Pipiens Pallens Mosquitoes Research Articles

Pipiserpin, a Culex factor Xa inhibitor, affects female reproductive capacity and serves as a potential target for mosquito control.

Mosquito control is still the main prevention and control measure for numerous mosquito-borne diseases causing millions of deaths each year. New strategies for mosquito control are in demand. Proteases play an important role in mosquito physiology, therefore this study explored the inhibition of a serpin (serine protease inhibitor) in mosquitoes and its effect on reproductive capacity. A factor Xa inhibitor homolog (named Pipiserpin) was amplified and identified in Culex pipiens pallens mosquitoes. We expressed a recombinant Pipiserpin protein in vitro against which a mouse antiserum was generated. We found that female mosquitoes expressed more Pipiserpin protein than male mosquitoes. After mating, female mosquitoes were fed with blood mixed with different amounts of antisera and results showed that consumption of Pipiserpin impeded ovary development and decreased eggs hatching rates compared to that of the pre-immune serum group. We identified a Culex mosquito factor Xa inhibitor, Pipiserpin, which affects female reproductive potential. Our results suggest that Pipiserpin may be a novel target for mosquito population control. The conclusions from our study on Cx. pipiens pallens might serve as a reference for the development of control measures for other mosquitoes as well. © 2022 Society of Chemical Industry.

Density and Distribution of the Mosquitoes Population Inhabiting Jeonbuk Region

Following the increase of vector-borne infectious diseases due to climate change and international exchange, the study collected mosquitoes using BL and BG twice a month for nine months, from March to November 2019, at cattle sheds, habitats for migratory birds, and city centers, after which they were classified, identified, and checked for the presence of flavivirus by using real time RT-PCR in order to classify and identify the species of disease vector mosquitoes in the Jeonbuk, estimate their population, and investigate their regional distribution and the presence of viral pathogen infection. A total of 8,234 mosquitoes were collected, which were categorized as seven genera and twelve species. In terms of environment types, the highest number of mosquitoes – 3,884 (Trap Index 215.8) – were collected at the Wanju cattle shed. In terms of mosquito populations, Anopheles spp. was the dominant species with 2,538 mosquitoes (30.9%) collected. As the dominant species, Anopheles spp. mosquitoes were first collected in April, and the greatest numbers were collected in the order of July, August, and June, with numbers gradually decreasing starting in August. In terms of the mosquito species collected in each environment, Anopheles spp. mosquitoes were collected in cattle sheds, Aedes vexans mosquitoes were collected in habitats for migratory birds, and Culex pipiens pallens mosquitoes were collected in city centers, revealing a difference in the species of mosquitoes collected depending on the environmental characteristics. The collected mosquitoes were tested for flavivirus using real time RT-PCR, but all tests turned out negative. The results of the collection confirmed the habitation of mosquitoes related to vector-borne diseases, predicting further results if an abnormal climate further progresses. The study is also expected to be used as comprehensive pest control standards for future mosquitoes.

Surveillance of Culex mosquitoes in Jiangsu Province from 2018 to 2019

To investigate the distribution and density of Culex mosquito populations and the resistance of Culex pipiens pallens to insecticides in Jiangsu Province in 2018 and 2019. During the period from June to October in 2018 and 2019, six counties (districts, cities) were sampled in southern, northern and central Jiangsu Province as surveillance sites. The density of Culex mosquitoes was measured overnight using the light trapping technique. In addition, Culex pipiens pallens mosquitoes were collected from Hai'an of Nantong City and Yandu District of Yancheng City, central Jiangsu Province, and the sensitivity of female first filial generations to dichlorodiphenyltrichloroethane (DDT), malation, proposur, beta cypermethrin and deltamethrin was tested using the standard WHO insecticide susceptibility test assay. A total of 104 423 Culex mosquitoes were captured in six surveillance sites of Jiangsu Province in 2018 and 2019, and Culex quinquefasciatus (49.11%), Culex pipiens pallens (28.38%), and Culex tritaeniorhynchus (21.04%) were predominant species. The density of Culex mosquitoes started to increase since early June, peaked in July and tended to be low in late October. Culex pipiens pallens mosquitoes captured from Hai'an was susceptible to malation, while those from Yandu District were moderately resistant to malation. Culex pipiens pallens mosquitoes from both Yandu and Hai'an were moderately resistant to proposur, and were resistant to DDT, beta cypermethrin and deltamethrin. Culex quinquefasciatus, Culex pipiens pallens and Culex tritaeniorhynchus are predominant Culex species in Jiangsu Province. Culex pipiens pallens is resistant to DT, beta cypermethrin and deltamethrin in central Jiangsu Province.

