Unfed Females Research Articles

Semi-field evaluation of electrocuting eave tubes for the control of endophagic mosquitoes in south-east Tanzania

BackgroundEave spaces are major entry points through which malaria vectors enter houses. Interventions that target mosquitoes at the eaves have recently been developed. However, most of these interventions are based on insecticides for which resistance has been reported. Here we evaluated the efficacy of mosquito electrocuting eave tubes (MEETs) against Anopheles gambiae sensu stricto (An. gambiae s.s.) and Anopheles funestus s.s. under semi-field conditions.MethodsExperiments were conducted in two semi-field chambers, each containing one experimental hut. Six electrocuting eave tubes were installed in each hut to assess their impact on laboratory-reared An. gambiae s.s. and An. funestus s.s.. Each species was assessed separately over 10 nights by releasing 200 unfed females per night into each chamber. One volunteer slept in each hut from 7 p.m. to 5 a.m. Mosquitoes were collected indoors and outdoors using mouth and Prokopack aspirators.ResultsThe placement of MEETs significantly reduced the nightly An. gambiae s.s. indoor and outdoor biting, by 21.1% and 37.4%, respectively. Indoor-biting An. funestus s.s. were reduced by 87.5% while outdoor-biting numbers of An. funestus s.s. declined by 10.4%.ConclusionsMEETs represent a promising tool for controlling mosquitoes at the point of house entry. Further validation of their potential under natural field conditions is necessary. Several advantages over insecticide-based eave tubes are indicated and discussed in this article.Graphical

The phenomenon of attachment and feeding of unfed ticks (Ixodoidea) on fed and feeding specimens of the same or different species: biological and epidemiological issues

Hyperparasitism, characterized by attachment and feeding of unfed ticks on engorged or feeding specimens of the same species (tick-to-tick attachment and feeding) has been extensively documented in laboratory colonies of ticks of the Ixodoidea superfamily. Existing literature generally assumes that hyperparasitism operates similarly across tick species in both main families, Argasidae and Ixodidae. However, a closer examination of the available data reveals distinct biological mechanisms underlying this phenomenon in different groups. In argasid ticks, hyperparasitism in laboratory colonies primarily involves unfed specimens stealing blood from their fed relatives, especially under stress of starvation or overcrowding. It remains uncertain whether this behavior of argasid ticks occurs under field conditions. If it does happen naturally, it may have originated as a consequence of the nidicolous lifestyle exhibited by soft ticks. In Ixodes ticks (Ixodinae or Prostriata), hyperparasitism of males on unfed or feeding females appears to be a side-effect in the male attempts to copulate while hyperparasitism in Amblyomminae (Metastriata) ticks is likely an aberration in feeding. This difference between Argasidae and Ixodidae may stem from independent adaptation to blood-feeding within the two Ixodoidea families. Experimental evidence of pathogen transmission between aggressor and victim during hyperparasitic feeding has only been demonstrated under laboratory conditions specifically in Ornithodoros species (Argasidae). The practical importance of this route of pathogen transmission is still unclear. Although there is an assumption in the literature that hyperparasitic pathogen transmission occurs in the taiga tick Ixodes persulcatus, it is important to know that no current data is available to support this presumption.

Fitness consequences of feeding-by-remating interactions in female Cerambyx welensii (Coleoptera: Cerambycidae: Cerambycinae)

Abstract The longhorn beetle, Cerambyx welensii (Küster) (Coleoptera: Cerambycidae), is considered a critical factor in oak decline in southwestern Iberia, but populations vary over space and time, depending on several ecological factors. Adults feed on ripe fruits and tree exudates, and evidence suggests that feeding could impact fitness in hot, dry summers, especially under climate change. In the present study, we assessed the impact of adult feeding (sugar-fed, water-fed, or unfed) and remating (monandrous versus polyandrous) on female reproductive output. Lifetime fecundity increased with female size in most feeding–remating combinations. Sugar-fed females achieved the highest longevity and fecundity, unfed females the lowest, and water-fed females had intermediate values. The daily fecundity pattern was strongly dependent on female feeding. Longevity and fecundity of once-mated and remated females were similar in both unfed and water-fed groups; however, in sugar-fed females, remating enhanced fecundity and shortened life span. Preoviposition, oviposition, and postoviposition periods were distinctly affected by both diet and remating. Results show that females require sugar to maximise reproductive output and that a water supply may partially mitigate the fitness decline of unfed females. We conclude that female feeding must be considered to explain C. welensii spatio-temporal occupancy–abundance patterns in oak woodlands.

