-panel/yellow-sphere presented no colour contrast to the flies. In 6 weeks of field trials, over 1500 flies were caught, almost exactly two-thirds of them being females. Overall, flies were more likely to be caught on the yellow panel than the sphere; but, for the commercial Ladd trap, proportionally more females were caught on the red sphere versus the yellow panel than would be predicted based on relative surface area of each component, a result also seen the field-cage trial. We determined that no modification of the trap was more effective than the commercially available Ladd trap and so consider that product suitable for more extensive field testing as a B. tryoni research and monitoring tool.

Less black than we're painted /

Acquisition made accessible thanks to the generous support of the Frederick J. and Margret L. Worden Endowment.