Coagulation-flocculation: a potential application for mosquito Larval Source Management (LSM)

Larval mosquitoes have a more limited home range and lower resistance to adverse environment than adults, thus can be ideal targets for vector control in some cases. Coagulation-flocculation technology, which could be used for water treatment in breeding sites of several vector mosquito species, can significantly change both the distribution of organic particles and surface sediment characteristics in water environment. The aim of this study was to explore the effect, principle and possibility of using coagulation-flocculation technology in immature mosquitoes killing. In this study, dechlorinated water was treated with Poly Aluminum Chloride (PACl, sewage treatment using), and we observed the impacts of PACl treatment on the development and survival of immature Culex pipiens pallens mosquitoes. When exposed to PACl treatment, physical effect is believed to be a main reason of coagulation-flocculation caused high larvae mortality: Ⅰ) alum floc layer increases the difficulty of larvae foraging, leads larvae starving to death; (Ⅱ) the little floc particles could attach to the lateral hair of larvae, which impede floatation process and then surface respiration by larval mosquitoes. The alum floc layer had a good killing effect on the mosquito larvae, presented the half lethal time (LT50) of 2d, the 90% lethal time (LT90) of 8.7±7.3 ∼ 14±4.5 d, and the pupation rate of 0 ∼ (6.5±0.5)%, respectively. Our results indicates alum floc, produced by PACl coagulation-flocculation, was shown to be highly active against 1st∼2nd instar larvae, the high mortality rate of immature mosquitoes as a result of physical effect. The observations suggest that coagulation-flocculation technology offers a novel potential approach to a sustainable and low-impact mosquito control method.

Diverse sensitivities of TRPA1 from different mosquito species to thermal and chemical stimuli

Temperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

Study on kdr allele mutation of Culex pipiens pallens in Zichuan District, Zibo City, Shandong Province

To study the seasonal dynamics of Culex pipiens pallens and the distribution of knockdown resistance (kdr) gene related sodium channel gene polymorphism in Zichuan District, Zibo City, Shandong Province. Cx. pipiens pallens mosquitoes were collected in Zichuan District during the peak period of mosquito vector activity from 2017 to 2018. The DNA from Cx. pipiens pallens was extracted, and the genotypes and frequencies of kdr allele mutation were detected by polymerase chain reaction. Totally 830 mosquitoes belonging to six species, including Cx. pipiens pallens, Armigeres subalbatus, Aedes albopictus, Ae. vexans, Anopheles sinensis, and Cx. tritaeniorhchus were collected in this study. The number of Cx. pipiens pallens accounted for 83.13% in total, with the density of 12.32 per lamp per night. The annual density monitoring curve of Cx. pipiens pallens showed a bimodal trend, and the peaks were observed in June and September respectively. In this study, five kdr alleles were detected at the 1 014 locus of kdr gene, with TTA (75.71%), TTT (10.00%), CTA (5.71%), TCA (4.29%), and TTC (4.29%). Two nonsynonymous nucleotide mutations were detected at site 1 014 of kdr gene, namely leucine (L1014) mutated to phenylalanine (L1014F) and serine (L1014S). The kdr gene mutation frequency (%) of Cx. pipiens pallens in Luochun Town and Taihe Town was 10.53% and 40.63%, respectively, and the difference was statistically significant ( χ2 = 8.559, P = 0.003). Cx. pipiens pallens is the dominant mosquito species in Zichuan District. In addition, two novel mutations, CTA and TTC, are identified in the voltage-dependent sodium channel gene of Cx. pipiens pallens. The kdr genotype of Cx. pipiens pallens in Zichuan area was polymorphic.

MosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

Larvicidal activity of Magnolia denudata seed hydrodistillate constituents and related compounds and liquid formulations towards two susceptible and two wild mosquito species.

