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Table of Contents
Year : 2018  |  Volume : 55  |  Issue : 2  |  Page : 89-97

Transcriptional responses of attractin gene in the mosquito Anopheles culicifacies: A synergistic neuro-olfactory regulation

1 Laboratory of Host-Parasite Interaction Studies, ICMR–National Institute of Malaria Research; Department of Biotechnology, Delhi Technological University, New Delhi, India
2 Department of Biotechnology, Delhi Technological University, New Delhi, India
3 Laboratory of Host-Parasite Interaction Studies, ICMR–National Institute of Malaria Research, New Delhi, India

Date of Submission27-Sep-2017
Date of Acceptance29-Nov-2017
Date of Web Publication1-Oct-2018

Correspondence Address:
Rajnikant Dixit
Scientist ‘D’, Laboratory of Host-Parasite Interaction Studies, ICMR–National Institute of Malaria Research, New Delhi–110 077
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.242569

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Background & objectives: Attractin, is a large multi-domain protein which has regulatory functions in multiple physiological processes and thus have strong therapeutic potential. In invertebrates, it was first identified as a water-borne protein pheromone that plays important role in chemical communication and coordinates reproductive activities. But its role in mosquitoes/insects remains unknown. Our unexpected discovery of attractin homolog from the olfactory tissue of Anopheles culicifacies mosquito prompted us to investigate the possible role of Ac-attractin (Ac-atrn) in diverse behavioural responses, e.g. feeding, mating and other non-genetic stresses.
Methods: A homology search analysis was performed to identify the full length attractin (Ac-atrn) gene of Anopheles culicifacies mosquito. To unravel its molecular function during external and internal stresses, extensive real-time PCR was performed in the neuro-olfactory tissues of the adult mosquitoes as well as in the larval stages. Further, a behavioural assay was conducted to elucidate its role in mosquitoes mating behaviour.
Results: The results indicated that Ac-atrn is a 3942 bp long transcript which encodes a 1313 amino acid protein, having multiple domains including CUB, EGF, Keltch, etc, with 80–90% homology to other insect/mosquito homologs. Ac-atrn gene was dominantly expressed in the young larvae and its expression was elevated in response to the fresh food supply in the starved larvae. Cold stress temporarily arrested the expression of Ac-atrn gene. In case of adult mosquitoes, olfactory and brain tissue showed relatively higher expression of Ac-atrn than reproductive organs. Although, starvation did not yield significant changes in olfactory tissues, but aging and nutritional stress modulated Ac-atrn expression in the brain tissue. Furthermore, a circadian rhythm dependent change in the expression of Ac-atrn of virgin and mated mosquitoes (both sexes), indicates that Ac-atrn might also have a pheromone guided role during swarm formation and mating behaviour.
Interpretation & conclusion: The relative expression profiling of Ac-atrn gene in the larvae during nutritional and cold stress suggested its possible role in mediating chemical communication towards the food source and in thermal regulation of young larvae. Similarly, it might have crucial regulatory role in the stress management and survival of adult mosquitoes. The results revealed that Ac-atrn gene is a global regulator of many physiological processes in mosquitoes including stress response and mating behaviour and thus might be a potential target to design novel intervention strategy against mosquitoes.

Keywords: Anopheles; attractin; malaria; mating; mosquito; olfactory; stress

How to cite this article:
Das De T, Hasija Y, Dixit R. Transcriptional responses of attractin gene in the mosquito Anopheles culicifacies: A synergistic neuro-olfactory regulation. J Vector Borne Dis 2018;55:89-97

How to cite this URL:
Das De T, Hasija Y, Dixit R. Transcriptional responses of attractin gene in the mosquito Anopheles culicifacies: A synergistic neuro-olfactory regulation. J Vector Borne Dis [serial online] 2018 [cited 2018 Oct 21];55:89-97. Available from: http://www.jvbd.org/text.asp?2018/55/2/89/242569

