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Identification and characterization of long noncoding RNAs and their association with acquisition of blood meal in Culex quinquefasciatus

Azlan A, Halim MA, Mohamad F, Azzam G

Insect Sci, 2021 Aug;28(4):917-928.
PMID: 32621332 DOI: 10.1111/1744-7917.12847

The Southern house mosquito, Culex quinquefasciatus (Cx. quinquefasciatus) is an important vector that transmit multiple diseases including West Nile encephalitis, Japanese encephalitis, St. Louis encephalitis and lymphatic filariasis. Long noncoding RNAs (lncRNAs) involve in many biological processes such as development, infection, and virus-host interaction. However, there is no systematic identification and characterization of lncRNAs in Cx. quinquefasciatus. Here, we report the first lncRNA identification in Cx. quinquefasciatus. By using 31 public RNA-seq datasets, a total of 4763 novel lncRNA transcripts were identified, of which 3591, 569, and 603 were intergenic, intronic, and antisense respectively. Examination of genomic features revealed that Cx. quinquefasciatus shared similar characteristics with other species such as short in length, low GC content, low sequence conservation, and low coding potential. Furthermore, compared to protein-coding genes, Cx. quinquefasciatus lncRNAs had lower expression values, and tended to be expressed in temporally specific fashion. In addition, weighted correlation network and functional annotation analyses showed that lncRNAs may have roles in blood meal acquisition of adult female Cx. quinquefasciatus mosquitoes. This study presents the first systematic identification and analysis of Cx. quinquefasciatus lncRNAs and their association with blood feeding. Results generated from this study will facilitate future investigation on the function of Cx. quinquefasciatus lncRNAs.

Matched MeSH terms: Culex/genetics*
First molecular genotyping of voltage gated sodium channel alleles in Culex quinquefasciatus populations in Malaysia

Low VL, Chen CD, Lim PE, Lee HL, Tan TK, Lim YA, et al.

Pestic Biochem Physiol, 2013 Sep;107(1):127-31.
PMID: 25149246 DOI: 10.1016/j.pestbp.2013.06.004

A nationwide investigation was performed to detect the presence of 1014 mutation(s) in voltage gated sodium channel (kdr) gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Molecular genotyping of kdr mutation was performed via a modified three tubes allele-specific-polymerase chain reaction (AS-PCR) and direct sequencing of kdr gene. Based on the results of AS-PCR, homozygous susceptible (SS) genotype was found in nine out of 14 populations with 38 individuals from a total sample size of 140. Heterozygous (RS) genotype was most predominant (99 individuals) and distributed across all study sites. Homozygous resistance (RR) genotype was detected in Perak (one individual) and Selangor (two individuals). The resistance kdr allele frequencies ranged from 0.1 to 0.55, with the highest being detected in Cx. quinquefasciatus population from Selangor. This study has documented the first field-evolved instance of 1014F mutation in Malaysian mosquitoes and the findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia.

Matched MeSH terms: Culex/genetics*
Gender beyond male and female: Occurrence of a gynandromorph in the Japanese encephalitis vector Culex sitiens (Diptera: Culicidae)

Low VL, Wong ML, Liew JWK, Pramasivan S, Jeyaprakasam NK, Vythilingam I

Acta Trop, 2020 Jan;201:105207.
PMID: 31586449 DOI: 10.1016/j.actatropica.2019.105207

A gynandromorph of Culex sitiens Wiedemann (Diptera: Culicidae) was attracted to a human during a mosquito surveillance programme conducted in Kuala Lipis, Pahang, Malaysia on July 20, 2019. Gynandromorphism was observed in antennae, maxillary palps, legs and wings of the specimen, with distinct male characters on the left and female characters on the right, though the left maxillary palp is slightly shorter than the proboscis of a typical male. The abdomen, however, displays well-developed male genitalia. This study represents the first report of oblique gynandromorphism in Cx. sitiens, one of the vectors of Japanese encephalitis in Asia.

