A membrane and blood-free approach to rear adult Aedes albopictus.

Most artificial dietary systems for feeding mosquitoes require a membrane feeder, host cues, phosphate saline buffers and a phagostimulant. These multicomponent feeders are complex, expansive and cumbersome that requires fully trained personnel. The objective of the present is to develop a simple sugar assisted protein (SAP) diet for the egg production of Aedes albopictus. The present study assessed the potential use of SAP dietary system on the engorgement, fecundity, preference of diet components and production of multiple generations of Ae. albopictus. Our data show that the female mosquitoes have strongly preferred a diet with: (i) a combination of sugar and protein over the individual component, and (ii) water over PBS (phosphate buffered saline) buffer as a carrier, whereas adenosine triphosphate (ATP) was not required as a phagostimulant. Based on our optimization data, the SAP diets (10-20% bovine serum albumin in 5% sucrose aqueous solution) do not require chemo-attractive lure, phagostimulant ATP, temperature and membrane feeding components. Female mosquitoes readily engorge on SAP diets and show similar rates of survival and fecundity compared to those when blood-fed on live animals. In addition, the number of eggs produced by female mosquitoes fed on SAP diets kept consistent for 10 consecutive generations. Our results indicate that SAP diet is a potential alternative against blood feeding that is simple and cost-effective diets for Ae. albopictus colony maintenance and to support large scale mass- production for experimental and other purposes.

Large-Scale Operational Pyriproxyfen Autodissemination Deployment to Suppress the Immature Asian Tiger Mosquito (Diptera: Culicidae) Populations.

Effective suppression of container-inhabiting Asian Tiger [Aedes albopictus (Skuse)] (Diptera: Culicidae) and yellow fever [Aedes aegypti (L.)] (Diptera: Culicidae) mosquitoes presents one of the most intractable problems for modern mosquito control. Traditional tools often fail to control populations of these mosquito species, and are prohibitively expensive or have negative environmental impacts. Novel approaches and tools are urgently needed for integrated container-inhabiting mosquito management programs. One of the most promising techniques is autodissemination. We present the results of a long-term large-scale study conducted in a temperate urbanized environment representing typical Ae. albopictus habitats. Three treatment sites with autodissemination stations and three nearby reference sites were monitored for eggs, immature, and adult mosquitoes over a period of 3 yr from 2014 to 2016. Elevated larval and pupal mortality of 12-19% on average was the most notable outcome in sentinel cups of the treatment sites. The number of eggs in the treatment sites was significantly reduced in 2014, but not in 2015 or 2016. Adult populations remained similar in treatment and reference sites throughout the study. The impact of autodissemination on mosquito populations was lower than reported by previous investigations. Technical and logistical problems associated with wider coverage and working in multiple urban neighborhoods contributed to reduced efficacy. Incorporating autodissemination with routine mosquito control operations and commercializing this methodology for general public use will require further research on combining this tool with other novel or conventional technologies.

Heterodissemination: precision targeting container Aedes mosquitoes with a cohabiting midge species carrying insect growth regulator.

BACKGROUND: Management of Aedes albopictus and Ae. aegypti is challenging in large part due to the cryptic nature of their larval habitats. Autodissemination, using conspecific species to transfer pesticide, is unable to provide proactive control. Here we report results from a new hypothesis, heterodissemination, wherein females of the cohabiting non-biting midge, Chironomus decorus, reared in the laboratory, treated with pyriproxyfen, and released to transfer lethal concentrations to shared mosquito larval habitats. RESULTS: Pyriproxyfen-impregnated oil and powder formulations were developed. The average payload for each female midge treated with oil followed by powder formulations was 5.07 ± 0.92 µg of active ingredient or 1660 times the median lethal concentration (LC50 ) for Ae. albopictus or Ae. aegypti in 200 mL of water. Subsequent residue analysis showed pyriproxyfen transference from chironomids, treated with oil formulation only, into water-holding containers up to 2.06 ppb or 171.7 times the LC50 . Releasing 20 laboratory reared and contaminated Chironomus decorus into a small room resulted in 80.42 ± 0.67% and 75.67 ± 3.14% Ae. albopictus pupal mortality in open and cryptic sentinel ovicups, respectively. Container water volumes ranging up to 4 L did not affect efficacy. In a large field cage, 90.3 ± 2.5% Ae. albopictus mortality was resulted from releasing 100 treated female midges. Releasing 400 contaminated midges into a residential backyard resulted in 74.3% pupal mortality in sentinel ovicups. CONCLUSIONS: Room, large field cage and field release trials demonstrated that adult midges reared and treated in the laboratory transfer highly lethal concentrations of pyriproxyfen to Ae. albopictus container habitats. Heterodissemination provides a potential approach for precision, proactive mosquito control, which may draw attention for further studies. © 2020 Society of Chemical Industry.

