Characterization of Solenopsis invicta virus 4, a polycipivirus infecting the red imported fire ant Solenopsis invicta.

Solenopsis invicta virus 4 (SINV-4), a new polycipivirus, was characterized in the host in which it was discovered, Solenopsis invicta. SINV-4 was detected in the worker and larval stages of S. invicta, but not in pupae, male or female alates, or queens. The SINV-4 titer was highest in worker ants, with a mean of 1.14 × 107 ± 5.84 ×107 SINV-4 genome equivalents/ng RNA. Electron microscopic examination of negatively stained samples from particles purified from SINV-4-infected fire ant workers revealed isometric particles with a mean diameter of 47.3 ± 1.4 nm. The mean inter-colony SINV-4 infection rate among S. invicta worker ants was 45.8 ± 38.6 in Alachua County, Florida. In S. invicta collected in Argentina, SINV-4 was detected in 22% of 54 colonies surveyed from across the Formosa region. There did not appear to be any seasonality associated with the SINV-4 infection rate among S. invicta nests. SINV-4 was successfully transmitted to uninfected S. invicta colonies by feeding. Among three colonies of S. invicta inoculated with SINV-4, two retained the infection for up to 72 days. The replicative genome strand of SINV-4 was detected in 18% (n = 11) of SINV-4-infected S. invicta colonies. Among 33 ant species examined, the plus genome strand of SINV-4 was detected in undetermined species of Dorymyrmex and Pheidole, Cyphomyrmex rimosus, Monomorium pharaonis, Pheidole obscurithorax, Solenopsis geminata, Solenopsis richteri, Solenopsis xyloni, and Solenopsis invicta. However, the replicative (minus) genome strand was only detected in S. invicta. SINV-4 infection did not impact brood production or queen fecundity in S. invicta. The mean brood rating (63.3% ± 8.8) after 31 days for SINV-4-infected colonies was not statistically different from that of uninfected colonies (48.3 ± 25.5). At the end of the 31-day test period, mean egg production was not significantly different between SINV-4-infected S. invicta colonies (287.7 ± 45.2 eggs laid/24 hours) and uninfected control colonies (193.0 ± 43.6 eggs laid/24 hours).

Insecticidal Activity and Free Radical Scavenging Properties of Isolated Phytoconstituents from the Saudi Plant Nuxia oppositifolia (Hochst.).

Chromatographic purification of the alcoholic extract from the aerial parts of the Saudi plant Nuxia oppositifolia (Hochst.), Benth., resulted in five isolated phenolic compounds. Two flavones, hispidulin (1) and jaceosidin (2), and the phenylethanoid glycosides, verbascoside (3), isoverbascoside (4), and conandroside (5), were identified and their chemical structures were determined by spectroscopic analyses. The insecticidal activity of compounds 1 and 2, in addition to 11 compounds isolated in a previous research (6-16), was evaluated against the Yellow Fever mosquito, Aedes aegypti. Four compounds displayed adulticidal activity with LD50 values of 2-2.3 µg/mosquito. Free radical scavenging properties of the plant extracts and compounds (1-5) were evaluated by measuring the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate radical cation (ABTS•+) scavenging activity. All compounds exhibited notable activity, compared with the positive control, l-Ascorbic acid. This study suggests that N. oppositifolia could be a promising source of secondary metabolites, some with lethal adulticidal effect against Ae. aegypti.

Chemical Treatments for Insect Cell Differentiation: The Effects of 20-Hydroxyecdysone and Veratridine on Cultured Spodoptera frugiperda (Sf21) Insect Cell Ultrastructure.

Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, methoxyfenozide. Those cell growing processes resemble nerve cells, and the present study evaluates the ultrastructure of these cultures by transmission electron microscopy. Sf21 cells treated with 20-hydroxyecdysone (with or without veratridine amendment) and subjected to ultrastructural analysis had a similar somatic appearance to control cells, with slight changes in organelles and organization, such as a greater number of cytoplasmic vacuoles and mitochondrial granules. Finger-like projections were observed between control and treated cells. However, no structural markers of synaptic contacts (e.g., vesicles or synaptic thickenings) were observed in controls, 20-hydroxyecdysone, or 20-hydroxyecdysone + veratridine treated cells. It is concluded that additional agents would be required to induce functional synaptogenesis in Sf21 cells.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes.

