SBV Journal of Basic, Clinical and Applied Health Science
Volume 3 | Issue 4 | Year 2020

Dynamics of Transferrin 2 (Tsf2) Expression in Various Developmental Stages of Culex quinquefasciatus

Vignesh Mariappan1, Azhagu Rani Manoharan2, Agieshkumar Balakrishna Pillai3

1,3Central Inter-disciplinary Research Facility (CIDRF), Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, India
2Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

Corresponding Author: Agieshkumar Balakrishna Pillai, Central Inter-disciplinary Research Facility (CIDRF), Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, India, Phone: +91 8220028031, e-mail: agiesh.b@gmail.com

How to cite this article Mariappan V, Rani Manoharan A, Balakrishna Pillai A. Dynamics of Transferrin 2 (Tsf2) Expression in Various Developmental Stages of Culex quinquefasciatus. J Basic Clin Appl Health Sci 2020;3(4):148–153.

Source of support: Nil

Conflict of interest: None


Aim: Transferrin is an acute-phase protein known to be involved in insect immune responses. In the present study, we have assessed the transcript levels of transferrin 2 (Tsf2) in various developmental stages of vector mosquito Culex quinquefasciatus.

Materials and methods: Culex quinquefasciatus mosquitoes were reared in laboratory conditions. Total RNA was extracted from larvae, pupae, and adult mosquitoes using the trizol method. Complementary DNA (cDNA) was synthesized and utilized for gene expression analysis using quantitative real-time PCR (qRT-PCR) and the result was expressed in the percentage fold change.

Results: In this study, we observed an elevation in the fold change expression of Tsf2 in the larval to pupal stages followed by a decline in the fold of the adult stage. We speculate that the upregulation may be because of the microbial challenge or the aquatic stages are programmed to synthesis more Tsf2 to encounter the upcoming microbes present in the aquatic environment. However, the same should be ascertained by screening for the presence of microorganisms if any.

Conclusion: The study presents a dynamic change in the expression of Tsf2 during the developmental process of mosquito. The role of the protein in vector competence deserves further studies.

Keywords: Culex quinquefasciatus, Transferrin, Transferrin 2, Vector immunity..


Transferrin (Tsf) is an iron-binding glycoprotein with two iron binding sites and each located within a separate and similar lobe of protein.1 In vertebrates, transferrin is predominantly employed in iron transport in the blood and finally utilized in heme production.1,2 It delivers iron to several cells via membrane transferrin receptor, which has a very low affinity toward apotransferrin, less affinity for the monoferric form, and a very high affinity for diferric transferrin at physiological pH. After binding to the membrane receptor, transferrin is taken inside the endocytotic vesicle, and iron dissociates from transferrin and is taken into the cytoplasm, while the transferrin receptor and apotransferrin are recycled in the plasma membrane, where the apotransferrin is released into the bloodstream.3 In vertebrates, serum transferrin is categorized as an acute-phase protein because, during the infection or stress condition, the level of transferrin will either rise or fall depending upon the animal host.4 For example, the human lactoferrin level is increased in plasma during infection and indicates that it might have an antibiotic role by interfering with the utilization of iron needed for pathogen proliferation.5 Similarly, ovotransferrin found in white avian eggs have protective action against the pathogen.6

Transferrin proteins, as well as complementary DNAs (cDNAs) encoding insect Tsfs, have been identified for several insect species.7 The role of transferrin and iron metabolism in insects and other organisms are studied very little, but enough information is known to indicate that there is a distinct difference between an insect and other organisms (specifically mammals). For example, insect transferrin has been demonstrated to play a vital role in iron transport and act as antibiotic agents, antioxidants, and vitellogenic protein.8 Similarly, transferrin can also contribute to the adaptation of insects to various stresses.911 Transferrin 1 of Aedes aegypti and Drosophila melanogaster was found to be expressed in larval, pupal, and adult stage but not in embryo, with expression in Ae. aegypti were highly in the fat body where it is secreted in the hemolymph.12,13 About this line, in most organisms, iron-containing protein ferritin is stored in the cytoplasm while insect ferritin is deficient in the cytoplasm but found in the export pathway of cell and hemolymph.14 Later, transferrin 2 (Tsf2) was founded in Ae. aegypti with low iron binding capacity due to the mutation in key amino acids in the binding site. Expression of these two genes in the adult female mosquitos differs with the AaTF1 expression elevated during 24-hour post blood meal and AaTF2 expression elevated during 72-hour post blood meal when compared to sugar-fed mosquitoes.15 Other than Tsf1, two additional putative transferrins (Tsf2 and Tsf3) were identified in D. melanogaster. Similar, in Ae. aegypti and Anopheles gambiae, three additional putative transferrin have been discovered.16 Authors have earlier reported the role of transferrin 1 (Tsf1) in the innate immune response of Culex quinquefasciatus to the filarial parasite Wuchereria bancrofti.17,18 This upregulation due to pathogen suggests that insect transferrin has a role in the vector immune system.4,12 However, to the best of our knowledge, no studies are available on Tsf2 in C. quinquefasciatus. Since the transferrin molecule could be a potential target for vector immunity the present study is designed to understand the expression dynamics of Tsf2 in filarial vector C. quinquefasciatus.


