Identification of nectar sources foraged by female mosquitoes in Canada.

For many mosquito species, the females must obtain vertebrate blood to complete a gonotrophic cycle. These blood meals are frequently supplemented by feeding on sugary plant nectar, which sustains energy reserves needed for flight, mating, and overall fitness. Our understanding of mosquito nectar foraging behaviors is mostly limited to laboratory experiments and direct field observations, with little research into natural mosquito-host plant relationships done in North America. In this study, we collected nectar-fed female mosquitoes over a 2-year period in Manitoba, Canada, and amplified a fragment of the chloroplast rbcL gene to identify the plant species fed upon. We found that mosquitoes foraged from diverse plant families (e.g., grasses, trees, ornamentals, and legumes), but preferred certain species, most notably soybean and Kentucky blue grass. Moreover, there appeared to be some associations between plant feeding preferences and mosquito species, date of collection, landscape, and geographical region. Overall, this study implemented DNA barcoding to identify nectar sources forage by mosquitoes in the Canadian Prairies.

Evaluation of the colchicine-like activity of Gloriosa superba-extracted fractions for mosquito (Diptera: Culicidae) cytogenetic study.

Four fractions of Gloriosa superba L., i.e., hexane fraction, dichloromethane fraction 1, dichloromethane fraction 2, and methanol fraction, were investigated for colchicine-like activity using a mosquito cytogenetic assay. The results revealed that the latter three fractions yielded promisingly high colchicine-like activity, whereas the hexane fraction yielded very low activity compared with 1% colchicine in a 0.85% sodium chloride solution. The metaphase rates and average number of metaphase chromosomes per positive brain ganglion (range) of Aedes aegypti L. larvae after incubation with 0.25-2% solutions of dichloromethane fraction 1, dichloromethane fraction 2, 0.5-2% solutions of methanol fraction, and 1% colchicine solution were 90-100% and 7 (2-19) to 22 (7-47); 90-100% and 4 (1-11) to 30 (4-73); 95-100% and 11 (1-28) to 17 (2-62); and 100% and 6 (2-11), respectively. The temperature stability tests of the three promising fractions were performed by heating 0.5% working solution at 121 degrees C for 15 min and preparing 0.5% working solution from stock frozen at -20 degrees C for 10 mo. These fractions also yielded satisfactory outcomes of metaphase rates and an average number of metaphase chromosomes per positive brain ganglia compared with 1% colchicine solution.

The application of ethanol-extracted Gloriosa superba for metaphase chromosome preparation in mosquitos.

The application of ethanol-extracted Gloriosa superba for metaphase chromosome preparation in adult and 4th larva Aedes aegypti revealed that 0.5-8% ethanol-extracted Gl. superba solution could be used instead of 1% colchicine in Hanks' balanced salt solution. For adult mosquitos, the metaphase rates and average number of metaphase chromosomes per positive mosquito after intrathoracic inoculation with 1-2% ethanol-extracted Gl. superba solution were 100% and 11.8 (2-16) -12.6 (3-28) in females, and 80-90% and 16.5 (1-52) - 29.89 (1-72) in males, whereas the inoculation with 1% colchicine solution yielded 80% and 50% metaphase rates, and 18.25 (1-40) and 16.5 (2-53) average number of metaphase chromosomes per positive mosquito in females and males, respectively. For 4th stage larvae, the metaphase rates and average number of metaphase chromosomes per positive mosquito after incubation with 0.5-8% ethanol-extracted Gl. superba solution were 90-100% and 14.42 (1-65) - 64 (19-137), while incubation with 1% colchicine solution yield 100% metaphase rate and 10.9 (7-15) average number of metaphase chromosomes per positive mosquito.

Screening of ten plant species for metaphase chromosome preparation in adult mosquitoes (Diptera: Culicidae) using an inoculation technique.