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite's life cycle, and this protocol provides detailed guidance and visual aids for clarification.

A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae).

DNA metabarcoding offers new perspectives, especially with regard to the high-throughput identification and diagnostics of pathogens. Microsporidia are an example of widely distributed, opportunistic and pathogenic microorganisms in which molecular identification is important for both environmental research and clinical diagnostics. We have developed a method for parallel detection of both microsporidian infection and the host species. We designed new primer sets: one specific for the classical Microsporidia (targeting the hypervariable V5 region of small subunit [ssu] rDNA), and a second one targeting a shortened fragment of the COI gene (standard metazoan DNA-barcode); both markers are well suited for next generation sequencing. Analysis of the ssu rDNA data set representing 607 microsporidian species (120 genera) indicated that the V5 region enables identification of >98% species in the data set (596/607). To test the method, we used microsporidians that infect mosquitoes in natural populations. Using mini-COI data, all field-collected mosquitoes were unambiguously assigned to seven species; among them almost 60% of specimens were positive for at least 11 different microsporidian species, including a new microsporidian ssu rDNA sequence (Microsporidium sp. PL01). Phylogenetic analysis showed that this species belongs to one of the two main clades in the Terresporidia. We found a high rate of microsporidian co-infections (9.4%). The numbers of sequence reads for the operational taxonomic units suggest that the occurrence of Nosema spp. in co-infections could benefit them; however, this observation should be retested using a more intensive host sampling. Our results show that DNA barcoding is a rapid and cost-effective method for deciphering sample diversity in greater resolution, including the hidden biodiversity that may be overlooked using classical methodology.

Failure of Permethrin-Treated Military Uniforms to Protect Against a Laboratory-Maintained Knockdown-Resistant Strain of Aedes aegypti.

Military forces and the recreational industry rely on the repellent properties of permethrin-treated fabrics and N,N-diethyl-meta-toluamide (deet)-based lotions to provide protection from disease vectors and hematophagous organisms. Concerns regarding efficacy have been raised as pyrethroid resistance becomes more common and recent publications present contradictory conclusions. In this preliminary study, consenting volunteers were exposed to pyrethroid-susceptible and pyrethroid-resistant Aedes aegypti strains while wearing sleeves of untreated or permethrin-treated army uniform fabric as well as with untreated or deet-treated exposed forearms. Deet was nearly 100% effective against both susceptible and resistant strains. However, permethrin treatment provided no significant protection against the resistant Puerto Rico strain relative to untreated control sleeves. These results confirm that pyrethroid-resistant vectors can negate the efficacy of permethrin-treated uniforms. Additional testing with resistant field strains is needed to better understand the risk to service members.

A formal redefinition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) and reassignment of species based on molecular phylogenetics.

The microsporidian genera Nosema and Vairimorpha comprise a clade described from insects. Currently the genus Nosema is defined as having a dimorphic life cycle characterized by diplokaryotic stages and diplosporoblastic sporogony with two functionally and morphologically distinct spore types ("early" or "primary" and "environmental"). The Vairimorpha life cycle, in addition to a Nosema-type diplokaryotic sporogony, includes an octosporoblastic sporogony producing eight uninucleate spores (octospores) within a sporophorous vesicle. Molecular phylogeny, however, has clearly demonstrated that the genera Nosema and Vairimorpha, characterized by the absence or presence of uninucleate octospores, respectively, represent two polyphyletic taxa, and that octosporogony is turned on and off frequently within taxa, depending on environmental factors such as host species and rearing temperature. In addition, recent studies have shown that both branches of the Vairimorpha-Nosema clade contain species that are uninucleate throughout their life cycle. The SSU rRNA gene sequence data reveal two distinct clades, those closely related to Vairimorpha necatrix, the type species for the genus Vairimorpha, and those closely related to Nosema bombycis, the type species for the genus Nosema. Here, we redefine the two genera, giving priority to molecular character states over those observed at the developmental, structural or ultrastructural levels and present a list of revised species designations. Using this approach, a series of species are renamed (combination novum) and members of two genera, Rugispora and Oligosporidium, are reassigned to Vairimorpha because of their phylogenetic position. Moreover, the family Nosematidae is redefined and includes the genera Nosema and Vairimorpha comprising a monophyletic lineage of Microsporidia.

