Assessments of Temporal Variations in Haplotypes of 'Candidatus Liberibacter solanacearum' and Its Vector, the Potato Psyllid, in Potato Fields and Native Vegetation.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), had been known for nearly a century to cause psyllid yellows of solanaceous crops. However, it has only been a decade since the insect was discovered to transmit the bacterium 'Candidatus Liberibacter solanacearum' (Lso), which putatively causes potato zebra chip disease. This project was initiated to quantify temporal incidences of haplotypes of the psyllid (Central, Southwestern, and Western) and Lso (A, B) in potato fields and in native vegetation. Psyllids were collected from native vegetation in Texas (2011-2014), and from potato fields in Texas and New Mexico (2014-2017). Psyllids were tested for Lso and haplotypes of both psyllid and Lso. In Texas, the Central psyllid haplotype was overwhelmingly dominant both in potato fields and in native vegetation regardless of location and time of collection. However, in New Mexico potato fields, although the Southwestern haplotype was overall dominant, the ratios of individual haplotypes varied among years and within a season. The Southwestern psyllid haplotype was greater in incidence than the Central early but declined later in the season in each of the 4 yr, while the Central haplotype was low in incidence early but increased over time. Lso was detected in all three psyllid haplotypes representing the first report in Southwestern psyllid haplotype. In Texas, Lso haplotype A was more frequently detected than B, but in New Mexico the incidence of positive psyllids was not high enough to make definitive conclusions regarding predominant Lso haplotype.

Importance of Transplanted Onions Contributing to Late-Season Iris yellow spot virus Epidemics in New York.

Iris yellow spot virus (IYSV) is an economically significant tospovirus of onion transmitted by onion thrips (Thrips tabaci Lindeman). IYSV epidemics in onion fields are common in New York; however, the role of various habitats contributing to viruliferous onion thrips populations and IYSV epidemics is not known. In a 2-year field study in New York, the abundance of dispersing onion thrips, including those determined to be viruliferous via reverse-transcriptase polymerase chain reaction, was recorded in habitats known to harbor both IYSV and its vector. Results showed that viruliferous thrips were encountered in all habitats; however, transplanted onion sites accounted for 49 to 51% of the total estimated numbers of viruliferous thrips. During early to midseason, transplanted onion sites had 9 to 11 times more viruliferous thrips than the other habitats. These results indicate that transplanted onion fields are the most important habitat for generating IYSV epidemics in all onion fields (transplanted and direct-seeded) in New York. Our findings suggest that onion growers should control onion thrips in transplanted fields early in the season to minimize risk of IYSV epidemics later in the season.

Driven to extinction? The ethics of eradicating mosquitoes with gene-drive technologies.

Mosquito-borne diseases represent a significant global disease burden, and recent outbreaks of such diseases have led to calls to reduce mosquito populations. Furthermore, advances in 'gene-drive' technology have raised the prospect of eradicating certain species of mosquito via genetic modification. This technology has attracted a great deal of media attention, and the idea of using gene-drive technology to eradicate mosquitoes has been met with criticism in the public domain. In this paper, I shall dispel two moral objections that have been raised in the public domain against the use of gene-drive technologies to eradicate mosquitoes. The first objection invokes the concept of the 'sanctity of life' in order to claim that we should not drive an animal to extinction. In response, I follow Peter Singer in raising doubts about general appeals to the sanctity of life, and argue that neither individual mosquitoes nor mosquitoes species considered holistically are appropriately described as bearing a significant degree of moral status. The second objection claims that seeking to eradicate mosquitoes amounts to displaying unacceptable degrees of hubris. Although I argue that this objection also fails, I conclude by claiming that it raises the important point that we need to acquire more empirical data about, inter alia, the likely effects of mosquito eradication on the ecosystem, and the likelihood of gene-drive technology successfully eradicating the intended mosquito species, in order to adequately inform our moral analysis of gene-drive technologies in this context.

Characterization and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies.