Allatotropic peptides modulate muscle contraction of the female reproductive system in Rhodnius prolixus (STÄL)

Allatotropin (AT) acts as a myoregulator at the level of the dorsal vessel (DV) and midgut (MG) in triatominae insects. Previous analyses of the expression of the AT receptor in Rhodnius prolixus showed that AT is expressed in the DV and MG, but also in the reproductive system in females. To further study the activity of AT on female reproductive organs we analyzed the response by adult females in different physiological conditions, including unfed (virgin and mated), and fed mated females (gravid), to doses ranging between 10-14 and 10-6M. Myoregulatory activity was evaluated in vivo, by recording independently the frequency of contractions of each organ after treatment. The results show that the effect of AT varies depending on the organs and on the physiological state of the female. Whilst unfed virgin females did not show response to the peptide for neither of the applied doses, the ovaries showed a differential response, presenting the highest frequency of contractions in gravid individuals. An increase in the frequency of contractions of the oviducts was only observed in mated females. Uterus and spermathecae responded in both gravid and mated females, with maximum activity in the latter. In the bursa, responses were only detected in gravid females. The differential response of the organs seems to be associated to particular moments along the reproductive cycle, such as with the spermathecae that reacted to AT in both unfed mated and gravid females, when the movement of spermatozoids is physiologically crucial. Testes and accessory glands of the male, expressed the mRNA of AT precursor, suggesting that the male would modulate the contractile behavior of the female reproductive system after copula. The ovaries also expressed AT mRNA suggesting the existence of a paracrine/autocrine system modulating muscle contraction.

Knockdown of carnitine palmitoyltransferase I (CPT1) reduces fat body lipid mobilization and resistance to starvation in the insect vector Rhodnius prolixus.

The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial β-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10days later, but no changes were observed in the flight muscle. β-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.

Changes in the Ixodes ricinus microbiome associated with artificial tick feeding.

Artificial tick feeding systems (ATFS) can be used to study tick biology and tick-pathogen interactions. Due to the long feeding duration of hard ticks, antibiotics are commonly added to the in vitro blood meal to prevent the blood from decaying. This may affect the ticks' microbiome, including mutualistic bacteria that play an important role in tick biology. This effect was examined by the consecutive feeding of Ixodes ricinus larvae, nymphs, and adults in vitro with and without the supplementation of gentamicin and in parallel on calves. DNA extracted from unfed females was analyzed by 16S rRNA sequencing. The abundance of Candidatus Midichloria mitochondrii, Rickettsia helvetica and Spiroplasma spp. was measured by qPCR in unfed larvae, nymphs, and adults. Larvae and nymphs fed on calves performed significantly better compared to both in vitro groups. Adults fed on blood supplemented with gentamicin and B vitamins had a higher detachment proportion and weight compared to the group fed with B vitamins but without gentamicin. The detachment proportion and weights of females did not differ significantly between ticks fed on calves and in vitro with gentamicin, but the fecundity was significantly higher in ticks fed on calves. 16S rRNA sequencing showed a higher microbiome species richness in ticks fed on calves compared to ticks fed in vitro. A shift in microbiome composition, with Ca. Midichloria mitochondrii as dominant species in females fed as juveniles on calves and R. helvetica as the most abundant species in females previously fed in vitro was observed. Females fed in vitro without gentamicin showed significant lower loads of Ca. M. mitochondrii compared to females fed in vitro with gentamicin and ticks fed on calves. Spiroplasma spp. were exclusively detected in female ticks fed on cattle by qPCR, but 16S rRNA sequencing results also showed a low abundance in in vitro females exposed to gentamicin. In conclusion, the employed feeding method and gentamicin supplementation affected the ticks' microbiome composition and fecundity. Since these changes may have an impact on tick biology and vector competence, they should be taken into account in studies employing ATFS.