Rearing of C. quinquefasciatus

Culex quinquefasciatus egg rafts (field-collected) were placed in a tray containing tap water and incubated at room temperature, under covering conditions. The temperature of about 27–30°C provides a suitable condition for the growth of mosquito eggs. Mosquito eggs were fed with organic material for their essential growth for a certain period as prescribed by Kauffman et al.19 After 2 days, mosquito eggs transformed into the larvae stage. The larvae pass through four larval instars, and toward the end of the fourth instar, they stop eating and undergo molting to give rise to pupae. After 36 hours (at 27°C), adults emerge from the pupal stage. It took 6–8 days for their complete development. Finally, the different developmental stages of C. quinquefasciatus, such as, larvae (L), pupae (P), and adults (A) were pooled stage-wise and stored in 1 mL of trizol −80°C until further use. All the samples were collected from one generation. The difference in expression patterns between developmental stages was observed in the present study.

Vector Base and Primer Designing

Culex quinquefasciatus mosquito’s Tsf2 gene sequence was searched in the vector base site (www.vectorbase.org). A particular sequence of Tsf2 gene sequences (XM001863377.1) was obtained and these sequences were used to design primers for quantitative real-time PCR (qRT-PCR). Primer 3 online software (http://bioinfo.ut.ee/primer3-0.4.0/) was used to design the specific primer for both the gene of interest (Tsf2) and endogenous genes (β-actin).

RNA Extraction and cDNA Synthesis

Total RNA was extracted using Trizol Method. NanoDrop spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) was used for assessing purity and concentration of RNA. One microgram of RNA was used to synthesize cDNA using PrimeScript RT Reagent Kit (Takara, Japan). The cDNA thus obtained was used to study the gene expression of specific endogenous genes (β-actin) and selected target gene (Tsf2) in a 40 cycle qRT-PCR.

Table 1: List of primers
Primer nameForward primerReverse primerProduct length (bp)

qRT-PCR Analysis

The primers of the endogenous gene and target gene are listed in Table 1. All reactions were performed in duplicates with a standard run protocol of initial denaturation at 95°C for 30 seconds followed by 40 cycles of denaturation (95°C for 10 seconds) and combined annealing and extension (60°C for 30 seconds). Fold change between the groups was determined using 2−∆∆CT values.

Multiple Sequence Alignment of Tsf2 between Different Species

A specific protein sequence of Tsf2 of different species (Table 2) was obtained and sequence similarity was analyzed using online CLUSTAL W software (https://www.genome.jp/tools-bin/clustalw).


The different developmental stages of C. quinquefasciatus, such as, larvae, pupae, and adults were collected and stored in a separate container. It took about 6–8 days for the complete development of adult C. quinquefasciatus from the larval stage. Early (larvae group I—1st instar) and late (larvae group II—4th instar) larvae were analyzed. Similarly, early- (pupae group I) and late-stage (pupae group II) of pupae were analyzed. Late pupae were transferred into closed test tubes. Adults upon eclosion were immediately used. (Adult insects were not maintained.)

Expression Level of Tsf2

The gene expression level of Tsf2 in different developmental stages of C. quinquefasciatus is represented in Table 3 and Figure 1. Number of animals pooled in each stage: Larvae group I (L1–10 larvae), Larvae group II (L2–10 larvae), Pupae group I (P1–10 pupae), Pupae group II (P2–10 pupae), and Adult group I (A1–10 adult). In the larval stage, the expression level of Tsf2 was less when compared to the pupal and adult stages. On the one hand, the Tsf2 expression in the pupal stage is highly elevated when compared to the adult stage. On the other hand, the expression of Tsf2 in the adult stage is decreased when compared to the pupal stage.