Anopheles sinensis, Aedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit malaria, dengue fever and West Nile virus diseases respectively. This study was conducted to determine the toxicity of 17 constituents from Magnolia denudata seed hydrodistillate (MD-SHD) and four experimental MD-SHD liquid formulations (10-50 mg L(-1) liquid) to third-instar larvae from insecticide-susceptible Cx. p. pallens and Ae. aegypti as well as wild Ae. albopictus and An. sinensis. 2,4-Di-tert-butylphenol was the most toxic constituent (LC50 1.98-3.90 mg L(-1)), followed by linoleic acid (7.19-10.49 mg L(-1)), towards larvae of the four mosquito species. High toxicity was also produced by nerolidol, (±)-limonene, α-terpinene and γ-terpinene (LC50 9.84-36.42 mg L(-1)). The toxicity of these compounds was virtually identical towards larvae of the four mosquito species, even though An. sinensis larvae were resistant to deltamethrin and temephos. The MS-SHD 50 mg L(-1) liquid resulted in 92-100% control towards larvae of the four mosquito species, while commercial temephos 200 g L(-1) emulsifiable concentrate was almost ineffective towards An. sinensis larvae (30% mortality). Reasonable mosquito control in the aquatic environment can be achieved by MD-SHD 50 mg L(-1) liquid as a potential larvicide.

Larvicidal activity and possible mode of action of four flavonoids and two fatty acids identified in Millettia pinnata seed toward three mosquito species.

BackgroundAedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit dengue fever and West Nile virus diseases, respectively. This study was conducted to determine the toxicity and mechanism of action of four flavonoids and two fatty acids from Millettia pinnata (Fabaceae) seed as well as six pure fatty acids and four fatty acid esters toward third instar larvae from insecticide-susceptible C. pipiens pallens and A. aegypti as well as wild A. albopictus. Efficacy of 12 experimental liquid formulations containing M. pinnata seed methanol extract and hydrodistillate (0.5–10.0% liquids) was also assessed.MethodsThe contact toxicities of all compounds and 12 formulations were compared with those of two larvicides, temephos and fenthion and the commercial temephos 200 g/L emulsifiable concentrate (EC). The possible mode of larvicidal action of the constituents was elucidated using biochemical methods. Larval mortality and cAMP level were analyzed by the Bonferroni multiple-comparison method.ResultsPotent toxicity was produced by karanjin, oleic acid, karanjachromene, linoleic acid, linolenic acid, pongamol, pongarotene, and elaidic acid toward C. pipiens pallens larvae (24 h LC50, 14.61–28.22 mg/L) and A. aegypti larvae (16.13–37.61 mg/L). Against wild A. albopictus larvae, oleic acid (LC50, 18.79 mg/L) and karanjin (35.26 mg/L) exhibited potent toxicity. All constituents were less toxic than either temephos or fenthion. Structure–activity relationship indicates that the degree of saturation, the side chain length, and the geometric isomerism of fatty acids appear to play a role in determining the fatty acid toxicity. Acetylcholinesterase (AChE) is the main site of action of the flavonoids, oleic acid, and palmitic acid. The mechanism of larvicidal action of elaidic acid, arachidic acid, and behenic acid might be due to interference with the octopaminergic system. Linoleic acid and linolenic acid might act on both AChE and octopaminergic receptor. M. pinnata seed extract or hydrodistillate applied as 10% liquid provided 100% mortality toward the three mosquito species larvae and the efficacy of the liquids was comparable to that of temephos 200 g/L EC.ConclusionFurther studies will warrant possible applications of M. pinnata seed-derived products as potential larvicides for the control of mosquito populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0848-8) contains supplementary material, which is available to authorized users.

Identification of TCT, a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na+ channel of Culex pipiens pallens from Shandong Province, China

The present study aimed to investigate deltamethrin resistance in Culex pipiens pallens (C. pipiens pallens) mosquitoes and its correlation with knockdown resistance (kdr) mutations. In addition, mosquito‑resistance testing methods were analyzed. Using specific primers in polymerase chain reaction (PCR) and allele-specific (AS)-PCR, kdr gene sequences isolated from wild C. pipiens pallens mosquitoes were sequenced. Linear regression analysis was used to determine the correlation between the mutations and deltamethrin resistance. A kdr allelic gene was cloned and sequenced. Analysis of the DNA sequences revealed the presence of two point mutations at the L1014 residue in the IIS6 transmembrane segment of the voltage‑gated sodium channel (VGSC): L1014F, TTA→TTT, replacing a leucine (L) with a phenylalanine (F); L1014S, TTA→TCA, replacing leucine (L) with serine (S). Two alternative kdr-like mutations, L1014F and L1014S, were identified to be positively correlated with the deltamethrin-resistant phenotype. In addition a novel mutation, TCT, was identified in the VGSC of C. pipiens pallens. PCR and AS-PCR yielded consistent results with respect to mosquito resistance. However, the detection rate of PCR was higher than that of AS-PCR. Further studies are required to determine the specific resistance mechanism. PCR and AS-PCR demonstrated suitability for mosquito resistance field tests, however, the former method may be superior to the latter.

Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens

Follicular atresia, the degeneration of developing follicles, is always incident to normal oogenesis in both oviparous and viviparous animals. Photo- and electron-microscopic observation of degenerating follicles within developing ovaries taken from blood-fed Culex pipiens pallens mosquitoes showed gradual degradation of the internal structures including yolk granules in the oocyte. The epithelial cells, which sometimes incorporated yolk granules from the oocyte along with the shrinkage of the follicle, gradually lost their uniform columnar shape, while their integrity as a covering layer remained. In situ active caspase analysis detected active enzymes in these epithelial regions. In the latest stages of atresia where either the nurse cells or oocyte were lost, the follicle was mainly comprised of irregularly shaped epithelial cells, and some of these cells’ nuclei contained condensed chromatin peripherally, one of the characteristics of apoptotic cells. Also terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling treatment indicated that DNA fragmentation occurred in these follicles. It seems likely that in atretic follicles the epithelial cells survive to play key roles in the event, and then finally undergo their own apoptotic cell death so as to give the developmental site to the next follicle in the same ovariole.

Ovarian development induced in decapitated female Culex pipiens pallens mosquitoes by infusion of physiological quantities of 20-hydroxyecdysone together with amino acids.

Infusion of 20-hydroxyecdysone into the hemocoel of unfed decapitated female Culex pipiens pallens mosquitoes, at a very low rate of 500–2000 pg per day, often stimulated oögenesis of this species, when the hormone was infused together with amino acids. The hormone alone or amino acids alone showed no such stimulatory effect. Previous reports that using an abdomen ligated immediately after a blood meal for hormone injection reduced the quantity of 20-hydroxyecdysone needed to activate unfed female Aedes aegypti by a few thousand times, are therefore due mainly to a sufficient supply of amino acids from the midgut in the isolated abdomen.

Autogenous egg development in anautogenous Culex pipiens pallens mosquitoes

Ovaries of 1- to 4-day-old adult anautogenous Culex pipiens pallens mosquitoes were transplanted into newly-emerged females of the same species. Ovaries taken from 1-day-old females were never activated. However, ovaries taken from 2-day-old adults were all activated and 12% matured eggs “autogenously”. Development to maturity occurred more frequently as the follicular development of transplanted ovaries was more advanced, and 39% of the ovaries removed from 4-day-old donors (follicles had reached the previtellogenic resting stage) formed mature eggs. When ovaries from 4-day-old females were transplanted into 1- to 3-day-old hosts, only 1-day-old hosts could activate the implanted ovaries, but matured no eggs, and those females older than 2 days had little stimulatory effect on the implanted ovaries.

Balanced amino acid composition essential for infusion-induced egg development in the mosquito ( Culex pipiens pallens)

Amino acid mixtures, in which one or more amino acids were omitted from a basal mixture of 17 amino acids, were infused into the hemocoels of female Culex pipiens pallens mosquitoes. The resulting oögenesis was compared with previous studies of oögenesis induced by the basal mixture to evaluate the oögenic stimulatory effect of individual amino acids. Infusion of solutions deficient in isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan or valine, which are known to be absolutely essential for producing mature eggs, failed even to initiate oögenesis. Removal of arginine (also absolutely essential for egg production), cysteine, histidine or methionine from the basal mixture resulted in decreased activation of oögenesis. Single elimination of other amino acids (alanine, glycine, proline and serine) resulted in high percentages of oögenesis and egg maturation, comparable to the levels produced by basal mixture infusion. The combination of asparagine and glutamine, which were reported to increase in the hemolymph of blood-fed females, can be substituted for asparagine and glutamate in the basal mixture to give a high level of oögenesis. On the other hand, infusion of a mixture comprised of the eight amino acids absolutely essential for egg production showed only a slight stimulation of oögenesis initiation. Single amino acid infusion of isoleucine or threonine also failed to stimulate mosquito oögenesis. Thus, we have concluded that initiation of mosquito oögenesis requires an appropriate balance in increased hemolymph amino acids rather than just one or a few special trigger amino acids.