The screening of 10 plant species (Aloe barbadensis Mill., Asparagus officinalis L., As. plumosus Bak., As. racemosus Willd., As. sprengeri Regel, Codyline fruticosa Goppert, Dracaena loureiri Gagnep., Gloriosa superba L., Hemerocallis flava L., and Sansevieria cylindrica Bojer) for colchicine-like substance(s) using a mosquito cytogenetic assay revealed that a 1% solution of dried Gl. superba rhizome extracted in 0.85% sodium chloride solution could be used instead of a 1% colchicine in Hanks' balanced salt solution. The metaphase rates and average number of metaphase chromosomes per positive mosquito of Aedes aegypti (L.) after intrathoracic inoculation with 1% Gl. superba-extracted solution were 100% and 29.80 in females, and 90% and 25.78 in males, whereas the inoculation with 1% colchicine solution yielded 100 and 90% metaphase rates, and 20.90 and 12.22 average number of metaphase chromosomes per positive mosquito in females and males, respectively. The application of Gl. superba-extracted solution for metaphase chromosome preparation in other mosquito genera and species [e.g., Culex quinquefasciatus Say, Toxorhynchites splendens (Wiedemann), and Anopheles vagus (Döenitz)] also has yielded the satisfactory results.

Similarities to a LINE element shared by Anopheline and Culicine mosquitos map to the distal end of dihydrofolate reductase amplicons in Aedes albopictus mosquito cells.

To extend our understanding of amplicon structure in methotrexate-resistant Mtx-5011-256 Aedes albopictus mosquito cells, we examined a series of cosmids containing genomic DNA corresponding to the unique 3'-end of the Type 1 dihydrofolate reductase amplicon. Cosmid pWED118 contained five EcoRI fragments ranging from 2 to 5 kb (A, B, C, F, G) that hybridized to cDNA from methotrexate-resistant cells. Of these, fragments B and F hybridized weakly to first-strand cDNA from sensitive cells and shared considerable sequence identity. Fragment G occurred twice in the map of pWED118; one copy mapped within a 10 kb BssHII core fragment from the Type I amplicon and a second copy mapped downstream in the 48 kb BssHII core fragment. Hybridization signals among fragments contained in overlapping cosmids suggested that a branch point defining two or more subtypes of the Type I amplicon occurs within or near the 10 kb BssHII genomic DNA fragment. A 1.8 kb sequence common to fragments B and F included an approximately 0.4 kb region that shared sequence similarities with a LINE element from Aedes aegypti and with a repeated sequence from Anopheles gambiae. In addition, these elements shared amino acid similarity to a reverse transcriptase from the nematode, Caenorhabditis elegans. Shared sequence between Aedes and Anopheles elements supports the hypothesis that an ancestral LINE-like element was active in mosquito genomes prior to the divergence of the subfamilies Culicinae and Anophelinae. The presence of homologies to LINE-like elements near a branch point in the dihydrofolate reductase amplicon is consistent with a possible role of repeated sequences in amplicon shortening.

Molecular cloning, characterization and tissue expression of prophenoloxidase cDNA from the mosquito Armigeres subalbatus inoculated with Dirofilaria immitis microfilariae.

A cDNA encoding mosquito Armigeres subalbatus prophenol oxidase (As-pro-PO) was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) after Dirofilaria immitis inoculation. The 2205 bp As-pro-PO cDNA contains a 32 bp 5'-noncoding region, a 2055 bp open reading frame (685 amino acids), and a 118 bp 3'-noncoding region. Hydrophobic signal peptide for the endoplasmic reticulum targeting is not found in the NH2-terminal region. Two potential copper-binding domains, amino acids 197-245 and 345-412, are highly homologous to those of the other insect pro-POs. A 2.2 kb As-pro-PO transcript was identified by Northern blot analysis using D. immitis microfilariae-inoculated A. subalbatus. Both in situ hybridization and Northern blot analysis demonstrated that As-pro-PO mRNA was synthesized in mosquito haemocytes but not in other tissues, i.e. fat bodies, midguts and ovaries, etc.