Distance from human dwellings differentially affects the efficacy of a synthetic cattle urine odour lure to trap malaria vectors

BackgroundCost–effective outdoor–based devices for surveillance and control of outdoor mosquito vector populations can substantially improve their efficacy when baited with synthetic human and animal odours. This study aimed at assessing the dose–dependent efficacy of a previously developed synthetic cattle urine odour to lure malaria vectors, and other mosquito species, to traps placed at different distances from human dwellings outdoors.MethodsThe efficacy of the cattle urine odour lure was assessed through a 5 × 5 Latin square design, using two sets of 5 Suna traps placed at either 1.5 m or 5 m from an adjacent human dwelling, in the rural village of Sagamaganga, Tanzania. Each trap was deployed with one of four doses of the synthetic cattle urine odour blend or a solvent control (heptane). Traps were rotated daily so that each dose and control visited each position twice over a period of 20 experimental nights. The relative attractiveness of each treatment dose and control was compared using a generalized linear mixed model for each species caught.ResultsA total of 1568 mosquitoes were caught, of which 783 were anophelines and 785 were culicines. Of the anophelines, 41.6 and 58.3% were primary and secondary vector species, respectively. Unfed and fed females of the primary vector, Anopheles arabiensis, were caught dose–dependently, close to human dwellings (1.5 m), whereas unfed, fed and gravid secondary vector Anopheles pharoensis females were caught dose–dependently, but at a farther distance from the dwellings (5 m). Females of Culex spp. were caught dose–dependently in similar numbers irrespective of the distance from human dwellings.ConclusionsThis study further clarifies the factors to be considered for the implementation of outdoor trapping using the synthetic cattle urine lure to target exophilic and exophagic malaria vectors, for which efficient surveillance and control tools are currently lacking. The findings resulting from this study make significant progress in providing the needed information to overcome the regulatory obstacles to make this tool available for integrated vector management programs, including registration, as well as evaluation and regulation by the World Health Organization.

Behavioral responses of sand fly Nyssomyia neivai (Psychodidae: Phlebotominae) to 1-hexanol and light

BackgroundThe search for attractive baits that may facilitate the capture of haematophagous insects has been epidemiologically relevant. Sand flies use chemical cues in different phases of their life cycles to find carbohydrate meals, mates, blood meals and oviposition sites. Few studies have related the behaviours of sand flies with volatile compounds that can influence their life cycles. Previous studies in our laboratory have shown that 1-hexanol released on filter paper is a good attractant for the sand fly Nyssomyia neivai, which is suspected in the transmission of the aetiologic agent of American cutaneous leishmaniasis. MethodsIn this study, we developed two release systems to modulated 1-hexanol release: system 1 contained gellan gum and pectin (4:1 ratio), 3% aluminium chloride and 1% glutaraldehyde; system 2 contained: gellan gum and pectin (4:1 ratio) and 3% aluminium chloride. After addition of 1-hexanol to each release system, trials were performed in a wind tunnel with Ny. neivai males and females (unfed, blood-fed and gravid) to evaluate activation and attraction responses. ResultsMales and unfed females showed the same response pattern to the systems. For both systems, the males and unfed females of the sand flies showed an activation response up to 24 h. The number of responsive gravid females was lower than unfed females for both systems. The blood-fed females showed no responses in any of the release systems. ConclusionsOur findings indicate that the state of the females (unfed, fed and gravid) can interfere with the sand fly responses to volatile compounds. Additionally, both systems evaluated with the compound showed effectiveness for sand fly attraction.

