Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Cryopreservation of Anopheles stephensi embryos

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

Spontaneous mutation rate estimates for the principal malaria vectors Anopheles coluzzii and Anopheles stephensi

Using high-depth whole genome sequencing of F0 mating pairs and multiple individual F1 offspring, we estimated the nuclear mutation rate per generation in the malaria vectors Anopheles coluzzii and Anopheles stephensi by detecting de novo genetic mutations. A purpose-built computer program was employed to filter actual mutations from a deep background of superficially similar artifacts resulting from read misalignment. Performance of filtering parameters was determined using software-simulated mutations, and the resulting estimate of false negative rate was used to correct final mutation rate estimates. Spontaneous mutation rates by base substitution were estimated at 1.00 × 10−9 (95% confidence interval, 2.06 × 10−10—2.91 × 10−9) and 1.36 × 10−9 (95% confidence interval, 4.42 × 10−10—3.18 × 10−9) per site per generation in A. coluzzii and A. stephensi respectively. Although similar studies have been performed on other insect species including dipterans, this is the first study to empirically measure mutation rates in the important genus Anopheles, and thus provides an estimate of µ that will be of utility for comparative evolutionary genomics, as well as for population genetic analysis of malaria vector mosquito species.

Detection of Zika virus in Anopheles stephensi Liston, 1901 (Diptera: Culicidae) in India - First report

Zika virus (ZIKV) a mosquito-borne, causing acute febrile illness associated with rash, arthralgia and conjunctivitis in the patient, was reported from Thiruvananthapuram, Kerala, as an outbreak with 83 cases. Entomological surveillance revealed the presence of aedine mosquitoes viz., Aedes aegypti (Linnaeus, 1762), Ae. albopictus (Skuse, 1894) and Ae. vittatus (Bigot, 1861) and nonaedine mosquitoes viz., Anopheles stephensi Liston,1901, Mansonia uniformis (Theobald, 1901), Culex tritaeniorhynchus Giles, 1901 and Cx. gelidus Theobald,1901. Aedes (Ae. aegypti, Ae. vittatus and Ae. Albopictus) mosquito larvae were high in the Zika affected areas. Moreover ZIKV was detected in An. stephensi mosquitoes collected from Parassala, Thiruvananthapuram (the native place of the first ZIKV confirmed case in the present outbreak in Kerala). Molecular diagnostics of Ae. Aegypti, Ae. vittatu and An. stephensi mosquitoes revealed that the species were loaded with ZIKV. Significantly this is the first ever report of ZIKV detecting in An. stephensi in the world. Aedes adults (male and female) and An. stephensi emerged from fourth instar larvae and pupae were found to have ZIKV, indicating transovarial transmission of the virus.

Current Situation of Malaria and Resistance of Main Vectors to WHO Recommended Insecticides in an Endemic Area, Southeastern Iran

Although malaria is endemic in some areas of southeastern Iran, following the successful national malaria elimination plan, the local transmission area has been shrunk. The main cases in Iran are due to Plasmodium vivax followed by P. falciparum. This study was aimed to determine the current situation of malaria in Kerman Province of Iran and evaluate the insecticide resistance of main vectors. The field study was conducted in 2019. Data of new malaria cases were obtained from the health centers for the period of 2009–2018. Susceptibility status of Anopheles stephensi and An. dthali was evaluated against dichlorodiphenyltrichloroethane, Dieldrin, Malathion, Bendiocarb, Deltamethrin, and Temephos at the diagnostic dose. A total of 522 malaria cases were recorded and divided into indigenous (33.14%) and imported (66.86%) categories. The highest incidence of the disease was reported from the southern areas of the province, where all indigenous cases occurred. Adults of An. stephensi were resistant to dichlorodiphenyltrichloroethane while its resistance to be confirmed to dieldrin, bendiocarb and deltamethrin. As An. dthali had less than 98% mortality against bendiocarb, the resistance status should be confirmed with more tests. Our findings showed both species had less than 98% mortality against bendiocarb and deltamethrin insecticides which are used in malaria vector control program in Iran. Due to the susceptibility of these vectors to temephos, larviciding can be advised for vector control in this area.

