Jennifer L. Gillett-Kaufman

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

BackgroundDetermination 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.MethodsWe compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success.ResultsPreservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor.ConclusionsOur data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided.

Education and training to increase adoption of IPM for western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae).

ABSTRACT The University of Florida, IFAS, IPM Florida and Southern Plant Diagnostic Network (SPDN) are cooperatively developing education and training to increase integrated pest management (IPM) of western flower thrips (WFT), Frankliniella occidentalis (Pergande). Management of WFT is exacerbated by difficulty in identifying thrips species and by their development of insecticide resistance. Education and training will emphasize workshops on identification of thrips; insecticide resistance management protocols; description of the effects of insecticides on natural enemies, i.e., Orius spp., and use of the “Growers IPM Guide for Florida Tomato and Pepper Production”. Thrips identification aids, such as a national field-based identification deck currently under development by the SPDN and the North Central IPM Center (NCIPMC), will be useful to Florida growers. This kind of information will be delivered through Extension programs, including workshops, in-service training, field days, and classroom education. A section of the IPM Florida website is devoted to thrips in several Florida crops (blueberry, cotton, greenhouse and nursery grown ornamentals, pepper, strawberry, tomato) and contains general information, such as (1) F. occidentalis biology and ecology, (2) management tactics incorporated into an IPM program that is crop and location specific, (3) identification of thrips and natural enemies, (4) practices that reduce damaging pest populations in space and time, (5) problems with managing F. occidentalis and other pests, (6) updated, crop and location specific information on population levels, and (7) resistance monitoring. Future needs for specific in-service or other educational programs, including advanced diagnostic training sessions, will be determined by clientele groups. Adoption of IPM for WFT will benefit growers by minimizing insecticide resistance and maximizing benefits of cultural practices and biological controls.

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

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.

Identification of Uranotaenia sapphirina as a specialist of annelids broadens known mosquito host use patterns

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, Uranotaenia sapphirina, specializes on annelid hosts (earthworms and leeches) while its sympatric congener, Uranotaenia lowii, feeds only on anurans (frogs and toads). Our results demonstrate that Ur. sapphirina feeds on annelid hosts (100% of identified blood meals; n = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding on Sparganophilus 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.Lawrence Reeves et al. report evidence that adult females of the mosquito species Uranotaenia sapphirina feed primarily on annelid hosts. This is the first known example of a mosquito species that specializes on invertebrate blood and suggests that mosquito host use patterns are more diverse than previously recognized.

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

ABSTRACT 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 Floridas 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.

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

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.

Attraction of thrips (Thysanoptera) to colored sticky traps in a Florida olive grove

Abstract A study was conducted in 4 plots within a newly established olive grove in Florida to assess surveillance methods for insects present around the period of olive bloom. Over 99% of thrips collected were Frankliniella bispinosa (Morgan) (Thysanoptera: Thripidae), with occasional collections of predacious Leptothrips pini (Watson) (Thysanoptera: Phlaeothripidae). Collections of thrips using sticky traps or in tap or brush samples were high at the time of bloom, with low numbers before bloom and very low numbers after bloom. No differences in collections were seen among plots for thrips numbers when sampled using sticky cards. However, one plot had higher thrips numbers when sampled using tap and brush samples. Overall, and especially during bloom, blue sticky traps were most attractive, followed by yellow and then white sticky traps. Clear (color-free) traps collected the fewest thrips. Using tap samples, more thrips were collected on the edges than in the middle of the grove. Highly localized high densities of thrips were detected by the tap samples. Although sticky traps were highly effective for collecting thrips, only tap samples detected the localized hot spots.

Effect of plant Growth Regulators on Blissus insularis (Hemiptera: Blissidae)

Summary Plant growth regulators are known to influence a plants characteristics and therefore can be used as an alternative strategy in making Blissus insularis Barber (Hemiptera: Blissidae) habitat unfavorable for this insects survival. The reduction of B. insularis population densities following the application of mefluidide and trinexapac-ethyl in field plots indicated the indirect effect of plant growth regulator application on B. insularis for its management in St. Augustinegrass.

Manduca rustica (Lepidoptera: Sphingidae) damage on olive (Olea europaea; Lamiales: Oleaceae) trees in Florida

There is a growing interest in cultivating olives Olea europaea L. (Lamiales: Oleaceae) in Florida, USA. Although some parts of Florida seem well suited for olive production, the pests and diseases associ ated with cultivated olives in Florida are generally unknown. During a 2014 survey of olives being grown at the commercial and homeowner level, a novel pest was identified. A hornworm (Fig. 1), now known to be the larval stage of the rustic sphinx Manduca rustica (F.) (Lepidoptera: Sphingidae) (Wagner 2010; D. L. Wagner, personal communication), was reported as a pest of concern by growers from the Florida Panhandle in the north to Polk County in the south. Tuttle (2007) described M. rustica as a broadly distributed Neotropical species that breeds in the mid-Atlantic states as far north as Delaware, USA. In Florida, Heppner (2003) reported M. rustica ranges as far south as Miami-Dade County, but the species has not been reported from Monroe County (the Florida Keys).Worldwide, there are 49 recorded lepidopteran pests of O. europaea, 9 of which are in the family Sphingidae, but none is within the genus Manduca (Robinson et al. 2010). The primary concern of growers was the defoliation caused by larval feeding. One larva can quickly denude a 2-yr-old olive tree, causing problems for nursery producers and for growers with new plantings. Although trees can recover from this damage, olive trees are slow growing, and feeding damage can remain apparent for several months depending on the time of year. Manduca rustica is listed as having a host range that includes plants in the family Oleaceae (Kitching & Cadiou 2000; Tuttle 2007; Robinson et al. 2010); however, we were unable to find any report that it has been recorded as feeding on O. europaea. The rustic sphinx moth is recognized as a pollinator in the southeastern United States (Graham 2010). The only notable report of the rustic sphinx as a pest comes from Clavijo & Chacin (1992), who reported M. rustica as a pest of sesame in Venezuela. This is important to note for growers who are interested in using sesame as a cover crop or an attractant for beneficial insects near olive production areas in Florida. Initial reports from growers included photos of specimens collected from orchards and nurseries. Many growers elected to control mechani