Evaluation of Lambda-Cyhalothrin and Pyriproxyfen Barrier Treatments for Aedes albopictus (Diptera: Culicidae) Management in Urbanized Areas of New Jersey.

Mosquito control programs in the United States are still searching for best management practices to control the Asian tiger mosquito, Aedes albopictus (Skuse; Diptera: Culicidae). Most intervention methods for this species are either labor intensive (e.g., source reduction) or short-term (e.g., ultra-low-volume adulticiding). We investigated the effectiveness of barrier spray pesticide applications within urban and suburban residential yards in New Jersey as a control strategy using a before-after-control-impact (BACI) approach. Applications of Demand CSR pyrethroid (9.7% AI lambda-cyhalothrin) only or combined Demand CSR and Archer IGR insect growth regulator (1.3% AI pyriproxyfen) applications resulted in significant and similar decreases in adult mosquito abundance post-treatment ranging from 78 to 74% respectively, compared with the untreated control. Both insecticides exceeded the 70% reduction threshold considered as effective for Ae. albopictus control for 2 to 4 wk. However, applications of Archer IGR alone did not reduce adult mosquito abundance. The field study results were supported by laboratory no-choice bioassays using treated leaf foliage. Our study is the first data driven evidence of the residual efficacy of barrier pesticide applications in New Jersey with lambda-cyhalothrin that provided significant reductions in adult Ae. albopictus populations for an extended duration.

Defective lipid metabolism associated with mutation in klf-2 and klf-3: important roles of essential dietary salts in fat storage.

BACKGROUND: Dietary salts are important factors in metabolic disorders. They are vital components of enzymes, vitamins, hormones, and signal transduction that act synergistically to regulate lipid metabolism. Our previous studies have identified that Krüppel-like factor -3 (KLF-3) is an essential regulator of lipid metabolism. However, it is not known if KLF-2 also regulates lipid metabolism and whether KLF-2 and -3 mediate the effects of dietary salts on lipid metabolism. METHODS: In this study, we used klf mutants [homozygous klf-2 (ok1043) V and klf-3 (ok1975) II mutants] to investigate the role of dietary salts in lipid metabolism. All gene expression was quantified by qRT-PCR. Localization of KLF-2 was analyzed by the expression of klf-2::gfp (in pPD95.75 vector) using a fluorescent microscope. Fat storage was measured by Oil Red O staining. RESULTS: Klf-2 was identified to express in the intestine during all stages of Caenorhabditis elegans development with peak expression at L3 stage. Mutation of klf-2 increased fat accumulation. Under regular growth media free of Ca2+, the expression of both klf-2 and -3 was inhibited slightly; further their expression reduced significantly in WT worms fed on 10X Ca2+ diet. When klf-3 was mutated, the expression of klf-2 increased under 10X Ca2+ diet; but when klf-2 was mutated, the expression of klf-3 was not altered under 10X Ca2+ diet. Overall, Mg2+ and K+ were less effective on the gene expression of klfs. KLF target gene Ce-C/EBP-2 showed elevated expression in WT and klf-3 (ok1975) worms with changed Ca2+ concentrations but not in klf-2 (ok1043) worms. However, high Ca+2 diet exhibited inhibitory effect on Ce-SREBP expression in WT worms. CONCLUSION: Dietary Ca2+ is most effective on fat storage and klf-2 expression, wherein high Ca2+ diet decreased klf-2 expression and reduced fat buildup. Mechanistic study identified Ce-C/EBP (C48E7.3; lpd-2) and Ce-SREBP (Y47D3B.7; lpd-1) as the target genes of klf-2 and/or klf-3 to mediate lipid metabolism. This study identifies a new function of klf-2 in inhibiting fat buildup and reveals the interplay between dietary salts and klf-2 and klf-3 in lipid metabolism.