Ovicidal, larvicidal and pupicidal efficacy of silver nanoparticles synthesized by Bacillus marisflavi against the chosen mosquito species

Microbial synthesis of silver nanoparticles is more advantageous and is eco-friendly to combat the various vectors that cause diseases in humans. Hence, in the present study a Bacillus strain is isolated from marine habitat and is evaluated for its ability to synthesize silver nanoparticles (AgNPs) and its efficacy evaluated against the immature stages of selected mosquito species. The effective candidate was confirmed to be Bacillus marisflavi after 16S rRNA sequencing. The synthesis of AgNPs was confirmed by UV-Vis spectrophotometer. Atomic Force Microscopic (AFM) analysis showed spherical nanoparticles. Size analysis using Scanning Electron Microscope (SEM) showed particles of nano size averaging 78.77 nm. The diameter of the particles analyzed by Dynamic Light Scattering (DLS) showed 101.6 nm with a poly-dispersive index of 0.3. Finally the elemental nature of the nanoparticles was identified by Fourier-transform infrared spectroscopy (FTIR). LC50 and LC90 values for the ovicidal, larvicidal and pupicidal efficacy of the AgNPs against the egg, larvae and pupae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi respectively were evaluated. The present study revealed that the nanoparticles have an excellent toxic effect against the disease transmitting vector mosquitoes. Hence, the rapid synthesis of AgNPs would be an appropriate eco-friendly tool for biocontrol of vector mosquitoes.

Preparation and Optimization of Peppermint (Mentha Pipertia) Essential Oil Nanoemulsion with Effective Herbal Larvicidal, Pupicidal, and Ovicidal Activity against Anopheles Stephensi

Objectives: The Plasmodium parasite is transmitted directly to humans through the Anopheles mosquito bite and causes vector-borne Malaria disease, which leads to the transmission of the disease in Southeast Asia, including India. The problem of persistent toxicity, along with the growing incidence of insect resistance, has led to the use of green pesticides to control the spread of the disease in a cost-effective and environment-friendly manner. Based on this objective, this work investigated the larvicidal, pupicidal, and ovicidal activity of Mentha pipertia using a natural nanoemulsion technique. Methods: GC-MS characterized essential oils of Mentha pipertia leaves were formulated as a nanoemulsion for herbal larvicidal, pupicidal, and ovicidal activities. Size of the nanoemulsion was analyzed by photon correlation spectroscopy. The herbal activities against Anopheles Stephensi of nanoemulsion were evaluated in terms of the lethal concentration for 50% (LC50) and 90% (LC90) to prove low cost, pollution free active effective formulation. Results: Chiral, keto, and alcohol groups attached Mentha pipertia leaves essential oil nanoemulsions demonstrated good results in the larvicidal probit analysis, with values of LC50=09.67 ppm and LC90=20.60 ppm. Activity results of the most stable nano formulation with 9.89 nm size showed a significant increase when compared to the bulk. Conclusion: The nanoemulsion of Mentha pipertia leaves can be a promising eco-friendly widely available, low-cost herbicide against the Anopheles mosquito.

Using marine cargo traffic to identify countries in Africa with greatest risk of invasion by Anopheles stephensi

Anopheles stephensi is an efficient malaria vector commonly found in South Asia and the Arabian Peninsula, but in recent years it has established as an invasive species in the Horn of Africa (HoA). In this region An. stephensi was first detected in a livestock quarantine station near a major seaport in Djibouti in 2012, in Ethiopia in 2016, in Sudan in 2018 and Somalia in 2019. Anopheles stephensi often uses artificial containers as larval habitats, which may facilitate introduction through maritime trade as has been seen with other invasive container breeding mosquitoes. If An. stephensi is being introduced through maritime traffic, prioritization exercises are needed to identify locations at greatest risk of An. stephensi introduction for early detection and rapid response, limiting further invasion opportunities. Here, we use UNCTAD maritime trade data to 1) identify coastal African countries which were most highly connected to select An. stephensi endemic countries in 2011, prior to initial detection in Africa, 2) develop a ranked prioritization list of countries based on likelihood of An. stephensi introduction for 2016 and 2020 based on maritime trade alone and maritime trade and habitat suitability, and 3) use network analysis to describe intracontinental maritime trade and eigenvector centrality to determine likely paths of further introduction on the continent if An. stephensi is detected in a new location. Our results show that in 2011, Sudan and Djibouti were ranked as the top two countries with likelihood of An. stephensi introduction based on maritime trade alone, and these were indeed the first two coastal countries in the HoA where An. stephensi was detected. Trade data from 2020 with Djibouti and Sudan included as source populations identify Egypt, Kenya, Mauritius, Tanzania, and Morocco as the top five countries with likelihood of An. stephensi introduction. When factoring in habitat suitability, Egypt, Kenya, Tanzania, Morocco, and Libya are ranked highest. Network analysis revealed that the countries with the highest eigenvector centrality scores, and therefore highest degrees of connectivity with other coastal African nations were South Africa (0.175), Mauritius (0.159), Ghana (0.159), Togo (0.157), and Morocco (0.044) and therefore detection of An. stephensi in any one of these locations has a higher potential to cascade further across the continent via maritime trade than those with lower eigenvector centrality scores. Taken together, these data could serve as tools to prioritize efforts for An. stephensi surveillance and control in Africa. Surveillance in seaports of countries at greatest risk of introduction may serve as an early warning system for the detection of An. stephensi, providing opportunities to limit further introduction and expansion of this invasive malaria vector in Africa.