Insecticidal and repellent properties of novel trifluoromethylphenyl amides III.

The adulticidal, larvicidal, and repellent activity of 18 trifluoromethylphenyl amides (TFMPAs) was determined against Aedes aegypti mosquitoes. The compounds studied are the third generation designed from active structures of the previous two generations. N-(3,5-Bis(trifluoromethyl)phenyl)-2-chloroacetamide (8f) and N-(3,5-bis(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (8h) were most active against 1st stage Ae. aegypti larvae with LC50 values of 125 and 2.53 µM; for comparative purposes, the published LC50 for fipronil is 0.014 µM. Compound 8h was the most toxic against adult female Ae. aegypti with an LD50 = 2.12 nmol/mg, followed by 8f, and N-(3,5-bis(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide (8g) with LD50 values of 4.27 and 4.73 nmol/mg, respectively, although these compounds were significantly less toxic than fipronil against adult female Ae. aegypti. Compounds N-(2-(trifluoromethyl)phenyl)butyramide (9c), N-(2-(trifluoromethyl)phenyl)pentanamide (9d) and N-(2-(trifluoromethyl)phenyl)hex-5-enamide (9e) were the best repellents for female Ae. aegypti, with minimum effective dosages (MEDs) of 0.026, 0.052, and 0.091 µmol/cm2, respectively, compared to DEET at 0.052 µmol/cm2. Out of 52 TFMPAs (total number of compounds from three generations of this research) compound 9c was the most active repellent along with two synthesized in our previous studies, 2-chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) and 2,2,2-trifluoro-N-(2-(trifluoromethyl)phenyl)acetamide (4c).

Chemical composition of the essential oil and n-hexane extract of Stachys tmolea subsp. Tmolea Boiss., an endemic species of Turkey, and their mosquitocidal activity against dengue vector Aesdes aegypti.

Stachys tmolea subsp. tmolea Boiss. is endemic to Turkey and is a species of the genus Stachys L. which is one of the largest genera of the family Lamiaceae with about 300 species. The aims of this study were to examine the chemical composition of the essential oil and n-hexane extract of S. tmolea subsp. tmolea as natural sources of insecticidal activity against the dengue vector, Aedes aegypti. Analysis of the essential oil by GC-FID and gas chromatography-mass spectrometry (GC-MS) systems identified hexahydrofarnesyl acetone (15%), viridiflorol (10%), hexadecanoic acid (7%) and 9-geranyl-p-cymene (6%) as major components. The volatile components of the n-hexane extract were extracted using headspace solid-phase microextraction (HS-SPME) and were analyzed using GC-MS. The principal constituents were 3,4-dimethyl decane (16%), 3-methyl-3-pentanol (15%), 2-methyl-2-pentanol (12%), 1,4-bis (1,1-dimethylethyl) benzene (12%), heptanal (10%), acetic acid (6%) and decane (4%). Bioassay of the n-hexane extract, at 5 µg/mosquito, produced 90% mortality against adult Ae. aegypti while the S. tmolea essential oil demonstrated 13% mortality. No larvicidal activity was observed both in essential oil and n-hexane extract. Further studies are needed to assess the adulticidal activity of the responsible compounds in the crude extract.

Assessment of selected Saudi and Yemeni plants for mosquitocidal activities against the yellow fever mosquito Aedes aegypti.