  Introduction Top

Attractin (atrn) is a member of the CUB family of type I glycosylated protein, having multiple domains. It is widely expressed in different types of tissues in the body and facilitate cell adhesion[1],[2]. In addition, it acts as a guidance molecule in regulating multiple physiological and pathological processes[1]. Attractin was first identified as an autosomal recessive gene, which suppresses Aγ-induced pigmentation in mice[3]. It primarily exists in two forms, a transmembrane and a secreted form[4]. Both the secreted and membrane type atrn protein contains two epidermal growth factor (EGF) domains, a CUB domain, a C-type lectin domain, and two laminin-like epidermal growth factor domains. It is predicted that these functional domains are present in the extracellular portion of the membrane type atrn[5]. It has been reported that the secreted form of atrn is more predominant in the body, that exists as serum glycoprotein. Earlier reports have suggested that atrn is also involved in crucial immune physiological process. It is synthesized by activated T-cells and released into the extracellular region, where it modulates the interaction between the monocytes and the T-cells and thus facilitates antigen presentation process effectively[1]. Membrane type atrn has a novel function in myelination and has been found to prevent neuronal damage during oxidative stress[2]. Mutation of this gene is responsible for the zitter mutant phenotypes in mouse which includes the symptoms of hypomyelination and vacuolation in the central nervous system (CNS)[6]. It is also involved in hair pigmentation[7],[8]. Both the membrane and the secreted form of atrn are found in humans, however, only the membrane type has been identified in mice, where it is also reported to regulate the proper functioning of sperm in the testes[5]. Age-dependent progressive loss in functioning of atrn are responsible for test is vacuolation and diminished sperm function[5]. Further, a knockdown study of atrn gene in mice showed that it might be responsible for regulating other associated proteins in the sertoli cells of testes, which work cumulatively for the proper functioning of sperms[9]. In invertebrates, it was first identified as water-borne pheromone in the mollusc Aplysia californica, which is synthesized and secreted by the female reproductive organ to guide and attract sperms and facilitate sperm-egg interaction for successful fertilization within a distance of 10 m[10]. In Aplysia, atrn is a 58–60 kDa secreted protein which lacks the transmembrane domain and belongs to a member of multiple pheromone proteins, facilitating chemical communication, mate attraction, egg-sperm fertilization and many other functions[11].

Mosquitoes that transmit many infectious diseases are central to the control and management of vector borne diseases, causing heavy human health toll annually. Interfering their behavioural activities such as feeding, mating, breeding with novel approaches is valuable to counter attack the disease transmission. Pheromones, that are small peptides secreted by many tissues play a crucial role in mosquito's social behaviour and consequently their life cycle maintenance[12],[13],[14]. So far, there is very limited knowledge on the mosquito pheromones characterization, in principal due to challenges in in vitro identification, purification and characterization. In the present study, a unique transcript encoding the atrn homolog was characterized from the olfactory tissue of Anopheles culicifacies mosquito, the main rural malarial vector in India[15]. A comprehensive in silico analysis and extensive transcriptional profiling in response to feeding, mating and other stress responses provided evidences that Ac-attractin might have an important role in the management of neuro-olfactory regulation and stress management, enabling mosquito's successful survival.

  Material & Methods Top

Mosquito rearing and maintenance

The cyclic colony of An. culicifacies, sibling species A was reared and maintained under standard laboratory conditions at 28 ± 2°C and 80% relative humidity in central insectary facility as mentioned in previous studies[16],[17].

Ethical statement: All protocols for rearing and maintenance of the mosquito culture were approved by ethical committee of the National Institute of Malaria Research, New Delhi (NIMR/IAEC/2017-1/07; dated 28/09/2017).

Tissue collection and RNA extraction

From the 5 min cold (4 °C) anesthesized, 5–6 day-old sugar fed adult male and female An. culicifacies mosquito's various tissues were collected according to the assays. The tissues included olfactory organs (antennae, maxillary palp, proboscis and labium), brain, and reproductive organs (male reproductive organs: Testes and male accessory gland; female reproductive organs: Spermathecae and atrium). The tissues were dissected and collected in TRIzol. The developmental stages of An. culicifacies, viz. egg, larvae (stages I–IV) and pupae were also collected in TRIzol reagent after removal of extra water through filter paper. Total RNA was isolated by the standard TRIzol method as described previously[16], [18].

