Alexandra Chaskopoulou

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India

Visceral Leishmaniasis (VL) is a neglected vector-borne disease. In India, it is transmitted to humans by Leishmania donovani-infected Phlebotomus argentipes sand flies. In 2005, VL was targeted for elimination by the governments of India, Nepal and Bangladesh by 2015. The elimination strategy consists of rapid case detection, treatment of VL cases and vector control using indoor residual spraying (IRS). However, to achieve sustained elimination of VL, an appropriate post elimination surveillance programme should be designed, and crucial knowledge gaps in vector bionomics, human infection and transmission need to be addressed. This review examines the outstanding knowledge gaps, specifically in the context of Bihar State, India.The knowledge gaps in vector bionomics that will be of immediate benefit to current control operations include better estimates of human biting rates and natural infection rates of P. argentipes, with L. donovani, and how these vary spatially, temporally and in response to IRS. The relative importance of indoor and outdoor transmission, and how P. argentipes disperse, are also unknown. With respect to human transmission it is important to use a range of diagnostic tools to distinguish individuals in endemic communities into those who: 1) are to going to progress to clinical VL, 2) are immune/refractory to infection and 3) have had past exposure to sand flies.It is crucial to keep in mind that close to elimination, and post-elimination, VL cases will become infrequent, so it is vital to define what the surveillance programme should target and how it should be designed to prevent resurgence. Therefore, a better understanding of the transmission dynamics of VL, in particular of how rates of infection in humans and sand flies vary as functions of each other, is required to guide VL elimination efforts and ensure sustained elimination in the Indian subcontinent. By collecting contemporary entomological and human data in the same geographical locations, more precise epidemiological models can be produced. The suite of data collected can also be used to inform the national programme if supplementary vector control tools, in addition to IRS, are required to address the issues of people sleeping outside.

Ecology of West Nile virus across four European countries: review of weather profiles, vector population dynamics and vector control response

West Nile virus (WNV) represents a serious burden to human and animal health because of its capacity to cause unforeseen and large epidemics. Until 2004, only lineage 1 and 3 WNV strains had been found in Europe. Lineage 2 strains were initially isolated in 2004 (Hungary) and in 2008 (Austria) and for the first time caused a major WNV epidemic in 2010 in Greece with 262 clinical human cases and 35 fatalities. Since then, WNV lineage 2 outbreaks have been reported in several European countries including Italy, Serbia and Greece. Understanding the interaction of ecological factors that affect WNV transmission is crucial for preventing or decreasing the impact of future epidemics. The synchronous co-occurrence of competent mosquito vectors, virus, bird reservoir hosts, and susceptible humans is necessary for the initiation and propagation of an epidemic. Weather is the key abiotic factor influencing the life-cycles of the mosquito vector, the virus, the reservoir hosts and the interactions between them. The purpose of this paper is to review and compare mosquito population dynamics, and weather conditions, in three ecologically different contexts (urban/semi-urban, rural/agricultural, natural) across four European countries (Italy, France, Serbia, Greece) with a history of WNV outbreaks. Local control strategies will be described as well. Improving our understanding of WNV ecology is a prerequisite step for appraising and optimizing vector control strategies in Europe with the ultimate goal to minimize the probability of WNV infection.

EFFICACY OF AERIAL ULTRA-LOW VOLUME APPLICATIONS OF TWO NOVEL WATER-BASED FORMULATIONS OF UNSYNERGIZED PYRETHROIDS AGAINST RICELAND MOSQUITOES IN GREECE

Abstract We assessed the efficacy of ultra-low volume aerial adulticiding with 2 new water-based, unsynergized formulations of Aqua-K-Othrin (2% deltamethrin) and Pesguard S102 (10% d-phenothrin) against the riceland mosquitoes of Greece. A helicopter with Global Positioning System (GPS) navigation, real-time weather recording, and spray dispersal modeling (AgDISP) was utilized to accurately treat the experimental blocks by adjusting spray line positions to changing meteorological conditions. Two application rates were applied per formulation that corresponded to 0.75 and 1.00 g AI/ha of deltamethrin and 7.50 and 10.00 g AI/ha of d-phenothrin. The mosquitoes used for the trials were the main nuisance species found in rice field areas of Thessaloniki, which were primarily Aedes caspius, followed by Culex modestus and Anopheles sacharovi. Overall mean mortality of caged mosquitoes was 69.2% and 64.8% for deltamethrin and d-phenothrin, respectively. Mean population decrease in wild mosquito populations within the treatment areas was 76.5% and 78% for deltamethrin and d-phenothrin, respectively. The AgDISP dispersal model, coupled with GPS navigation and real-time weather recording, enabled accurate placement of the spray cloud such that the majority of the treatment area received sufficiently high droplet densities to result in uniform caged-mosquito mortality across all sampling sites.