Mosquitoes (Diptera: Culicidae) serve as important vectors for a wide number of parasites and pathogens of huge medical and veterinary importance. Aedes aegypti is a primary dengue vector in tropical and subtropical urban areas. There is an urgent need to develop eco-friendly mosquitocides. In this study, silver nanoparticles (AgNP) were biosynthesized using neem cake, a by-product of the neem oil extraction from the seed kernels of Azadirachta indica. AgNP were characterized using a variety of biophysical methods, including UV-vis spectrophotometry, FTIR, SEM, EDX, and XRD analyses. Furthermore, the neem cake extract and the biosynthesized AgNP were tested for acute toxicity against larvae and pupae of the dengue vector Ae. aegypti. LC50 values achieved by the neem cake extract ranged from 106.53 (larva I) to 235.36 ppm (pupa), while AgNP LC50 ranged from 3.969 (larva I) to 8.308 ppm (pupa). In standard laboratory conditions, the predation efficiency of a Carassius auratus per day was 7.9 (larva II) and 5.5 individuals (larva III). Post-treatment with sub-lethal doses of AgNP, the predation efficiency was boosted to 9.2 (larva II) and 8.1 individuals (larva III). The genotoxic effect of AgNP was studied on C. auratus using the comet assay and micronucleus frequency test. DNA damage was evaluated on peripheral erythrocytes sampled at different time intervals from the treatment; experiments showed no significant damages at doses below 12 ppm. Overall, this research pointed out that neem cake-fabricated AgNP are easy to produce, stable over time, and can be employed at low dosages to reduce populations of dengue vectors, with moderate detrimental effects on non-target mosquito natural enemies.

Genetic and host-associated differentiation within Thrips tabaci Lindeman (Thysanoptera: Thripidae) and its links to Tomato spotted wilt virus-vector competence.

Of eight thelytokous populations of onion thrips (Thrips tabaci) collected from potato (three populations), onion (four) or Chrysanthemum (one) hosts from various regions of Australia, only those from potato were capable of transmitting Tomato spotted wilt virus (TSWV) in controlled transmission experiments. Genetic differentiation of seven of these eight populations, and nine others not tested for TSWV vector competence, was examined by comparison of the DNA sequences of mitochondrial cytochrome oxidase subunit 1 (COI) gene. All Australian populations of T. tabaci grouped within the European 'L2' clade of Brunner et al. (2004). Within this clade the seven populations from potato, the three from onion, and the four from other hosts (Chrysanthemum, Impatiens, lucerne, blackberry nightshade) clustered as three distinct sub-groupings characterised by source host. Geographical source of thrips populations had no influence on genetic diversity. These results link genetic differentiation of thelytokous T. tabaci to source host and to TSWV vector capacity for the first time.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties.

BACKGROUND: Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. METHODS: A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. RESULTS: A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. CONCLUSIONS: Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.

Transmission blocking activity of a standardized neem (Azadirachta indica) seed extract on the rodent malaria parasite Plasmodium berghei in its vector Anopheles stephensi.

BACKGROUND: The wide use of gametocytocidal artemisinin-based combination therapy (ACT) lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid) abundant in neem (Azadirachta indica, Meliaceae) seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal(R), an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi. METHODS: Anopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal(R) on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined. RESULTS: NeemAzal completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications) did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post-zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post-zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality of the microtubule organizing centres and with the assembly of cytoskeletal microtubules, which are both fundamental processes in Plasmodium gametogenesis and ookinete formation. CONCLUSIONS: This work demonstrated in vivo transmission blocking activity of an azadirachtin-enriched neem seed extract at an azadirachtin dose compatible with 'druggability' requisites. These results and evidence of anti-plasmodial activity of neem products accumulated over the last years encourage to convey neem compounds into the drug discovery & development pipeline and to evaluate their potential for the design of novel or improved transmission-blocking remedies.

The role of research in molecular entomology in the fight against malaria vectors.

The text summarizes the principal current fields of investigation and the recent achievements of the research groups presently contributing to the Molecular Entomology Cluster of the Italian Malaria Network. Particular emphasis is given to the researches with a more direct impact on the fight against malaria vectors.

Monitoring pyrethroid insecticide resistance in major malaria vector Anopheles culicifacies: comparison of molecular tools and conventional susceptibility test.