Targeting a Hidden Enemy: Pyriproxyfen Autodissemination Strategy for the Control of the Container Mosquito Aedes albopictus in Cryptic Habitats.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is a vector of dengue, Chikungunya, and Zika viruses. This mosquito inhabits a wide range of artificial water-holding containers in urban and suburban areas making it difficult to control. We tested the hypothesis that female-driven autodissemination of an insect growth regulator could penetrate cryptic oviposition habitats difficult to treat with conventional insecticidal sprays. METHODOLOGY: Oviposition preferences of Ae. albopictus females for open and cryptic cups were tested in semi-field experiments. Two conventional larvicidal sprayers were tested to determine droplet penetration and larvicidal efficacy in open and cryptic habitats using Bacillus thuringiensis var. israelensis (Bti) in the field. Finally, the efficacy of pyriproxyfen autodissemination stations was assessed in cryptic and open cups in residential areas during 2013 and 2014. PRINCIPAL FINDINGS: Gravid females strongly preferred cryptic (53.1±12.9 eggs/cup) over open (10.3±4.3 eggs/cup) cups for oviposition. Cryptic cups showed limited droplet penetration and produced 0.1-0.3% larval mortality with a conventional backpack and low-volume sprays of Bti. The autodissemination stations effectively contaminated these cryptic cups (59.3-84.6%) and produced 29.7-40.8% pupal mortality during 2013-2014. Significant pupal mortality was also observed in open cups. CONCLUSIONS: The autodissemination station effectively exploits the oviposition behavior of wild gravid females to deliver pyriproxyfen to targeted oviposition habitats. Although the pupal mortality in cryptic cups was relatively lower than expected for the effective vector control. Autodissemination approach may be a suitable supporting tool to manage Ae. albopictus immatures in the cryptic habitats those are less accessible to conventional larvicidal sprays.

Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India.

BACKGROUND: Microbiota inhabiting midguts of mosquitoes play a key role in the host - parasite interaction and enhance vectorial capacity of viral diseases like dengue and chikungunya fevers. Mosquito midgut is considered to be an important site for host-pathogen interaction and pathogen survival is thought to be an outcome of this interaction. In the present study we examined the bacterial community in the midgut of Aedes mosquitoes in Arunanchal Pradesh, India, a subtropical zone where dengue fever is reported to be emerging. METHOD: Larvae and pupa of Aedes mosquitoes were collected from a biodiversity hotspot, Bhalukpong, Arunachal Pradesh, India. 16S rRNA gene sequences were used for identification of isolated bacterial population from each species of mosquitoes. We used various diversity indices to assess the diversity and richness of the bacterial isolates in both mosquito species. RESULT: On the basis of 16S rRNA gene sequence analysis a total of 24 bacterial species from 13 genera were identified belonging to 10 families of four major phyla. Phylum Proteobacteria was dominant followed by Firmicutes, Bacteroidetes and Actinobacteria. The midgut bacteria belonging to the phylum Proteobacteria and Firmicutes were isolated from both Ae. albopictus and Ae. aegypti, whereas, bacteria belonging to phylum Bacteroidetes and Actinobacteria were isolated only from Ae. albopictus and Ae. aegypti respectively. Enterobacter cloacae was the dominant bacterial species in both Ae. albopictus (33.65%) and Ae. aegypti (56.45%). Bacillus aryabhattai (22.78%) was the second most common bacterial species in Ae. albopictus whereas, in Ae. aegypti the second most common bacterial species was Stenotrophomonas maltophilia (7.44%). CONCLUSION: The family Enterobacteriaceae of phylum Proteobacteria was dominant in both species of Aedes mosquitoes. To the best of our knowledge, this is the first attempt to study midgut microbiota from a biodiversity hotspot in Northeastern India. Some bacterial genera Enterobacter and Acinetobacter isolated in this study are known to play important roles in parasite-vector interaction. Information on midgut microflora may lead towards the development of novel, safe, and effective strategies to manipulate the vectorial capacity of mosquitoes.

Isolation and characterization of Vagococcus sp from midgut of Culex quinquefasciatus (Say) mosquito.