Toxicities of 31 Volatile Low Molecular Weight Compounds Against Aedes aegypti and Culex quinquefasciatus

ABSTRACT This research studied 31 volatile compounds for indoor control of the medically important mosquitoes Aedes aegypti L. and Culex quinquefasciatus Say. Only adult female mosquitoes were tested. The test compounds were from six families that included five heterobicyclics, eight formate esters, formic acid (a hydrolyzed metabolite of formate esters), eight acetate esters, four propionate esters, three butyrate esters, and two valerate esters. Also, the organophosphate compound dichlorvos (DDVP) was tested as a positive control. Cx. quinquefasciatus was generally more susceptible than Ae. aegypti. Cx. quinquefasciatus was most susceptible to a subset of heterobicyclics and formate esters (rank: n-butyl formate > hexyl formate = dihydrobenzofuran = menthofuran = heptyl formate = ethyl formate). Ae. aegypti was most susceptible to a subset of formate esters (rank: methyl > n-butyl > propyl = ethyl = hexyl). The most active materials against both species had LC50s of 0.4–1 mg active ingredient per 0.5 liter of air volume (0.8–2 mg/liter), which is 50- to 60-fold less toxic than dichlorvos (an organophosphate insecticide that is being phased out from indoor use). In relation to Drosophila melanogaster Meigen, both mosquito species were generally more susceptible to formate esters but more tolerant of heterobicyclics. Generally, the most toxic compound against all dipterans tested to date is n-butyl formate, whereas menthofuran is additionally toxic against Cx. quinquefasciatus and D. melanogaster. Finally, the toxicity differences between species point to the potential for differential toxicity among mosquito genera/species, suggesting that further studies of a number of mosquito species might be warranted.

Species composition, activity patterns and blood meal analysis of sand fly populations (Diptera: Psychodidae) in the metropolitan region of Thessaloniki, an endemic focus of canine leishmaniasis.

Species composition, activity patterns and blood meal analysis of sand fly populations were investigated in the metropolitan region of Thessaloniki, North Greece from May to October 2011. Sampling was conducted weekly in 3 different environments (animal facilities, open fields, residential areas) along the outskirts of the city in areas of increased canine leishmania transmission. Six sand fly species (Phlebotomus perfiliewi, Phlebotomus tobbi, Phlebotomus simici, Plebotomus papatasi, Sergentomya minuta and Sergentomya dentata) were identified using both classical and molecular techniques. DNA barcodes were characterized for the first time for two (P. simici and S. dentata) of the six recorded species. Phylogenetic analysis based on the COI gene sequences confirmed the grouping of P. tobbi, P. perniciosus and P. perfiliewi (subgenus Larrousius) and the monophyly of P. simici (subgenus Adlerius). By far the most prevalent species was P. perfiliewi, followed by P. simici and P. tobbi. The largest populations of sand flies were collected from animal facilities, followed by residential areas and open agricultural fields. Peak activity of sand flies overall occurred mid-August to mid-September and then declined sharply in October. Blood meal analysis showed that P. perfiliewi and P. simici feed preferentially on humans (88% & 95%, respectively) but also feed on chickens and goats. When designing a control strategy to alleviate sand fly nuisance in the region of Thessaloniki the following conclusions can be reached from this study: a) August and September are high risk months due to increased sand fly activity levels, b) animal facilities within or adjacent to urban settlements are high risk areas and may act as a maintenance and amplification foci for the vector as well as the parasite, and c) the abundance, ubiquity and feeding behavior of P. perfiliewi and P. simici establishes them as potentially important vectors of Leishmania in the region.

Vapor Toxicity of Three Prototype Volatile Insecticidal Compounds to House Fly (Diptera: Muscidae)

ABSTRACT Vapor toxicities of two prototype formate esters, ethylene glycol di-formate (EGDF) and heptyl formate, and the prototype heterobicyclic menthofuran were determined against laboratory-reared adult house flies, Musca domestica L. (Diptera: Muscidae). Toxicities were compared between house flies and other dipteran species tested previously. The organophosphate fumigant insecticide dichlorvos (DDVP) was used as a positive control. Although less toxic than DDVP, all three prototype compounds were toxic to house flies. Most toxic was menthofuran (LC50 = 3.70 mg/liter air), followed by EGDF (LC50 = 9.27 mg/liter air) and heptyl formate (LC50 = 32.62 mg/liter air). The toxicity of menthofuran when applied after piperonyl butoxide increased 1.5-fold. The toxicities of heptyl formate and EGDF when applied after S,S,S-tributyl phosphorotrithioate decreased by 1.5-and 2-folds, respectively. These results are slightly different from results obtained for other dipterans, further supporting previous hypotheses that toxicity may have some species specificity. Ceramic porous rods impregnated with heptyl formate were used to evaluate the effectiveness of controlled vapor release for a single dose of heptyl formate against house flies. Controlled vapor release of heptyl formate can be used successfully to provide prolonged house fly mortality. Even though the prototype compounds did not exhibit toxicity as high as that of DDVP, their toxicities to dipterans, along with their reduced mammalian toxicities relative to DDVP, make them potential DDVP replacement candidates.

Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin

Background Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE) genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos. Principal findings Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida): one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries. Significance The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies.

Nontarget Effects of Aerial Mosquito Adulticiding with Water-Based Unsynergized Pyrethroids on Honey Bees and other Beneficial Insects in an Agricultural Ecosystem of North Greece

ABSTRACT We assessed the nontarget effects of ultra-low-volume (ULV) aerial adulticiding with two new water-based, unsynergized pyrethroid formulations, Aqua-K-Othrine (FFAST antievaporant technology, 2% deltamethrin) and Pesguard S102 (10% d-phenothrin). A helicopter with GPS navigation technology was used. One application rate was tested per formulation that corresponded to 1.00 g (AI)/ha of deltamethrin and 7.50 g (AI)/ha of d-phenothrin. Three beneficial nontarget organisms were used: honey bees (domesticated hives), family Apidae (Apis mellifera L.); mealybug destroyers, family Coccinellidae (Cryptolaemus montrouzieri Mulsant); and green lacewings, family Chrysopidae (Chrysoperla carnea (Stephens)). No significant nontarget mortalities were observed. No bees exhibited signs of sublethal exposure to insecticides. Beehives exposed to the insecticidal applications remained healthy and productive, performed as well as the control hives and increased in weight (25–30%), in adult bee population (14–18%), and in brood population (15–19%).

Analysis of population structure and insecticide resistance in mosquitoes of the genus Culex, Anopheles and Aedes from different environments of Greece with a history of mosquito borne disease transmission

Greece has been recently affected by several mosquito borne diseases with the West Nile Virus (WNV) outbreak in 2010 being one of the largest reported in Europe. Currently at the epicenter of an economic and refugee crisis and visited by over 16 million tourists a year the integrated management of diseases transmitted by mosquitoes is a public health and economic priority. Vector control programs rely mainly on insecticides, however data on insecticide resistance and the mosquito fauna is essential for successful applications. We determined the mosquito species composition and population dynamics in areas of increased vulnerability to vector borne disease transmission, as well as investigated the resistance status of major nuisance and disease vectors to insecticides. High mosquito densities were recorded in Thessaloniki and Evros, with Aedes caspius, a nuisance species, Culex pipiens, a known vector of WNV and Anopheles hyrcanus a potential vector of malaria being among the most prevalent species. Both vector species populations reached their peak in late summer. Aedes albopictus was recorded at high densities in Thessaloniki, but not in Evros. Notably, Cx. pipiens hybrids, which show an opportunistic biting behavior and are suspected to be involved in the transmission of the WNV, were recorded in considerable numbers in Thessaloniki and Attica. Culex pipiens and An. hyrcanus, but not Ae. caspius mosquitoes, showed moderate levels of resistance to deltamethrin. The presence of resistance in areas not exposed to vector control indicates that other factors could be selecting for resistance, i.e. pesticide applications for agriculture. Both L1014F and L101C kdr mutations were detected in Cx. pipiens populations. Anopheles hyrcanus resistance was not associated with mutations at the L1014 site. The Ace-1 mutations conferring insensitivity to organophosphates and carbamates were detected at low frequencies in all Cx. pipiens populations. Increased activity of P450s and esterases was found in Cx. pipiens individuals from Thessaloniki. Our study contributes evidence for sustainable and efficient vector control strategies and the prevention of disease outbreaks.

Mild-Vectolysis: A Nondestructive DNA Extraction Method for Vouchering Sand Flies and Mosquitoes

Abstract Nondestructive techniques allow the isolation of genomic DNA, without damaging the morphological features of the specimens. Though such techniques are available for numerous insect groups, they have not been applied to any member of the medically important families of mosquitoes (Diptera: Culicidae) and phlebotomine sand flies (Diptera: Psychodidae). This study presents Mild-Vectolysis, the first nondestructive DNA extraction methodology for vouchering taxa of mosquitoes and sand flies, which provided sufficient amounts of DNA, tested in a verified barcode (cytochrome oxidase I gene), while preserving their morphological integrity. Application of the method to sand flies allowed successful insect identification post DNA extraction, as all basic taxonomical structures necessary for identification (pharynx, cybarium, and genitalia) remained intact. The development of the methodology was more challenging in mosquitoes, due to the fragility of key morphological characters (scales and color). A small modification of the lysis buffer concentration, in combination with the adjustment of the incubation time, a postlysis freezing stage, and the avoidance of ethanol, achieved the extraction of sufficient DNA quantity, while preserving the integument of the mosquitoes, although a small proportion of the scales and the color still appeared to have been lost. In addition to the practicality and efficiency of our methodology, preserving of the original insect specimen post DNA extraction is highly advantageous, as it allows for 1) utilization of the specimen for further analysis and 2) storage for vouchering.