Table 2: Sequence ID of Tsf2 of difference species
S. noSpeciesSequence IDAmino acid
1Culex quinquefasciatusXP_001841784.1820
2Aedes aegyptiXP_021699709.1823
3Aedes albopictusA0A182GKF4783
4Anopheles gambiaeXP_315107.4813
5Drosophila melanogasterNP_524044.1819
Table 3: Expression level of transferrin 2
Sample (n = 10)Tsf2 meanActin mean∆CT∆∆CT2−∆∆CT% Fold change
Larvae group I27.29517.579.725  01100
Larvae group II26.93517.819.125−0.61.5157151
Pupae group I24.65517.6557−2.7256.6116661
Pupae group II25.01517.8757.14−2.5856.0001600
Adult group I24.816.997.81−1.9153.7711377

(% Fold change calculated using CT mean value of Tsf2 and Actin gene)

Fig. 1: Expression level of Tsf2 in larval, pupa and adult stages of C. quinquefasciatus

Homology of Tsf2—CLUSTAL W

We found that the Tsf2 of C. quinquefasciatus shares 82.75, 80.73, 70.23, and 58.45% of sequence similarity with Aedes albopictus, Ae. aegypti, An. gambiae, and D. melanogaster, respectively. The multiple sequence alignment output represents in Figure 2.


We have earlier demonstrated the role of iron-binding Tsf2, lipid-binding proteins lipophorin, other effector molecules like defensins in serpins in regulating the development of filarial parasite Wuchereria bancrofti.17,20,21 The importance of these molecules in functional genomics studies in vector competence was recently reviewed.22 In this context, the present study was designed to assess the levels of Tsf2 in C. quinquefasciatus. Molecular studies on Tsf protein from several infects has shown that, like their vertebrates homolog, they are multifunctional.7 The present study exhibited the expression of Tsf2 in the different developmental stages of C. quinquefasciatus, such as, larvae, pupae, and adults (Fig. 3). The results reveal that there was an elevation in the fold change expression of Tsf2 in the larval to pupal stages followed by a decline in the fold of the adult stage as shown in Figure 1. Based on our previous report,17 we speculate that this may be because of the microbial challenge or the aquatic stages are programed to synthesis more Tsf2 to encounter the upcoming microbes present in the aquatic environment. This shows that there is a distinct role of the Tsf2 gene and iron metabolism in the different development stages of C. quinquefasciatus. Several studies reported the elevation of mosquito Tsf when treated with heat-killed bacteria and filarial worms.4,23 Later studies were done with honeybees,24 moths,25,26 termites,27 and Drosophila12 exhibit that Tsf is upregulated upon wounding and bacterial/fungal infection. Together indicates that transferrin plays a major role in vector immunity. In our result, the expression level of Tsf2 is very high in the pupal stage of C. quinquefasciatus indicates that the pupae stage may be highly resistant to bacterial/fungal infection when compared to larvae and adult stage. Similarly, the upregulation of Tsf in mosquito in blood-fed female mosquito follows closely the decrease in juvenile hormones (JH).28 This suggests that JH suppresses the mosquito TSF expression has shown in cockroaches, the bean bug (Riptortus pedestris), and honeybees.24,2830 Together shows that transferrin is regulated by JH31,32 and JH may be one of the reasons the upregulation and downregulation of Tsf protein during the different developmental stages of C. quinquefasciatus accompanied by microbial challenges.

Fig. 2: Homology sequence alignment output of Tsf2 in five different species of Diptera

Fig. 3: Developmental stages of C. quinquefasciatus


The present study showed the dynamics of Tsf2 expression in the different developmental stages of C. quinquefasciatus. The elevation in the fold change expression of Tsf2 in the larval to pupal stages followed by a decline in the fold of the adult stage may be because of the microbial challenge or the aquatic stages are programd to synthesis more Tsf2 to encounter the upcoming microbes present in the aquatic environment via JH. However, the same should be ascertained by screening for the presence of microorganisms if any. Together based on the dynamic changes in the gene expression if Tsf2 during various developmental stages shows that the protein may be involved in innate immune responses of the vector mosquito. In addition, this difference in the expression of Tsf2 in C. quinquefasciatus may be also due to a distinct role of the transferrin gene in iron metabolism. However, the precise role of this gene remains to be determined.


The authors would like to thank Prof Adithan C, for providing “Dr Vany Adithan Ph.D. Fellowship” to carry out the research work. We would like to thank Sri Balaji Vidyapeeth (Deemed to be University) for providing the basic facility and instrumentation.


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