Matched MeSH terms: Culex/genetics*
Fulltext Characterization on malathion and permethrin resistance by bioassays and the variation of esterase activity with the life stages of the mosquito Culex quinquefasciatus

Selvi S, Edah MA, Nazni WA, Lee HL, Azahari AH

Trop Biomed, 2007 Jun;24(1):63-75.
PMID: 17568379 MyJurnal

Larvae and adults of Culex quinquefasciatus were used for the test undertaken for malathion resistant strain (F61 - F65) and permethrin resistant strain (F54 - F58). The results showed that the LC50 for both malathion (F61 - F65) and permethrin (F54 - F58) resistant Cx. quinquefasciatus increased steadily throughout the subsequent five generations, indicating a marked development of resistance. The adult female malathion resistant strain have developed a high resistance level to malathion diagnostic dosage with a resistance ratio of 9.3 to 17.9 folds of resistance compared with the susceptible Cx. quinquefasciatus. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developed at a higher rate in adult females compared to permethrin. Enzyme-based metabolic mechanisms of insecticide resistance were investigated based on the biochemical assay principle. From the results obtained obviously shows that there is a significant difference (p < 0.05) in esterase level in both malathion and permethrin selected strains. Female malathion selected strain has the higher level of esterase activity compared to the female permethrin selected strain at (0.8 to 1.04) alpha-Na micromol/min/mg protein versus (0.15 to 0.24) alpha-Na micromol/min/mg protein respectively. This indicated increased level of non-specific esterase is playing an important role in resistance mechanism in female malathion selected strain. Permethrin selected strain exhibited non-specific esterase activity at a very low level throughout the different life stages compared to malathion selected strain. This study suggests that life stages play a predominant role in conferring malathion and permethrin resistance in Cx. quinquefasciatus.

Matched MeSH terms: Culex/genetics
Fulltext Determination of homozygous susceptible strain in Culex quinquefasciatus (Say), using single raft sib-selection method

Hidayati H, Nazni WA, Mohd SA

Trop Biomed, 2008 Apr;25(1):75-9.
PMID: 18600207 MyJurnal

The standard laboratory strain was found to be heterozygous for susceptibility. Hence, an attempt was made to obtain a homozygous susceptible strain in Culex quinquefasciatus (Say) using single raft sib-selection method. Lab-bred females of Cx. quinquefasciatus from insectariums, Unit of Medical Entomology were used in the experiment. After blood feeding Cx. quinquefasciatus mosquitoes laid eggs in raft form, ten rafts selected randomly for the test. Each egg raft was introduced into a plastic tray from number one to number ten. Twenty-five third stage larvae from each tray were exposed to 17.5 microl from 500mg/l malathion in a paper cup label number 1 to number ten. In the bioassay, which had 100% mortality, the respective larva in that particular tray was bred to adult stage for the following generation. Less than 7days old female mosquitoes that emerged from F(0) were used in the test. The F(0) and the subsequent adult and larval stage generations were subjected to adult and larval bioassay. After selection for about 10 generations, a homozygous susceptible strain in Cx. quinquefasciatus was obtained.

Matched MeSH terms: Culex/genetics
Fulltext Resistance development and insecticide susceptibility in Culex quinquefasciatus against selection pressure of malathion and permethrin and its relationship to cross-resistance towards propoxur

Selvi S, Endah MA, Nazni WA, Lee HL, Azahari AH

Trop Biomed, 2005 Dec;22(2):103-13.
PMID: 16883275

To determine resistance level and characterize malathion and permethrin resistance in Culex quinquefasciatus, two methods were used namely: WHO procedures of larval bioassay to determine the susceptibility of lethal concentration (LC) and adult bioassay to determine the lethal time (LT) which are resistant to malathion and permethrin. These mosquito strains were bred in the Insectarium, Division of Medical Entomology, IMR. Thousands of late fourth instar larvae which survived the selection pressure to yield 50% mortality of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected to the subsequent 10 generations in the test undertaken for malathion resistant strain (F61 - F70) and permethrin resistant strain (F54 - F63). Selection pressure at 50% - 70% mortality level was applied to the larvae of each successive generation. The rate of resistance development and resistance ratio (RR) were calculated by LC5 0 for larval bioassay and LT50 value for adult bioassay. The lab bred Cx. quinquefasciatus was used as a susceptible strain for comparison purpose. The adult bioassay test was carried out by using diagnostic dosages of malathion 5.0%, permethrin 0.75% and with propoxur 0.1%. All bioassay results were subjected to probit analysis. The results showed that LC5 0 for both malathion (F61 - F70) and permethrin (F54 - F63) resistant Cx. quinquefasciatus increased steadily to the subsequent 10 generations indicating a marked development of resistance. The adult female malathion resistant strain have developed high resistance level to malathion diagnostic dosage with resistance ratio 9.3 to 9.6 folds of resistance. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developing at a higher rate in adult females compared to permethrin. Female adults exposed to 2 hours of exposure period for propoxur 0.1% showed presence of cross-resistance among the both strains of mosquitoes towards propoxur and it was indicated by 70%-100% mortality at 24 hours post-recovery period.

