rapid-collection-floral-fragrance-volatiles-using-headspace-volatile

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important ...

University of Florida

Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program.

Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding.

South Florida is home to a number of native species of orchids. The Florida Panther National Wildlife Refuge has 27 known species, including Prosthechea cochleata, the clamshell orchid, which is listed as endangered on Florida's Regulated Plant Index. In a prior study done on this species in Mexico, P. cochleata was found to produce no floral fragrance at the particular study location. However, blooming orchids of this species at the University of Florida in Gainesville, were noted to be fragrant. In this paper, we document the presence of floral fragrance compounds from P. cochleata by using by gas chromatography mass spectrometry (GC/MS) analysis of headspace volatile collection. The orchids sampled were found to be consistently producing eight volatiles that are common in floral fragrances, including those of previous orchid species studied. By knowing the fragrance compounds produced, we can better understand the pollination biology of this endangered orchid. This information could be used to help future conservation efforts for P. cochelata by increasing pollination and subsequent seed capsule production.

Floral fragrance analysis of Prosthechea cochleata (Orchidaceae), an endangered native, epiphytic orchid, in Florida.

The Burmese python, Python bivittatus Kuhl, is a well-established invasive species in the greater Everglades ecosystem of southern Florida, USA. Most research on its ecological impacts focuses on its role as a predator and its trophic interactions with native vertebrate species, particularly mammals. Beyond predation, there is little known about the ecological interactions between P. bivittatus and native faunal communities. It is likely that established populations of P. bivittatus in southern Florida serve as hosts for native mosquito communities. To test this concept, we used mitochondrial cytochrome c oxidase subunit I DNA barcoding to determine the hosts of blood fed mosquitoes collected at a research facility in northern Florida where captive P. bivittatus and Argentine black and white tegu, Salvator merianae (Duméril and Bibron), are maintained in outdoor enclosures, accessible to local mosquitoes. We recovered python DNA from the blood meals of three species of Culex mosquitoes: Culex erraticus (Dyar and Knab), Culex quinquefasciatus Say, and Culex pilosus (Dyar and Knab). Culex erraticus conclusively (P = 0.001; Fisher's Exact Test) took more blood meals from P. bivittatus than from any other available host. While the majority of mosquito blood meals in our sample were derived from P. bivittatus, only one was derived from S. merianae. These results demonstrate that local mosquitoes will feed on invasive P. bivittatus, a recently introduced host. If these interactions also occur in southern Florida, P. bivittatus may be involved in the transmission networks of mosquito-vectored pathogens. Our results also illustrate the potential of detecting the presence of P. bivittatus in the field through screening mosquito blood meals for their DNA.

Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA.

The transmission dynamics of mosquito-vectored pathogens are, in part, mediated by mosquito host-feeding patterns. These patterns are elucidated using blood meal analysis, a collection of serological and molecular techniques that determine the taxonomic identities of the host animals from which blood meals are derived. Modern blood meal analyses rely on polymerase chain reaction (PCR), DNA sequencing, and bioinformatic comparisons of blood meal DNA sequences to reference databases. Ideally, primers used in blood meal analysis PCRs amplify templates from a taxonomically diverse range of vertebrates, produce a short amplicon, and avoid co-amplification of non-target templates. Few primer sets that fit these requirements are available for the cytochrome c oxidase subunit I (COI) gene, the species identification marker with the highest taxonomic coverage in reference databases. Here, we present new primer sets designed to amplify fragments of the DNA barcoding region of the vertebrate COI gene, while avoiding co-amplification of mosquito templates, without multiplexed or nested PCR. Primers were validated using host vertebrate DNA templates from mosquito blood meals of known origin, representing all terrestrial vertebrate classes, and field-collected mosquito blood meals of unknown origin. We found that the primers were generally effective in amplifying vertebrate host, but not mosquito DNA templates. Applied to the sample of unknown mosquito blood meals, > 98% (60/61) of blood meals samples were reliably identified, demonstrating the feasibility of identifying mosquito hosts with the new primers. These primers are beneficial in that they can be used to amplify COI templates from a diverse range of vertebrate hosts using standard PCR, thereby streamlining the process of identifying the hosts of mosquitoes, and could be applied to next generation DNA sequencing and metabarcoding approaches.

Barcoding blood meals: New vertebrate-specific primer sets for assigning taxonomic identities to host DNA from mosquito blood meals.

Feeding upon vertebrate blood by mosquitoes permits transmission of diverse pathogens, including viruses, protozoa, and nematodes. Despite over a century of intensive study, no mosquito species is known to specialize on non-vertebrate hosts. Using molecular analyses and field observations, we provide the first evidence, to our knowledge, that a mosquito, , specializes on annelid hosts (earthworms and leeches) while its sympatric congener, , feeds only on anurans (frogs and toads). Our results demonstrate that  feeds on annelid hosts (100% of identified blood meals;  = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding on  worms and freshwater leeches. These findings indicate that adult mosquitoes utilize a much broader range of host taxa than previously recognized, with implications for epidemiology and the evolution of host use patterns in mosquitoes.

Article	Year
Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding. Parasites & vectors\| PubMed ID: 27629021	2016
Floral fragrance analysis of Prosthechea cochleata (Orchidaceae), an endangered native, epiphytic orchid, in Florida. Plant signaling & behavior\| PubMed ID: 29297748	2018
Interactions between the invasive Burmese python, Python bivittatus Kuhl, and the local mosquito community in Florida, USA. PloS one\| PubMed ID: 29342169	2018
Barcoding blood meals: New vertebrate-specific primer sets for assigning taxonomic identities to host DNA from mosquito blood meals. PLoS neglected tropical diseases\| PubMed ID: 30161128	2018
Identification of as a specialist of annelids broadens known mosquito host use patterns. Communications biology\| PubMed ID: 30271973	2018

Jennifer L. Gillett-Kaufman

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