The life cycle of Hyalomma scupense (Acari: Ixodidae) under laboratory conditions

The life cycle of Hyalomma scupense on rabbit hosts was investigated under laboratory conditions. Hy. scupense exhibited one- and two-host life cycles of 163.2 and 161.4 days, respectively. The incubation of eggs required an average period of 52 days, which was the longest period among the four developmental stages. The average time for pre-feeding of larvae was 3.5 days. It took 20 days for larvae to become engorged nymphs and 52.3 days to become engorged females. The duration of the pre-feeding, feeding, pre-oviposition, and oviposition stages of female adults was 2.3, 13.5, 27.5, and 27.9 days, respectively. The average weight of engorged females was 390.0 mg (ranging from 129.3 mg to 828.6 mg), which was 28.95 times the weight of unfed females. There was a positive relationship between the weight and the number of eggs laid by engorged females (r = 0.927). The reproductive efficiency index (REI) was 8.63.

Characterization and manipulation of the bacterial community in the midgut of Ixodes ricinus

BackgroundTicks are obligate hematophagous arthropods transmitting a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, primarily in the ovaries and the midgut. In the previous study on Ixodes ricinus, we used a culture-independent approach and showed a diverse but quantitatively poor midgut bacterial microbiome. Our analysis also revealed the absence of a core microbiome, suggesting an environmental origin of the tick midgut microbiota.MethodsA bacterial analysis of the midgut of adult females collected by flagging from two localities in the Czech Republic was performed. Using the culture-independent approach, we tested the hypothesis that the midgut microbiome is of the environmental origin. We also cultured indigenous bacteria from the tick midgut and used these to feed ticks artificially in an attempt to manipulate the midgut microbiome.ResultsThe midgut showed a very low prevalence and abundance of culturable bacteria, with only 37% of ticks positive for bacteria. The culture-independent approach revealed the presence of Borrelia sp., Spiroplasma sp., Rickettsia sp., Midichloria sp. and various mainly environmental Gram-positive bacterial taxa. The comparison of ticks from two regions revealed that the habitat influenced the midgut bacterial diversity. In addition, the midgut of ticks capillary fed with the indigenous Micrococcus luteus (Gram-positive) and Pantoea sp. (Gram-negative) could not be colonized due to rapid and effective clearance of both bacterial taxa.ConclusionsThe midgut microbiome of I. ricinus is diverse but low in abundance, with the exception of tick-borne pathogens and symbionts. The environment impacts the diversity of the tick midgut microbiome. Ingested extracellular environmental bacteria are rapidly eliminated and are not able to colonize the gut. We hypothesize that bacterial elimination triggered in the midgut of unfed adult females is critical to maintain low microbial levels during blood-feeding.Graphical

Geographic distribution of deltamethrin resistance in malaria vectors from 2010 to 2020 in Republic of Benin, West Africa

The current study aims to establish the cartography of deltamethrin resistance in malaria vectors from 2010 to 2020 in Republic of Benin. An. gambiae s.l. populations were collected from March to July during the great rainy season in Ouémé in 2010, Littoral in 2012, Atlantic in 2012, Couffo in 2020 and Collines in 2012. An. gambiae s.l. populations were also collected from May to October during the rainy season in Alibori in 2012 and in 2015. Susceptibility tests were done following WHO protocol on unfed females’ mosquitoes aged 2-5 days old reared from larval and pupal collections with papers impregnated with deltamethrin (0.05%). The results showed that the populations of Anopheles gambiae s.l. from Adjara, Dangbo and Missérété were resistant to deltamethrin in 2010 whereas the populations of Anopheles gambiae s.l. from Kandi, Malanville, Cotonou, Allada and Dassa-Zoumè were resistant to this product in 2012. The populations of Anopheles gambiae s.l. from Parakou were resistant to deltamethrin in 2015 whereas the populations of Anopheles gambiae s.l. from Dogbo were resistant to the same product in 2020. The establishment of mapping or geographic distribution of deltamethrin resistance in malaria vectors from 2010 to 2020 in Republic of Benin is useful as this will properly inform control programs of the most suitable insecticides to use and facilitate the design of appropriate future resistance management strategies.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks

BackgroundTicks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick’s central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied.ResultsWe carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls).ConclusionsWe established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

Blood meal profile and positivity rate with malaria parasites among different malaria vectors in Sudan

BackgroundMalaria is a life-threatening public health problem globally with particularly heavy burden in the sub-Saharan Africa including Sudan. The understanding of feeding preference of malaria vectors on different hosts is a major challenge for hindering the transmission cycle of malaria. In this study, blood meals taken by blood-fed Anopheles mosquitoes collected from the field in malaria endemic areas of Sudan were analysed for source of blood meal and malaria parasite presence.MethodsAnopheles mosquitoes were collected from different regions in Sudan: Khartoum state, Sennar state, Northern state, and El Gedarif state between September 2020 and February 2021. Anopheles mosquitoes were collected using the standard pyrethrum spray catch and back-pack aspirator. Mosquito samples were sorted and morphologically identified to species level using international identification keys. Morphologically identified mosquito species were also confirmed using PCR. Genomic DNA was extracted from mosquitoes for molecular identification of blood meal source and parasite detection. The presence of Plasmodium species DNA in each mosquito sample was investigated using semi-nested PCR. Frequency of each blood meal source, Anopheles mosquito vector, and malaria parasite detected was calculated. Positivity rate of each fed female Anopheles mosquito was calculated for each species.ResultsA total of 2132 Anopheles mosquitoes were collected. 571 (26.8%) were males and 1561 (73.2%) were females classified based on their abdominal status into 1048 (67.1%) gravid, 274 (17.6%) fed, and 239 (15.3%) unfed females. Among the blood fed Anopheles mosquitoes, 263 (96.0%) were morphologically identified and confirmed using PCR to Anopheles arabiensis, 9 (3.3%) to Anopheles stephensi, and 2 (0.7%) to Anopheles rufipes. Of 274 blood-fed An. arabiensis, 68 (25.9%) fed on mixed blood meals from human and cattle, 8 (3.0%) fed on cattle and goat, and 13 (4.8%) fed on human, cattle and goat. For single blood meal sources, 70 (26.6%) fed on human, 95 (36.1%) fed on cattle, 8 (3.0%) fed on goat, and 1 (0.4%) fed on dog. While An. rufipes and An. stephensi fed on dog (2; 0.75%) and cattle (9; 3.3%), respectively. Plasmodium parasite detection in the blood meals showed that 25/274 (9.1%) An. arabiensis meals were positive for Plasmodium vivax and 19/274 (6.9%) An. arabiensis meals were positive for Plasmodium falciparum. The rate of positivity of An. arabiensis with any Plasmodium species was 16.7%. However, the positivity rate with P. falciparum only was 7.2%, while P. vivax was 9.5%. Both An. rufipes and An. stephensi were having positivity rates of 0.0% each.ConclusionsThis study which was mainly on blood-fed Anopheles mosquitoes showed a diversity in the type of diet from human, cattle, and goat. Anopheles mosquitoes especially An. arabiensis in Sudan, are opportunistic blood feeders and can feed broadly on both human and cattle. The application of blood meal identification is not only important in malaria vector epidemiological surveillance but also is very useful in areas where arthropods exhibit zoophilic feeding behaviour for mammals.

Evaluating sampling strategies for enzootic Venezuelan equine encephalitis virus vectors in Florida and Panama.

