Transferrin is a defence protein known to be up-regulated upon infection of parasites/pathogens in Aedes aegypti mosquito. However, no information is available on its up-regulation in Culex quinquefasciatus, the vector of bancroftian filarial parasite. In the present study, enhancement of transferrin in C. quinquefasciatus infected with Wuchereria bancrofti is demonstrated through amplification of the specific mosquito transcript, its sequencing, cloning, and expression. By using two oligonucleotide primers, a 950-bp polymerase chain reaction product was obtained from the first strand cDNA made from RNA of C. quinquefasciatus infected with W. bancrofti. A 707-bp sequence encoding the mature portion of transferrin was confirmed by sequencing the product. This is the first report of transferrin expression in C. quinquefasciatus. The deduced amino acid sequence shared 85% homology with A. aegypti transferrin precursor molecule. Western blot analysis of haemolymph proteins of infected C. quinquefasciatus with antibodies raised against recombinant transferrin protein showed binding to a 66-kDa protein, confirming its identity as transferrin. Hence, this molecule also could be added to the list of immune molecules of C. pipiens group, such as the defensin, gambicin, and cecropin, which are already known.

The mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is the vector of the filarial parasite Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), which causes human bancroftian filariasis. Information on the mosquito humoral response against the filarial parasite during the process of its infection and development is important, as it decides the vector competence of the mosquito. Visible changes in the haemocyte population of mosquito, if any, will be an indicator of the possible humoral factors. The present study was aimed at investigating changes in the populations of various types of haemocytes of Cx. quinquefasciatus following infection with W. bancrofti. On day 2 post-feeding on microfilaraemic blood, the haemolymph perfusate of infected mosquitoes with L1 stage of the parasite showed 44.1% granulocytes, 42% prohaemocytes and 13.9% plasmatocytes, whereas that of the control mosquitoes fed on amicrofilaraemic blood showed 63.4% plasmatocytes, 22.2% prohaemocytes and 14.4% granulocytes. Differences in the population numbers of haemocyte types between the infected and control were significant (P > 0.05). However, the mosquitoes examined on day 6 post-feeding, when the parasite was in L2 stage, did not show any such changes. But, similar changes reappeared on day 12 in mosquitoes with L3 stage of the parasite. The observed haemocyte population changes indicate the possibility of some amount of humoral immune response, through the production of certain immune molecules, in Cx. quinquefasciatus infected with W. bancrofti. The nature and exact role of such a response on the filarial parasite development need further investigation.

Bacillus sphaericus is a bio-control agent effective against Culex quinquefasciatus, the vector of bancroftian filariasis. Apart from its larvicidal effect, there are reports of reduced infection of filarial parasites in mosquitoes exposed to it. In the present study, adults of Cx. quinquefasciatus emerged from B. sphaericus treated larvae were fed on blood samples positive for microfilariae of Wuchereria bancrofti and examined at various time intervals to assess the infection level. The rate of infection was reduced from 95% on day 1 post-feeding to 75% on day 13, when fed with blood sample containing 41 mf/20 μl. The mean parasite burden was also reduced from 4.9 per mosquito on day 1 to 2.15 on day 13. When fed with another sample (30 mf/20 μl), the infection was reduced from 100% on day 1 to 80% on day 13. Reduction in parasite burden was 4.0 to 1.75. Abnormally developed second-stage larvae of the parasite were seen in treated mosquitoes. Thus, the results indicated adverse effect of B. sphaericus treatment on infection and development of the filarial parasite in mosquitoes. The possible reason for the parasite regulation was studied through the assessment of the carryover of the bacterium as well as its toxins to the surviving mosquitoes. The presence of B. sphaericus was determined through plating of homogenate of survived mosquitoes on NYSM agar. Toxic protein was detected through immunoblotting. The bacterium as well as its 41.9-kDa toxic protein was found to be transmitted from larvae to adults and affected the parasite development, directly by the toxin or indirectly by eliciting humoral immune response of the mosquito.

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Several antimicrobial/parasitic peptides are known to be upregulated in mosquitoes upon infection with parasites. The aim of this study was to identify immune-responsive genes in the vector mosquito, Culex quinquefasciatus Say (Diptera: Culicidae) against the human lymphatic filarial parasite, Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae). Suppression subtractive hybridization was performed using RNA from filarial infected and non-infected mosquitoes to obtain differentially expressed transcripts, and their identities were confirmed through reverse transcriptase polymerase chain reaction (RT-PCR). Out of 23 clones selected from the suppression subtractive library, three corresponded to antimicrobial peptide genes, defensins, and four corresponded to regulatory serpin peptide genes. RT-PCR using defensin-specific primers and sequencing of the product showed a 284-bp defensin cDNA. Sequence alignment with defensins of the mosquitoes Anopheles gambiae s.s. Giles and Aedes aegypti (L.) showed maximum homology with the former. Similarly, that of serpin-specific primers showed a 406-bp cDNA encoding serpins. Sequence alignment showed maximum homology with that of An. gambiae, as in the case of defensins. Hence, this investigation revealed upregulation of defensins and serpins in Cx quinquefasciatus infected with W. bancrofti. Antimicrobial peptide genes such as defensins may have limited or no specific role in regulating parasite development. Serpins may prove to be facilitating molecules, by regulating melanization of the parasite. However, the exact functions of these molecules in the immune system of the vector mosquito are yet to be investigated.

In mosquitoes, including Culex quinquefasciatus, immune molecules are known to be upregulated or produced de novo upon exposure to parasites or pathogens. These molecules are regulatory in nature acting against parasite or pathogen infection and development. Similarly, there are molecules that are upregulated to facilitate parasite development in the vector mosquitoes. Lipophorin, a major lipid transporting lipoprotein in the hemolymph of insects, is implicated as a helper molecule in the clotting mechanism and facilitator of parasite and pathogen development in mosquitoes. In the present study, upregulation of a 240 kDa protein was detected in C. quinquefasciatus infected with the human lymphatic filarial parasite, Wuchereria bancrofti. It was identified as a lipophorin through nano-Lc-MS/MS analysis. Transcription of the lipophorin receptor gene also was identified through RACE-PCR. C. quinquefasciatus is the vector of W. bancrofti, and it allows successful development of the parasite. The role of upregulated lipophorin and transcription of its receptor gene in this mosquito could be implicated as a facilitator for the parasite development.

Multiple Sequence Alignment (MSA) is a very effective tool in bioinformatics. It is used for the prediction of the structure and function of the protein, locating DNA regulatory elements like binding sites, and evolutionary analysis. This research paper proposed an optimization method for the improvement of multiple sequence alignment generated through progressive alignment. This optimization method consists of a fusion of two problem-solving techniques, divide-conquer and genetic algorithms in which the initial population of MSAs was generated through progressive alignment. Each multiple alignment was then divided vertically into four parts, three genetic operators were applied on each part of the MSA, recombination was done to reconstruct the full MSA. To estimate the performance of the method the results generated through the method are compared with well-known existing MSA methods named Clustal Ω, MUSCLE, PRANK, and Clustal W. Experimental results showed an 11-26% increase in sum_of_pair score (SP score) in the proposed method in comparison to the above-mentioned methods. SP score is the cumulative score of all possible pairs of alignment within the MSA.