James J. Becnel

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.

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.

Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on the importance of integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, can be the best option available 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, 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 (RRAe. 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 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. Author Summary: Aedes aegypti and Aedes albopictus can vector a variety of arboviruses that cause diseases and are thus a public health concern. Pyrethroid insecticide resistance is common in Aedes aegypti in many locations worldwide and can adversely affect vector control operations. However, the resistance status of these vectors in Florida is largely unreported and recent local transmission of dengue and Zika viruses has made this information critical for effective control operations. In this study, we showed that permethrin resistance and two common SNPs of the voltage gated sodium channel (V1016I and F1534C) previously associated with pyrethroid resistance were widely present in Florida Aedes aegypti strains. We also observed a strong correlation between the IICC genotype and RR as determined by topical application, which suggests, as have others, that kdr frequency may be a useful indicator of resistance in Aedes aegypti .

Aedes albopictus Skuse (Diptera: Culicidae) was tested for resistance to permethrin, bifenthrin, and malathion using Centers for Disease Control and Prevention (CDC) bottle bioassays and topical toxicology assays on adults and larval bioassays. Eggs were collected from 3 locations across St. Johns County, Florida, raised to the F3 generation and compared with an insecticide susceptible laboratory strain. Results from CDC bottle bioassays with permethrin indicate no significant differences between the 3 wild-type strains and the laboratory strain but suggest the possibility of resistance in 1 strain. Bottle bioassay results for malathion were inconclusive. Topical toxicological results for adults and bioassays for larvae showed a significant difference in permethrin resistance between the control strain and 1 of the wild-type strains. Results from this project indicate that insecticide susceptibility testing should be a regular part of mosquito surveillance programs. Upon detection of resistance, ... Resumen Aedes albopictus Skuse (Diptera: Culicidae) fue sometida a pruebas de resistencia a permetrina, bifentrina y malatión utilizando bioensayos con botellas del CDC (Centro de Control de Enfermedades y Prevención) y ensayos de toxicología en larvas y adultos. Se recolectaron huevos de tres localidades del condado de St. Johns, Florida, los cuales fueron criados hasta la generación F3 y fueron probardos contra una cepa de laboratorio susceptible al. Los resultados de los bioensayos con botellas de CDC no indican diferencias significativas entre las tres cepas de tipo salvaje y la cepa de laboratorio, pero sugieren la posibilidad de resistencia a la permetrina en una cepa. Los resultados del bioensayo de botella para el malatión fueron inconclusivos. Los resultados toxicológicos para adultos y larvas mostraron diferencias significativas entre la cepa control y una de las cepas silvestres para la resistencia a la permetrina. Los resultados de este proyecto indican que las pruebas de susceptibilidad a los...

Puerto Rico (PR) has a long history of vector-borne disease and insecticide-resistant Aedes aegypti (L.). Defining contributing mechanisms behind phenotypic resistance is critical for effective vector control intervention. However, previous studies from PR have each focused on only one mechanism of pyrethroid resistance. This study examines the contribution of P450-mediated enzymatic detoxification and sodium channel target site changes to the overall resistance phenotype of Ae. aegypti collected from San Juan, PR, in 2012. Screening of a panel of toxicants found broad resistance relative to the lab susceptible Orlando (ORL1952) strain. We identified significant resistance to representative Type I, Type II, and nonester pyrethroids, a sodium channel blocker, and a sodium channel blocking inhibitor, all of which interact with the sodium channel. Testing of fipronil, a chloride channel agonist, also showed low but significant levels of resistance. In contrast, the PR and ORL1952 strains were equally susceptible to chlorfenapyr, which has been suggested as an alternative public health insecticide. Molecular characterization of the strain indicated that two common sodium channel mutations were fixed in the population. Topical bioassay with piperonyl butoxide (PBO) indicated cytochrome P450-mediated detoxification accounts for approximately half of the resistance profile. Transcript expression screening of cytochrome P450s and glutathione-S-transferases identified the presence of overexpressed transcripts. This study of Puerto Rican Ae. aegypti with significant contributions from both genetic changes and enzymatic detoxification highlights the necessity of monitoring for resistance but also defining the multiple resistance mechanisms to inform effective mosquito control.

Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cell culture. An f homologue gene is absent in gammabaculoviruses. Here we characterized the putative F-homologue (Cuni-F), encoded by (ORF) 104 of Culex nigripalpus nucleopolyhedrovirus (CuniNPV), the only deltabaculovirus member. When expressed alone, this protein seems to locate on the cell surface and is able to induce cell–cell fusion. When expressed by an alphabaculovirus (Autographa california nucleopolyhedrovirus), it was found to be incorporated into BVs. Western blot analyses detected the uncleaved Cuni-F0 and the furin-cleaved F1 forms. Treatment of infected cells with tunicamycin showed that Cuni-F contains N-glycans. Mutagenesis analysis identified the canonical furin cleavage site 126RARR129 as being responsible for the cleavage of Cuni-F in insect cells. The collective evidence suggests that CuniNPV encodes a functional F protein.

Aedes aegypti L. is the major vector of the arboviruses responsible for dengue fever, one of the most devastating human diseases. Some bacterial, fungal and plant metabolites belonging to different chemical subgroups, including Amaryllidaceae alkaloids, anthracenes, azoxymethoxytetrahydropyrans, cytochalasans, 2,5-diketopiperazines, isochromanones, naphthoquinones, organic small acids and their methyl esters, sterols and terpenes including sesquiterpenes and diterpenes, were tested for their larvicidal and adulticidal activity against Ae. aegypti . Out of 23 compounds tested, gliotoxin exhibited mosquitocidal activity in both bioassays with an LC ₅₀ value of 0.0257 ± 0.001 µg/µL against 1 ^st instar Ae. aegypti and LD ₅₀ value of 2.79 ± 0.1197 µg/mosquito against adult female Ae. aegypti . 2-Methoxy-1,4-naphthoquinone and cytochalasin A showed LC ₅₀ values of 0.0851 ± 0.0012 µg/µL and 0.0854 ± 0.0019 µg/µL, respectively, against Ae. aegypti larvae. In adult bioassays, fusaric acid (LD ₅₀ = 0.8349 ± 0.0118 µg/mosquito), 3-nitropropionic acid (LD ₅₀ = 1.6641 ± 0.0494 µg/mosquito) and α-costic acid (LD ₅₀ = 2.547 ± 0.0835 µg/mosquito) exhibited adulticidal activity. Results from the current study confirm that compounds belonging to cytochalsin, diketopiperazine, naphthoquinone and low molecular weight organic acid groups are active and may stimulate further SAR investigations.