Determining effective sampling methods for mosquitoes are among the first objectives in elucidating transmission cycles of vector-borne zoonotic disease, as the effectiveness of sampling methods can differ based on species, location, and physiological state. The Spissipes section of the subgenus Melanoconion of Culex represents an understudied group of mosquitoes which transmit Venezuelan equine encephalitis viruses (VEEV) in the Western Hemisphere. The objective of this study was to determine effective collection methods that target both blood-engorged and non-engorged females of the Spissipes section of Culex subgenus Melanoconion to test the hypothesis that favorable trapping methods differ between species and by physiological status within a species. Mosquitoes were collected using two commercially available traps, (CDC-light trap and BG-Sentinel trap), two novel passive traps (a novel mosquito drift fence and pop-up resting shelters), and two novel aspirators, (a small-diameter aspirator and a large-diameter aspirator) in Darién, Panama, and Florida, USA. The total number of female mosquitoes collected for each species was compared using rarefaction curves and diversity metrics. We also compared the utility of each trap for collecting total females and blood-engorged females of four Spissipes section mosquito species in Florida and Darién. In Darién, it was found that both blood-engorged and unfed females of Cx. pedroi were most effectively collected using the mosquito drift fence at 57.6% and 61.7% respectively. In contrast, the most unfed Cx. spissipes were collected using the mosquito drift fence (40.7%) while blood-engorged females were collected effectively by pop-up resting shelters (42.3%). In Florida, the best sampling technique for the collection of blood-engorged Cx. panocossa was the large diameter aspirator at 41.9%, while the best trap for collecting Cx. cedecei was the pop-up resting shelter at 45.9%. For unfed female Spissipes section mosquitoes in Florida, the CDC light trap with CO2 collected 84.5% and 98.3% of Cx. cedecei and Cx. panocossa respectively in Florida. Rarefaction analysis, and both the Shannon and Simpsons diversity indices all demonstrated that the mosquito drift fence was capable of collecting the greatest diversity of mosquito species regardless of location. The finding that the proportions of unfed and blood-engorged mosquitoes collected by traps differed both among and between species has implications for how studies of VEEV vectors will be carried out in future investigations. In Florida a combination of pop-up resting shelters and use of a large-diameter aspirator would be optimal for the collection of both VEEV vectors for host-use studies. Results demonstrate that traps can be constructed from common materials to collect mosquitoes for VEEV vector studies and could be assessed for their utilization in vectors of other systems as well. Unfortunately, no single method was effective for capturing all species and physiological states, highlighting a particular need for assessing trap utility for target species of a study.

Insecticidal efficacy against Phlebotomus perniciosus in dogs treated orally with fluralaner in two different parallel-group, negative-control, random and masked trials

BackgroundDogs are the reservoir host of Leishmania infantum, the agent of zoonotic visceral leishmaniasis (VL), which is transmitted by the bite of phlebotomine sand flies. The sand fly Phlebotomus perniciosus is the main vector of zoonotic VL in the western Mediterranean region. Fluralaner has been shown to effectively kill this vector. The aim of this study was to evaluate the insecticidal efficacy of oral fluralaner in dogs bitten by P. perniciosus.MethodsTwo parallel-group, negative-controlled, randomized, masked laboratory trials with equivalent designs were performed in two different locations using two different pathogen-free laboratory-bred P. perniciosus strains for the challenge. In each trial, 12 purpose-bred beagles, initially ranked on natural attractiveness to sand flies, were randomly allocated to two groups (6 animals/group). Dogs in one group received fluralaner orally at the approved dose on day 0, and dogs in the control group were not treated. Each dog was subsequently exposed to an average of 70 unfed live sand fly females on days 1, 28, 56 and 84. Viability of blood-fed females was then evaluated for up to 96 h after exposure, and insecticidal efficacy was measured as the survival rate of flies fed on the fluralaner-treated dogs versus that of dogs in the control group. Significance was calculated for the proportion of live fed sand fly counts from treated versus control group dogs.ResultsComparison of the survival proportions between treated and control groups showed that fluralaner insecticidal efficacy was highly significant in both trials (P < 0.001 or P < 0.01 in different assessments) through to day 56. In the first trial, efficacy reached 100% on days 1 and 28, and 99.1% on day 56; in the second trial, the insecticidal efficacy was 98.5, 100 and 85.9%, respectively on the same days. On day 84, efficacy was in the range of 53–57% (P < 0.05) in the first trial and 0% in the second trial.ConclusionA single oral fluralaner administration to dogs under laboratory conditions results in strong and reproducible insecticidal efficacy against P. perniciosus for at least 8 weeks.Graphical