BACKGROUND: Anopheles culicifacies is a main malaria vector in southeastern part of Iran, bordring Afghanistan and Pakistan. So far, resistance to DDT, dieldrin, malathion and partial tolerance to pyrethroids has been reported in An. stephensi, but nothing confirmed on resistance status of An. culicifacies in Iran. METHODS: In current study, along with WHO routine susceptibility test with DDT (4%), dieldrin (0.4%), malathion (5%), permethrin (0.25%), lambadacyhalothrin (0.1%), and deltamethrin 0.025, we cloned and sequenced segment VI of domain II (SII6) in voltage-gated sodium channel (vgsc) gene of An. culicifacies specimens collected in Sistan and Baluchistan province (Iran). RESULTS: A 221-bp amplified fragment showed 91% and 93% similarity with exon I and exon II of An. gambiae. The size of intron II in An. culicifacies is 62 bp, while in An. gambiae is 57 bp. The major difference within An. culicifacies specimens and also with An. gambiae is in position 29 of exon I, which led to substitution of Leu to His amino acid. CONCLUSION: This data will act as first report on partial sequence of vgsc gene and its polymorphism in An. culicifacies. A Leu to His amino acid substitution detected upstream the formerly known knockdown resistance (kdr) mutation site could be an indication for other possible mutations related to insecticide resistance. However, the result of WHO susceptibility test carried out in Baluchistan of Iran revealed a level of tolerance to DDT and dieldrin, but almost complete susceptibility to pyrethroids in An. culicifacies. We postulate that the molecular diagnostic tool developed for detection and identification of kdr-related mutations in An. culicifacies, could be useful in monitoring insecticide resistance in Iran and neighbouring countries such as Pakistan and Afghanistan. A phylogenetic tree also constructed based on the sequence of exon I and II, which readily separated An. culicifacies populations from An. stephensi, An. fluviatilis and An. gambiae.

Experimental hut evaluation of bednets treated with an organophosphate (chlorpyrifos-methyl) or a pyrethroid (lambdacyhalothrin) alone and in combination against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes.

BACKGROUND: Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments. METHODS: Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1R). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments. RESULTS AND CONCLUSION: All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1R and Ace.1S genes did not differ significantly from mosquitoes that carried only Ace.1S genes on any of the treated nets, indicating that the Ace.1R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.

Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae).

Numerous Culicoides spp. are important vectors of livestock or human disease pathogens. Transcriptome information from midguts and salivary glands of adult female Culicoides sonorensis provides new insight into vector biology. Of 1719 expressed sequence tags (ESTs) from adult serum-fed female midguts harvested within 5 h of feeding, twenty-eight clusters of serine proteases were derived. Four clusters encode putative iron binding proteins (FER1, FERL, PXDL1, PXDL2), and two clusters encode metalloendopeptidases (MDP6C, MDP6D) that probably function in bloodmeal catabolism. In addition, a diverse variety of housekeeping cDNAs were identified. Selected midgut protease transcripts were analysed by quantitative real-time PCR (q-PCR): TRY1_115 and MDP6C mRNAs were induced in adult female midguts upon feeding, whereas TRY1_156 and CHYM1 were abundant in midguts both before and immediately after feeding. Of 708 salivary gland ESTs analysed, clusters representing two new classes of protein families were identified: a new class of D7 proteins and a new class of Kunitz-type protease inhibitors. Additional cDNAs representing putative immunomodulatory proteins were also identified: 5' nucleotidases, antigen 5-related proteins, a hyaluronidase, a platelet-activating factor acetylhydrolase, mucins and several immune response cDNAs. Analysis by q-PCR showed that all D7 and Kunitz domain transcripts tested were highly enriched in female heads compared with other tissues and were generally absent from males. The mRNAs of two additional protease inhibitors, TFPI1 and TFPI2, were detected in salivary glands of paraffin-embedded females by in situ hybridization.

Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly Bemisia tabaci.

To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) - a whitefly-transmitted geminivirus - was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.

[Recent observations on the sensitivity to pyrethroids and DDT of Anopheles arabiensis and Anopheles funestus in the central Highlands of Madagascar; preliminary results on the absence of the kdr mutation in An. arabiensis]. / Données récentes sur la sensibilité d'Anopheles arabiensis et d'Anopheles funestus aux pyréthrinoïdes et au DDT sur les Hautes Terres Centrales de Madagascar--résultats préliminaires montrant une absence de la mutation kdr chez An. arabiensis.