BACKGROUND & OBJECTIVES: Mosquito gut is a rich source of microorganisms. These microorganisms exhibit close association and contribute various physiological processes taking place in mosquito gut. The present study is aimed to characterize two bacterial isolates M19 and GB11 recovered from the gut of Culex quinquefasciatus mosquito collected from Bhuj and Jamnagar districts of Gujarat, India. METHODS: Both the strains were characterized using polyphasic approach including, phenotypic characterization, whole cell protein profiling and sequencing of 16S rRNA gene and groESL region. RESULTS: Sequences of 16S rRNA gene of M19 and GB11 were 99% similar to Vagococcus carniphilus and Vagococcus fluvialis. But phenotypic profile, whole cell protein profile and sequence of groESL region of both isolates were found to be similar to V. fluvialis. CONCLUSION: Based on phenotypic, genotypic and protein profiling, both the strains were identified as V. fluvialis. So far this species was known from domestic animals and human sources only. This is the first report of V. fluvialis inhabiting midgut of Cx. quinquefasciatus mosquito collected from Arabian sea coastal of India.

Midgut microbial community of Culex quinquefasciatus mosquito populations from India.

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.

Chryseobacterium culicis sp. nov., isolated from the midgut of the mosquito Culex quinquefasciatus.

A yellow-pigmented bacterial strain, R4-1A(T), isolated from the midgut of the mosquito Culex quinquefasciatus (a vector of lymphatic filariasis), was studied using a polyphasic approach. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of this organism with sequences of type strains of the most closely related species clearly showed an allocation to the genus Chryseobacterium, with the highest sequence similarities (all 97.9 %) to Chryseobacterium jejuense JS17-8(T), C. indologenes ATCC 29897(T), C. arthrosphaerae CC-VM-7(T) and C. aquifrigidense CW9(T). 16S rRNA gene sequence similarities to type strains of other Chryseobacterium species were below 97.5 %. The fatty acid profile of strain R4-1A(T) included the major fatty acids iso-15 : 0, summed feature 4 (comprising iso-15 : 0 2-OH and/or 16 : 1ω7c), iso-17 : 1ω9c and iso-17 : 0 3-OH. DNA-DNA hybridizations with C. jejuense KACC 12501(T), C. indologenes CCUG 14556(T), C. arthrosphaerae CC-VM-7(T) and C. aquifrigidense KCTC 12894(T) resulted in relatedness values of 38.3 % (reciprocal 30.5 %), 29.4 % (32.1 %), 23.2 % (37.2 %) and 29.5 % (47.1 %), respectively. These results and the differentiating biochemical and chemotaxonomic properties show that strain R4-1A(T) represents a novel species, for which the name Chryseobacterium culicis sp. nov. is proposed. The type strain is R4-1A(T) (=LMG 25442(T) =CCM 7716(T)).

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase.

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 degrees C with 0.5mM isopropyl beta-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 degrees C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 micromol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k(cat) [(3.2+/-0.02)x10(4)] with 3-acetylpyridine adenine dinucleotide (APAD+). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.

Amplification of LDH gene from indian strains of Plasmodium vivax.

BACKGROUND & OBJECTIVES: Plasmodium vivax is geographically widespread and responsible for > 50% of malaria cases in India. Increased drug resistance of the parasite highlights the immediate requirement of early and accurate diagnosis as well as new therapeutics. In view of this, the present study was undertaken to amplify P. vivax (Indian strains) lactate dehydrogenase gene (PvLDH) which has been identified as a good target for antimalarials as well as diagnostics. METHODS: P. vivax infected clinical blood samples were collected from southern part of India and were tested with established diagnostic parameters (ICT, Giemsa staining). Total DNA was extracted from blood samples and subjected to PCR using two sets of primers, one for the amplification of full PvLDH gene (951 bp) and the other for a partial PvLDH gene fragment (422bp), covering a variable antigenic region (140aa) as compared to other plasmodial species. RESULTS & CONCLUSION: PCRs for both the full and partial gene targets were optimised and found to be consistent when tested on several P. vivax positive clinical samples. In addition, full gene PCR was found to specifically detect only P. vivax DNA and could be used as a specific molecular diagnostic tool. These amplified products can be cloned and expressed as a recombinant protein that might be useful for the development and screening of antimalarials as well as for diagnostic purposes.