Molecular investigation on transstadial/transovarial transmission of Hepatozoon canis in Rhipicephalus sanguineus sensu lato ticks

ABSTRACT To investigate the possibility of transovarial and transstadial transmission of Hepatozoon canis, a total of 953 Rhipicehlalus sanguineus sensu lato ticks at different stages were collected from 231 stray dogs housed at Blue cross, and 10 dogs presented at small animal clinics. The engorged nymphs collected from the cracks and crevices of Blue cross were allowed to moult in the laboratory. The ticks collected from the host and moulted ticks were pooled according to their sex and feeding status as fed, unfed males and females. Each tick was dissected individually and examined microscopically, then pooled as H. canis positive and negative. Microscopic examination of the 953 ticks revealed H. canis oocysts in 228 (89.76%) unfed adult males and females which moulted from the nymphs collected from cracks and crevices. Positivity of 74% was observed in unfed females while all unfed males (100%) were positive. Amongst the 213 fed males, 174 (81.69%) were positive, while only 210 (43.20%) of the 486 fed females were positive. No oocyst was detected from ticks collected from dogs presented to clinics. The occurrence of H. canis DNA in the tick was confirmed by PCR assay based on the amplification of 666 bp fragment of partial 18S rRNA gene. Amplification was observed in pooled samples collected from Blue cross, whereas no amplification was observed in ticks collected from dogs presented to clinics. The DNA samples from tick larvae did not show amplification, which indicated that H. canis is not transmitted transovarially.

Novel sampling methods for monitoring Anopheles arabiensis from Eritrea.

BackgroundStudies comparing novel collection methods for host seeking and resting mosquitoes A. arabiensis were undertaken in a village in Eritrea. Techniques included an odor baited trap, a novel tent-trap, human landing collection and three methods of resting collection. A technique for the collection of mosquitoes exiting vegetation is also described. Pre-gravid rates were determined by dissection of host seeking insects and post-prandial egg development among insects collected resting.ResultsOverall 5,382 host-seeking, 2,296 resting and 357 A. arabiensis exiting vegetation were collected. The Furvela tent-trap was the most efficient, risk-free method for the collection of outdoor host-seeking insects, whilst the Suna trap was the least effective method. Mechanical aspirators (the CDC backpack or the Prokopack aspirator) were superior to manual aspiration in a dark shelter but there was no advantage over manual aspiration in a well-lit one. An estimated two-thirds of newly-emerged mosquitoes went through a pre-gravid phase, feeding twice before producing eggs. Mosquitoes completed gonotrophic development in a dark shelter but left a well-lit shelter soon after feeding. One blood-fed female marked in the village was recaptured 2 days after release exiting vegetation close to the oviposition site and another, shortly after oviposition, attempting to feed on a human host 3 days after release. Exit rates of males from vegetation peaked 3 min after the initial male had left. Unfed and gravid females exited approximately 6 min after the first males.ConclusionsFurvela tent-traps are suitable for the collection of outdoor biting A. arabiensis in Eritrea whilst the Prokopack sampler is the method of choice for the collection of resting insects. Constructing well-lit, rather than dark, animal shelters, may encourage otherwise endophilic mosquitoes to leave and so reduce their survival and hence their vectorial capacity.

New materials for improving catches of horseflies (Diptera: Tabanidae) in Nzi traps.