Anopheles arabiensis and Anopheles funestus are the principal vectors of malaria on the central highlands of Madagascar. These two species of mosquito are directly or indirectly the targets of indoor insecticide spread. The survey of the susceptibility of these two vectors to insecticides is essential specifying for the anti-vectorial current directives and for the future programs. This paper describes: --the recent tests of susceptibility and the study of the knock-down effect of four pyrethroids (deltamethrin 0.50%, permethrin 0.250%, alpha-cypermethrin 0.025%, cyfluthrin 0.150%) and DDT 4% on An. arabiensis collected from December 2002 to May 2003 in three villages on the district of Tsiroanomandidy and in Alasora, a rural area near the capital, Antananarivo. --the susceptibility tests of An. funestus realised in Morafeno, against cyfluthrin 0.150%, deltaméthrin 0.050%, lambda-cyhalothrin 0.050% and DDT 4%. --the preliminary study of the kdr gene mutation, which might account for the pyrethrinoid and DDT resistance, of An. arabiensis in Alasora. The data indicate a decrease in the efficiency of alpha-cypermethrin 0.025% (Tkd99 = 21 mn) and of DDT 4% (Tkd99 = 191.5 mn) on An. arabiensis in Analamiranga. Nevertheless, the effectiveness of permethrin 0.250% has been notified. In Soanierana, alpha-cypermethrin 0.025% was effective against An. arabiensis, whilst the ineffectiveness of the DDT 4% (Tkd99 = 116 mn) in Andranonahoatra was observed. In Alasora, a fall in the effectiveness of alpha-cypermethrin 0.025% (Tkd99 = 21 mn) and the resistance to DDT 4% (Tkd99 = 6894 mn) was noted. No kdr mutation was detected on the kd gene of An. arabiensis resistant to the alpha-cypermethrin 0.025% and to DDT 4%, collected in Alasora. An. funestus collected in the district of Miandrivazo is susceptible to pyrethroids and to DDT. To conclude, in the Malagasy Central Highlands, An. arabiensis is ordinary sensitive to pyrethroid and poorly sensitive, or even resistant to DDT (as observed in Antananarivo plain). However, An. funestus remains sensitive to all tested insecticides; this observation is crucial because this anopheles vector is the first target of the campaigns of indoor spraying insecticides.

Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vector Anopheles gambiae.

A cDNA clone for prophenoloxidase was isolated from the most important human malaria vector, Anopheles gambiae. The clone encoded a polypeptide of 79341 Da that contains the two copper binding domains common to all invertebrate prophenoloxidases and haemocyanins. Expression of the prophenoloxidase gene was detected throughout all life stages from egg to imago in two strains of A. gambiae; however, the strongest expression was observed in developing embryos in eggs. The prophenoloxidase gene was mapped to the inversion rich region of the right arm of chromosome-2 in region 13B.

Molecular cloning of cDNAs for two pro-phenol oxidase subunits from the malaria vector, Anopheles gambiae.

Phenol oxidase exists in insect hemolymph as a zymogen, pro-phenol oxidase (pro-PO), which is activated by specific proteolysis in response to infection or wounding. Phenol oxidase catalyses the synthesis of quinones that polymerize to form melanin deposits, which encapsulate parasites and help to seal wounds. Antibodies to pro-PO from Manduca sexta bound to 76, 72, and 71 kDa polypeptide bands from hemolymph of Anopheles gambiae larvae. This antiserum was used to screen a cDNA library from A. gambiae fourth-instar larvae. Full-length clones were isolated for two different pro-POs, designated A. gambiae proPO-p1 and proPO-p2, which are 67% identical in nucleotide sequence and 66% identical in deduced amino acid sequence. The A. gambiae pro-PO sequences are more similar to pro-PO from Drosophila melanogaster than to lepidopteran or crustacean pro-PO sequences in the GenBank database. Like the other arthropod pro-POs, the A. gambiae pro-PO sequences lack a signal peptide and have two conserved regions predicted to bind two copper atoms in the active site of the enzyme. The availability of these pro-PO cDNAs should be useful in examining the biochemical differences between A. gambiae strains that are refractory or susceptible to Plasmodium infection, and differ in their ability to encapsulate the parasites.