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.

Effects of Peganum harmala L. Seed Extract on Culex pipiens (Diptera: Culicidae)

Mosquito-borne diseases result in the loss of life and economy, primarily in subtropical and tropical countries, and the emerging resistance to insecticides is increasing this threat. Botanical insecticides are promising substitutes for synthetic insecticides. This study evaluated the larvicidal and growth index of Culex pipiens of four solvent extracts of Terminalia chebula, Aloe perryi, and Peganum harmala against Cx. pipiens. None of the 12 extracts exhibited larvicidal potential against third instars except the ethyl acetate extract of P. harmala. After 24 h of exposure, the LC50 value was 314.88 ppm, and the LC90 value was 464.19 ppm. At 320 ppm, the hatchability was 25.83%, and it resulted in 100% mortality. In addition, the eggs treated with the EtOAc extract of P. Harmala exhibited a long larval period compared with the control. The larval period continued for 12 days, and the pupal period took three days in the treatment groups. The growth index data also exhibited a decrease (0.00–7.53) in the treated groups compare with 8.5 in the control. The transformation of eggs into adults decreased with increasing concentrations. This paper is the first report on the development and growth index of Cx. pipiens potential using P. harmala seeds.

Immunohistochemical Characterization of the Nervous System of Culex pipiens (Diptera, Culicidae)

Arthropod-borne diseases represent one of the greatest infection-related threats as a result of climate change and globalization. Repeatedly, arbovirus-infected mosquitoes show behavioral changes whose underlying mechanisms are still largely unknown, but might help to develop control strategies. However, in contrast to well-characterized insects such as fruit flies, little is known about neuroanatomy and neurotransmission in mosquitoes. To overcome this limitation, the study focuses on the immunohistochemical characterization of the nervous system of Culex pipiens biotype molestus in comparison to Drosophila melanogaster using 13 antibodies labeling nervous tissue, neurotransmitters or neurotransmitter-related enzymes. Antibodies directed against γ-aminobutyric acid, serotonin, tyrosine-hydroxylase and glutamine synthetase were suitable for investigations in Culex pipiens and Drosophila melanogaster, albeit species-specific spatial differences were observed. Likewise, similar staining results were achieved for neuronal glycoproteins, axons, dendrites and synaptic zones in both species. Interestingly, anti-phosphosynapsin and anti-gephyrin appear to represent novel markers for synapses and glial cells, respectively. In contrast, antibodies directed against acetylcholine, choline acetyltransferase, elav and repo failed to produce a signal in Culex pipiens comparable to that in Drosophila melanogaster. In summary, present results enable a detailed investigation of the nervous system of mosquitoes, facilitating further studies of behavioral mechanisms associated with arboviruses in the course of vector research.

West nile fever case in neurological practice

West Nile fever is a zoonotic arbovirus mosquito- and tick-borne infection, which occurs in humans in the form of an acute febrile illness with symptoms of general intoxication, in severe cases – with the central nervous system damage. West Nile virus belongs to the Flaviviridae family.After the start of Russians’ mass tourism to tropical and subtropical regions, number of cases of the disease in Russia has increased, especially in the south, where the virus is more viable. The infection mainly affects birds but also people and many mammals (bats, cats, dogs, skunks, squirrels, rabbits, etc.), which become infected after the bite of a mosquito of the genus Culex (Culex pipiens) carrying the disease, ixodid or argas mite. A clinical case of hemorrhagic stroke caused by West Nile fever is presented in the article.

First Evidence of West Nile Virus Overwintering in Mosquitoes in Germany

Mosquitoes collected from mid-December 2020 to early March 2021 from hibernacula in northeastern Germany, a region of West Nile virus (WNV) activity since 2018, were examined for WNV-RNA. Among the 6101 mosquitoes tested in 722 pools of up to 12 specimens, one pool of 10 Culex pipiens complex mosquitoes collected in early March 2021 in the cellar of a medieval castle in Rosslau, federal state of Saxony-Anhalt, tested positive. Subsequent mosquito DNA analysis produced Culex pipiens biotype pipiens. The pool homogenate remaining after nucleic acid extraction failed to grow the virus on Vero and C6/36 cells. Sequencing of the viral NS2B-NS3 coding region, however, demonstrated high homology with virus strains previously collected in Germany, e.g., from humans, birds, and mosquitoes, which have been designated the East German WNV clade. The finding confirms the expectation that WNV can overwinter in mosquitoes in Germany, facilitating an early start to the natural transmission season in the subsequent year. On the other hand, the calculated low infection prevalence of 0.016–0.20%, depending on whether one or twelve of the mosquitoes in the positive pool was/were infected, indicates a slow epidemic progress and mirrors the still-hypoendemic situation in Germany. In any case, local overwintering of the virus in mosquitoes suggests its long-term persistence and an enduring public health issue.