As part of our continuing investigation for interesting biological activities of native medicinal plants, thirty-nine plants, obtained from diverse areas in Saudi Arabia and Yemen, were screened for insecticidal activity against yellow fever mosquito Aedes aegypti (L.). Out of the 57 organic extracts, Saussurea lappa, Ocimum tenuiflorum, Taraxacum officinale, Nigella sativa, and Hyssopus officinalis exhibited over 80% mortality against adult female Ae. aegypti at 5 µg/mosquito. In the larvicidal bioassay, the petroleum ether extract of Aloe perryi flowers showed 100% mortality at 31.25 ppm against 1st instar Ae. aegypti larvae. The ethanol extract of Saussurea lappa roots was the second most active displaying 100% mortality at 125 and 62.5 ppm. Polar active extracts were processed using LC-MS/MS to identify bioactive compounds. The apolar A. perryi flower extract was analyzed by headspace SPME-GC/MS analysis. Careful examination of the mass spectra and detailed interpretation of the fragmentation pattern allowed the identification of various biologically active secondary metabolites. Some compounds such as caffeic and quinic acid and their glycosides were detected in most of the analyzed fractions. Additionally, luteolin, luteolin glucoside, luteolin glucuronide and diglucuronide were also identified as bioactive compounds in several HPLC fractions. The volatile ketone, 6-methyl-5-hepten-2-one was identified from A. perryi petroleum ether fraction as a major compound.

Overexpression of a glutathione S-transferase (Mdgst) and a galactosyltransferase-like gene (Mdgt1) is responsible for imidacloprid resistance in house flies.

BACKGROUND: Neonicotinoids are the largest class of insecticides and are used for control of house fly populations at animal production facilities throughout the world. There have been several reports of neonicotinoid resistance in house fly populations, but identification of the factors involved in resistance has proven challenging. The KS8S3 population of house flies is highly resistant to the neonicotinoid insecticide imidacloprid due to two factors: one on chromosome 3 and one on chromosome 4. RESULTS: A comparative transcriptomic approach was used, followed by validation using transgenic Drosophila melanogaster to investigate the genes responsible for resistance in the KS8S3 strain. Overexpression of a microsomal glutathione S-transferase (Mdgst) was identified as the factor likely responsible for resistance on chromosome 3. Resistance on chromosome 4 appears to be due to an unidentified trans-regulatory gene which causes overexpression of a galactosyltransferase-like gene (Mdgt1). No single nucleotide polymorphisms were found that could be associated with imidacloprid resistance. CONCLUSION: Identification of the underlying processes that cause imidacloprid resistance is an important first step towards the development of novel and sensitive resistance monitoring techniques. It will be valuable to investigate if overexpression of Mdgst and Mdgt1 are found in other imidacloprid resistant populations. © 2018 Society of Chemical Industry.

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies.

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds. Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts. This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

Quantification of permethrin resistance and kdr alleles in Florida strains of Aedes aegypti (L.) and Aedes albopictus (Skuse).

Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR<1.6) even when collected from the same containers producing resistant Ae. aegypti. Characterization of two sodium channel kdr alleles associated with pyrethroid-resistance showed widespread distribution in 62 strains of Ae. aegypti. The 1534 phenylalanine to cysteine (F1534C) single nucleotide polymorphism SNP was fixed or nearly fixed in all strains regardless of RR. We observed much more variation in the 1016 valine to isoleucine (V1016I) allele and observed that an increasing frequency of the homozygous V1016I allele correlates strongly with increased RR (Pearson corr = 0.905). In agreement with previous studies, we observed a very low frequency of three kdr genotypes, IIFF, VIFF, and IIFC. In this study, we provide a statewide examination of pyrethroid resistance, and demonstrate that permethrin resistance and the genetic markers for resistance are widely present in FL Ae. aegypti. Resistance testing should be included in an effective management program.

ICTV Virus Taxonomy Profile: Baculoviridae.

The family Baculoviridae comprises large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. The virions consist of enveloped, rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15-5 µm. The occlusion bodies consist of a matrix composed of a single viral protein expressed at high levels during infection. Members of this family infect exclusively larvae of the insect orders Lepidoptera, Hymenoptera and Diptera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Baculoviridae, which is available at www.ictv.global/report/baculoviridae.

Error-prone protein synthesis in parasites with the smallest eukaryotic genome.