Matched MeSH terms: Culex/genetics
Fulltext Rate of resistance development in wild Culex quinquefasciatus (Say) selected by malathion and permethrin

Nazni WA, Lee HL, Sa'diyah I

Southeast Asian J Trop Med Public Health, 1998 Dec;29(4):849-55.
PMID: 10772575

Wild caught female Culex quinquefasciatus (Say) from Kuala Lumpur were blood fed and reared in the insectarium. The late third stage of the F1 larvae which survived the high selection pressure of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected in the subsequent 9 generations to higher selection pressure. The rate of resistance development were measured by LC50 value of larval bioassay, LT50 value of adult bioassay and the frequency of the elevated esterase levels. In another set of experiments using the same batch of Culex mosquitos, the larvae were not exposed to any insecticides and the decrease in resistance rate was monitored in each subsequent 9 generations by using similar methods. The heterozygous standard laboratory strain was selected for susceptibility using the single raft sib-selection method. The result showed that the field collected F1 generation was 96.0 and 6.3 fold more resistant to malathion and permethrin, respectively. After selection for about 9 generations the resistance ratio to malathion and permethrin was 6.2 and 767.3 fold more compared to the LC50 values of F1 generations, respectively. Esterase in F1 larvae was 6.0 fold more than the standard laboratory strain.

Matched MeSH terms: Culex/genetics
Fulltext Monitoring resistance gene frequencies in Malaysian Culex quinquefasciatus Say adults using rapid non-specific esterase enzyme microassays

Lee HL, Tadano T

Southeast Asian J Trop Med Public Health, 1994 Jun;25(2):371-3.
PMID: 7855659

The ability to identify the occurrence of different resistance genotypes in field populations of mosquito is considered important for the purpose of optimising chemical control operations. The recent development of rapid microassays of enzymes responsible for resistance has provided a means for rapidly assessing the genetic background of target mosquito populations. This concept is the topic of investigation in this study. Non-specific esterase activity, which is responsible for the resistance to organophosphates in Malaysian Culex quinquefasciatus Say adults, was determined in 3 field populations from Kuala Lumpur City using rapid enzyme assay. The optical density results were used to estimate the genotypic frequencies of the populations. Subsequently, time-dependent changes in the various frequencies were determined. Such techniques allowed rapid assessment of resistance genotypes for decision-making and its possible use in insect control merits further investigation.

Matched MeSH terms: Culex/genetics*
First molecular genotyping of insensitive acetylcholinesterase associated with malathion resistance in Culex quinquefasciatus Say populations in Malaysia

Low VL, Chen CD, Lim PE, Lee HL, Lim YA, Tan TK, et al.

Pest Manag Sci, 2013 Dec;69(12):1362-8.
PMID: 23404830 DOI: 10.1002/ps.3512

Given that there is limited available information on the insensitive acetylcholinesterase in insect species in Malaysia, the present study aims to detect the presence of G119S mutation in the acetylcholinesterase gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia.

Matched MeSH terms: Culex/genetics
Mitochondrial DNA analyses reveal low genetic diversity in Culex quinquefasciatus from residential areas in Malaysia

Low VL, Lim PE, Chen CD, Lim YA, Tan TK, Norma-Rashid Y, et al.

Med Vet Entomol, 2014 Jun;28(2):157-68.
PMID: 23848279 DOI: 10.1111/mve.12022

The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1-A3) and four haplotypes (B1-B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1-AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1-AA4) by the COI gene and nine haplotypes (BB1-BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus.

Matched MeSH terms: Culex/genetics*
Fulltext New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Ang JXD, Kadir KA, Mohamad DSA, Matusop A, Divis PCS, Yaman K, et al.

Parasit Vectors, 2020 Sep 15;13(1):472.
PMID: 32933567 DOI: 10.1186/s13071-020-04345-2

BACKGROUND: Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak.
METHODS: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis.
RESULTS: Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo.
CONCLUSIONS: Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

Matched MeSH terms: Culex/genetics

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