New materials in 85 configurations were tested relative to standard fabric Nzi traps to improve catches of tabanids based on artificial visual cues that mimic those used in nature for locating hosts or water. Colour-fast synthetic fabrics and photo-selective horticultural mesh were tested to facilitate insecticide impregnation and for improved durability. Many plastics were explored to document how flies react to polarized and visible vs. ultraviolet light. Lastly, sticky horizontal, linearly polarizing ground-level shiny plastic targets were tested for capturing water-seeking tabanids relative to traps and vertical fabric targets. Based on the capture of 281 786 female Tabanidae (Tabanus Linnaeus, Hybomitra Enderlein, Chrysops Meigen), four alternative formats provided higher catches and/or durability. They were substituting: (1) phthalogen turquoise for phthalogen blue, (2) solution-dyed fabrics for blue and black cotton, (3) clear/white horticultural mesh for mosquito netting, and (4) clear, reflective plastic for some or all netting. A Sunbrella/BioNet fabric trap (portable) and a blue-black Plexiglas®/netting trap (fixed applications) consistently performed best (1.5-3 × higher catches). Ground-level targets captured many male and gravid female Hybomitra but were ineffective for Tabanus. Traps and vertical fabric or transparent sticky targets captured mainly unfed females.

Autogenic capacity in Triatoma infestans (Klug, 1834) (Hemiptera: Reduviidae) susceptible and resistant to deltamethrin.

The insecticide resistance in Triatoma infestans (Klug, 1834) was detected in different areas of its geographical distribution. The mechanisms of resistance involved can affect different biological processes in addition to toxicological ones. Previous studies showed that reproductive efficiency was modified in resistant females compared to susceptible ones. The objective of this study was to compare the autogenic capacity and subsequent reproductive potential between deltamethrin-resistant and susceptible T. infestans. For each toxicological phenotype, pairs were formed between unfed adult females and recently fed adult male, which were separated after confirming copulation. Females were observed weekly until death, and reproductive parameters (initiation of mating, initiation of oviposition, fecundity, fertility and period between mating and initiation of oviposition) were recorded. Females from both toxicological phenotypes showed autogenic capacity. However, a lower proportion of deltamethrin-resistant unfed females laid eggs. Autogenic females showed a higher nutritional status than non-autogenic ones. No other differences in reproductive parameters were found between resistant and susceptible autogenic females. The possible mechanisms underlying the differences observed and their consequences on the spread of resistance are discussed. This is the first report describing the effect of pyrethroid resistance on T. infestans autogeny.

Entomological study in an anthroponotic cutaneous leishmaniasis focus in Morocco: Fauna survey, Leishmania infection screening, molecular characterization and MALDI‐TOF MS protein profiling of relevant Phlebotomus species

In Morocco, leishmaniases are a major public health problem due to their genetic diversity and geographical distribution. Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted typically by bite of phlebotomine sand flies. This study identifies sand fly fauna in Ibaraghen village, province of Azilal, which is a focus of CL, by combination of morphological and molecular methods (sequencing of COI gene, MALDI-TOF MS protein profiling). Nested-kDNA PCR was used to detect and identify Leishmania species within potential vector species. 432 CDC light traps were placed at different heights above ground level at four capture sites during a whole year. Traps at 1.5m above the ground yielded capture of sand flies almost double compared to above ground level (29.33%), while the collection reached 55.09% when the traps were placed 2.5m above ground. A total of 2,830 sand flies were collected, 2,213 unfed specimens were morphologically identified, 990 males (44.73%) and 1,223 females (55.26%) of 13 species; ten Phlebotomus species and three Sergentomyia species. Six species were analysed by MALDI-TOF MS protein profiling (4 Phlebotomus and 2 Sergentomiya species), and their identification was confirmed by COI sequencing. 1,375 unfed females were screened for the presence of Leishmania by nested-kDNA PCR in pools, 11/30 pools of P. sergenti showing a single band of 750bp corresponding to L. tropica. Our results confirm the role of P. sergenti as a proven vector in Azilal focus of cutaneous leishmaniasis; however, the relative abundance of other species known as vectors of Leishmania species emphasizes the risk of introduction of L. infantum and L. major in this province. For the first time in Morocco, a combined approach to identify sand flies by both morphology and molecular methods based on DNA barcoding and MALDI-TOF MS protein profiling was applied.