Microsporidia are parasitic fungi-like organisms that invade the interior of living cells and cause chronic disorders in a broad range of animals, including humans. These pathogens have the tiniest known genomes among eukaryotic species, for which they serve as a model for exploring the phenomenon of genome reduction in obligate intracellular parasites. Here we report a case study to show an apparent effect of overall genome reduction on the primary structure and activity of aminoacyl-tRNA synthetases, indispensable cellular proteins required for protein synthesis. We find that most microsporidian synthetases lack regulatory and eukaryote-specific appended domains and have a high degree of sequence variability in tRNA-binding and catalytic domains. In one synthetase, LeuRS, an apparent sequence degeneration annihilates the editing domain, a catalytic center responsible for the accurate selection of leucine for protein synthesis. Unlike accurate LeuRS synthetases from other eukaryotic species, microsporidian LeuRS is error-prone: apart from leucine, it occasionally uses its near-cognate substrates, such as norvaline, isoleucine, valine, and methionine. Mass spectrometry analysis of the microsporidium Vavraia culicis proteome reveals that nearly 6% of leucine residues are erroneously replaced by other amino acids. This remarkably high frequency of mistranslation is not limited to leucine codons and appears to be a general property of protein synthesis in microsporidian parasites. Taken together, our findings reveal that the microsporidian protein synthesis machinery is editing-deficient, and that the proteome of microsporidian parasites is more diverse than would be anticipated based on their genome sequences.

Mosquitocidal Activity of a Naturally Occurring Isochroman and Synthetic Analogs from the Plant Pathogenic Fungus, Diaporthe eres Against Aedes aegypti (Diptera: Culicidae).

The culture filtrate of a plant pathogenic fungus that infects English ivy (Hedera helix L., Araliaceae) was investigated for mosquitocidal constituents by bioassay-guided isolation. The fungus responsible for pathogenic effects on the plant H. helix has been identified as Diaporthe eres Nitschke by molecular techniques. The mosquito adulticidal constituent in the culture filtrate was identified as 3,4-dihydro-8-hydroxy-3,5-dimethylisocoumarin (1) by spectroscopic techniques. Laboratory bioassays showed that (1) had larvicidal activity against permethrin-susceptible and -resistant Aedes aegypti strains. This compound was not active as an adulticide when tested by topical bioassay. Several analogs of (1) were synthesized and had better mosquitocidal activities than the naturally occurring (1) constituent.

Reduction in Musca domestica fecundity by dsRNA-mediated gene knockdown.

House flies (Musca domestica) are worldwide agricultural pests with estimated control costs at $375 million annually in the U.S. Non-target effects and widespread resistance challenge the efficacy of traditional chemical control. Double stranded RNA (dsRNA) has been suggested as a biopesticide for M. domestica but a phenotypic response due to the induction of the RNAi pathway has not been demonstrated in adults. In this study female house flies were injected with dsRNA targeting actin-5C or ribosomal protein (RP) transcripts RPL26 and RPS6. Ovaries showed highly reduced provisioning and clutch reductions of 94-99% in RP dsRNA treated flies but not in actin-5C or GFP treated flies. Gene expression levels were significantly and specifically reduced in dsRNA injected groups but remained unchanged in the control dsGFP treated group. Furthermore, injections with an Aedes aegypti conspecific dsRNA designed against RPS6 did not impact fecundity, demonstrating species specificity of the RNAi response. Analysis of M. domestica tissues following RPS6 dsRNA injection showed significant reduction of transcript levels in the head, thorax, and abdomen but increased expression in ovarian tissues. This study demonstrates that exogenous dsRNA is specifically effective and has potential efficacy as a highly specific biocontrol intervention in adult house flies. Further work is required to develop effective methods for delivery of dsRNA to adult flies.

Synthesis and structure-activity relationships of carbohydrazides and 1,3,4-oxadiazole derivatives bearing an imidazolidine moiety against the yellow fever and dengue vector, Aedes aegypti.

