Approaches based on organismal DNA found in the environment (eDNA) have become increasingly utilized for ecological studies and biodiversity inventories as an alternative to traditional field survey ...methods. Such DNA-based techniques have largely been used to establish the presence of free-living organisms, but have much potential for detecting and quantifying infectious agents in the environment, which is necessary to evaluate disease risk. We developed an eDNA method to examine the distribution and abundance of the trematode
Ribeiroia ondatrae
, a pathogenic parasite known to cause malformations in North American amphibians. In addition to comparing this eDNA approach to classical host necropsy, we examined the detectability of
R. ondatrae
in water samples subject to different degradation conditions (time and temperature). Our test exhibited high specificity and sensitivity to
R. ondatrae
, capable of detecting as little as 14 fg (femtograms) of this parasite's DNA (1/2500th of a single infectious stage) from field water samples. Compared to our results from amphibian host necropsy, quantitative PCR was ~90% concordant with respect to
R. ondatrae
detection from 15 field sites and was also a significant predictor of host infection abundance. DNA was still detectable in lab samples after 21 days at 25°C, indicating that our method is robust to field conditions. By comparing the advantages and disadvantages of eDNA vs. traditional survey methods for determining pathogen presence and abundance in the field, we found that the lower cost and effort associated with eDNA approaches provide many advantages. The development of alternative tools is critical for disease ecology, as wildlife management and conservation efforts require reliable establishment and monitoring of pathogens.
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BFBNIB, FZAB, GIS, IJS, INZLJ, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZRSKP
Estimated direct and indirect losses of grains and grain-based products caused by stored-product insects range from about 10% in temperate regions to almost 50% in humid tropical areas. Pest ...management strategies in bulk grains include the use of fumigants such as phosphine and sulfuryl fluoride, and grain protectants, which are sprayed directly on commodities as they are loaded into storage. Fumigants, aerosols, and contact sprays are also used as structural treatments in mills, processing plants, and food warehouses. Some older organophosphate protectants and contact sprays have been phased out worldwide and have been replaced by safer insecticides, including pyrethroids and insect growth regulators (IGRs). These IGRs include juvenile hormone analogues (JHAs), ecdysteroids and chitin synthesis inhibitors, and are considered safe due to their insect specificity. Methoprene is the JHA that has been used most extensively in stored-product pest management. The formulations of methoprene originally introduced into the stored-product market in the 1980s contained the racemic mixture with both R- and S- forms, but now only the purified S-methoprene isomer is used. Methoprene has received broad attention and has been tested over decades for its direct lethal effects, but many recent studies focus more on sub-lethal effects. Although methoprene has been used for more than four decades, there has not been a recent and comprehensive synopsis or review of this IGR on stored-product insects. This review addresses the history and present use of methoprene with special emphasis on stored-product protection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, ...and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double‐stranded (ds) RNA construct, DWV‐dsRNA, which is specific to DWV in DWV‐inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV‐dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein‐dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV‐dsRNA did not affect larval survival rates, which suggests that dsRNA is non‐toxic to larvae. Feeding adult workers with DWV‐dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Carbon dioxide (CO2) is an important long‐range chemosensory cue used by blood‐feeding female mosquitoes to find their hosts. The CO2 receptor in Drosophila melanogaster was previously determined to ...be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)‐mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO2 sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi‐mediated knockdown of AaGr2 expression had no impact on CO2 reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO2 receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO2 receptor in different species are discussed. The functional identification of the CO2 receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
The
maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to
mariner and
Tc transposons, with a distribution thus far limited to a few ...invertebrate species. In the nematode
Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in
C. elegans and that the
CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of
C. elegans is comparable to that of the
Tc1 transposon. However, unlike
Tc transposons in mutator strains of
C. elegans,
maT transposons do not exhibit increased frequencies of mobility, suggesting that
maT is not regulated by the same factors that control
Tc activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the
C. elegans genome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 ...were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few ...invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
We examined the relative efficiency of microinjection, electroporation and particle bombardment for introducing DNA into the embryos of the Kuruma prawn,
Penaeus japonicus. The amount of DNA that ...could be delivered into one- to four-celled embryos and subsequently recovered was examined using plasmids with selectable antibiotic resistance. Microinjection proved to be the most reliable technique. The mean value for plasmid recovery from injected embryos ranged from 225 to 243 plasmids per embryo. Electroporation was effective in delivering small, but measurable amounts of DNA into embryos with values ranging from 0.5 to 0.9 plasmids per embryo. Bombardment failed to introduce any significant amount of DNA into viable embryos.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Malathion resistance in a strain of Culex tarsalis mosquitoes is due primarily to the activity of a malathion carboxylesterase (MCE). The resistant strain was 150 times more resistant to malathion ...than the susceptible strain and was weakly resistant to malaoxon and carbaryl, but not to any other insecticide tested. The phenotype could be reversed with the carboxylesterase inhibitor triphenylphosphate, but no synergism was observed with either the phosphatase or polysubstrate monooxygenase inhibitors, NaF and piperonyl butoxide. MCE is expressed throughout development and is most concentrated in the gut tissues of the larvae. Subcellular fractionation indicated that MCE was localized primarily in the mitochondria of resistant insects and the cytoplasm of susceptible insects. The enzyme was purified to homogeneity from both strains, and has a molecular weight of 59,000. However, chromatofocusing indicated that resistant insects have two MCEs with pIs of 6.8 and 6.2, while susceptible insects possessed only one MCE with a pI of 6.8. The MCE unique to the resistant strain hydrolysed malathion 18 times faster than the MCE common to both strains, suggesting that malathion resistance in C. tarsalis is due to the presence of a qualitatively different esterase in the resistant strain.
Members of the hAT transposable element family are mobile in non‐host insect species and have been used as transformation vectors in some of these species. We report that the Queensland fruit fly, ...Bactrocera tryoni, contains at least two types of insect hAT elements called Homer and a Homer‐like element (HLE). The Homer element is 3789 bp in size and contains 12‐bp imperfect inverted terminal repeats. The Homer element contains a long open reading frame (ORF) that encodes a putative transposase. Three different copies of this long ORF were recovered from the B. tryoni genome and, upon transcription and translation in an in vitro system, all produced transposase. The HLE is an incomplete element since no 3′ inverted terminal repeat (ITR) was found. Homer and the HLE are as related to one another as either is to the other insect hAT elements such as Hermes, hobo, hermit and hopper. The structure and distribution of these two Homer elements is described.
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