BACKGROUND: 1,3,4-Oxadiazole and imidazolidine rings are important heterocyclic compounds exhibiting a variety of biological activities. In this study, novel compounds with oxadiazole and imidazolidine rings were synthesized from 3-(methylsulfonyl)-2-oxoimidazolidine-1-carbonyl chloride and screened for insecticidal activities. The proposed structures of the 17 synthesized compounds were confirmed using elemental analysis, infrared (IR), proton nuclear magnetic resonance (1 H-NMR), and mass spectroscopy. RESULTS: None of the compounds showed larvicidal activity at the tested concentrations against first-instar Aedes aegypti larvae. However, nine compounds exhibited promising adulticidal activity, with mortality rates of ≥80% at 5 µg per mosquito. Further dose-response bioassays were undertaken to determine median lethal dose (LD50 ) values. Compounds 1, 2b, 2c, 2d, 2 g, 3b, 3c, 3 g, and 3 h were effective, with typical LD50 values of about 5 - 10 µg per mosquito against female Ae. aegypti. Compounds 2c (bearing a nitro group on the aromatic ring; LD50 = 2.80 ± 0.54 µg per mosquito) and 3 h (double halogen groups at 2,4 position on the phenyl ring; LD50 = 2.80 ± 0.54 µg per mosquito) were the most promising compounds. CONCLUSION: Preliminary mode of action studies failed to show consistent evidence of either neurotoxic or mitochondria-directed effects. Further chemical synthesis within this series may lead to the development of new effective insecticides. © 2017 Society of Chemical Industry.

Nosema maddoxi sp. nov. (Microsporidia, Nosematidae), a Widespread Pathogen of the Green Stink Bug Chinavia hilaris (Say) and the Brown Marmorated Stink Bug Halyomorpha halys (Stål).

We describe a unique microsporidian species that infects the green stink bug, Chinavia hilaris; the brown marmorated stink bug, Halyomorpha halys; the brown stink bug, Euschistus servus; and the dusky stink bug, Euschistus tristigmus. All life stages are unikaryotic, but analysis of the consensus small subunit region of the ribosomal gene places this microsporidium in the genus Nosema, which historically has been characterized by diplokaryotic life stages. It is also characterized by having the reversed arrangement of the ribosomal gene (LSU -ITS- SSU) found in species within the "true Nosema" clade. This microsporidium is apparently Holarctic in distribution. It is present in H. halys both where it is native in Asia and where it is invasive in North America, as well as in samples of North American native C. hilaris collected prior to the introduction of H. halys from Asia. Prevalence in H. halys from mid-Atlantic, North America in 2015-2016 ranged from 0.0% to 28.3%, while prevalence in C. hilaris collected in Illinois in 1970-1972 ranged from 14.3% to 58.8%. Oral infectivity and pathogenicity were confirmed in H. halys and C. hilaris. Morphological, ultrastructural, and ecological features of the microsporidium, together with a molecular phylogeny, establish a new species named Nosema maddoxi sp. nov.

Insecticidal and repellent properties of novel trifluoromethylphenyl amides II.

This project focused on the design, synthesis, and testing of trifluoromethylphenyl amides (TFMPAs) as potential mosquitocides and repellents. Fourteen compounds were evaluated for toxicity against larvae and adults of Aedes aegypti. Several compounds were toxic against Aedes aegypti (larval, adult and feeding bioassays) and Drosophila melanogaster (glass-surface contact assay), but were much less toxic than fipronil, with toxicity ratios ranging from 100-fold in the larval assay to 100,000-fold for topical application to adult insects. In repellency bioassays to determine minimum effective dosage (MED), compound N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (7b) repelled Ae. aegypti females at lower concentration, 0.017 (±0.006) µmol/cm2, than N, N-diethyl-meta-toluamide (DEET) 0.026 (±0.005) µmol/cm2. 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) performed better than DEET against two species of mosquitoes: it repelled Ae. aegypti females at 0.013 (±0.006) µmol/cm2 and Anopheles gambiae females (in a warm body repellent assay), at a standard exposure of 2 nmol/cm2. These studies revealed novel active structures that could further lead to compounds with better repellent activity.