Bioinformatic analysis

The putative Ac-atrn gene was identified as a partial contig during analysis of olfactory tissue transcriptome data from the naïve sugar fed adult female mosquitoes[19]. Initial BLASTX analysis against the NCBI NR database showed significant hits to the putative atrn like proteins of multiple mosquitoes, insects and other invertebrate species. To retrieve full-length Ac-atrn transcript, BLASTN analysis was performed against the genome predicted transcript database of the An. culicifacies mosquito, which is available from www.vectorbase.org. Domain prediction, multiple sequence alignment, and phylogenetic analysis were done using multiple softwares as described earlier[18].

External stress response of I instar larvae

To understand the response of Ac-atrn under the external stressed condition, the I instar larvae were kept overnight at 4 °C. For heat treatment, the larvae were exposed to 42 °C for 4 h. Then, to track the Ac-atrn expression under the nutritional stressed condition, the freshly hatched I instar larvae were taken and divided into three groups, each containing 30–40 larvae. The first group of larvae were collected immediately after hatching as control batch, the second group of larvae were kept overnight in starvation, without any food supply; and the third group was provided with 20–30 mg of a mixture of fish food and dog biscuit. After 24 h, 30 starved larvae were collected in TRIzol and the remaining starved larvae were provided with food to recover from the nutritional stress. Following 24 h of maintenance, these remaining larvae were collected in TRIzol for relative gene expression analysis.

External stress response of adult mosquitoes

To expose the adult male and female mosquitoes with extrinsic stress, the adult mosquitoes were kept starved for overnight. Next day, the olfactory and brain tissue were dissected from starved and their respective sugarfed mosquitoes (control). Further, a detailed time course experiment of the starvation assay was performed, and the brain tissue was dissected and collected in TRIzol to analyze the Ac-atrn expression in brain tissue during starvation.

Behavioural and molecular assay

To track the possible role of Ac-atrn in mating behaviour, an assay was designed monitoring the sex-specific changes of the behavioural activities occurring in response to day/night cycle in the 5–6 days-old mosquitoes. As per the assay design, olfactory and reproductive tissues were collected from either virgin and/or mixed cage mosquitoes of both the sexes. For the assurance of mating success, an equal number of male and female virgin mosquitoes were mixed in a single cage at morning (1000 hrs), and the tissues (both olfactory and reproductive) were collected at evening (1700 hrs) and next morning (1000 hrs) from the cage.

cDNA preparation and gene expression analysis

A 1 μg of total RNA was used to synthesize the first strand cDNA using Verso cDNA synthesis kit (Thermo Scientific, USA) as described in the manufacturer's protocol. Routine differential gene expression analysis was carried out by RT-PCR. Relative gene expression analysis was done using SYBR green qPCR (Thermo Scientific, USA) master mix and Illumina eco real-time PCR machine. The four step PCR cycle included an initial denaturation at 95 °C for 5 min, 40 cycles of 10 sec at 95 °C, 15 sec at 52 °C, and 22 sec at 72 °C. Fluorescence reading was recorded at 72 °C after each cycle. In final steps, PCR at 95 °C for 15 sec followed by 55 °C for 15 sec and again 95 °C for 15 sec were completed before generating melting curve. Reproducibility of the result was ensured by repeating the experiments with three independent biological replicates. Throughout the experiment, RpS7 and actin genes were used as an internal control (as they are constitutively expressed in all conditions) and the relative quantification data were analyzed by 2-ΔΔCt method[18]. Statistical analysis of differential gene expression was done using student's t-test.

  Results Top

Identification, annotation and molecular characterization of Ac-atrn

A 1070 bp long unique transcript (Accession# MF599469) was identified during annotation of largescale RNA-Seq transcriptomic database originating from the olfactory tissues of the adult female An. culicifacies mosquitoes[19]. A detailed in-silico analysis predicted a 3942 bp long full-length Ac-atrn transcript (ACUA00-4165-RA), encoding 1313 amino acid long peptide. A function prediction analysis unravelled that putative Ac-atrn is a multidomain protein containing at least five domains. These include two calcium binding EGF domain, one cysteine rich Plexin repeat-PSI domain, one nitrile specific-PLN01293 and one extracellular CUB domain [Figure 1]a and [Table 1]. A conserved domain architecture retrieval (CDART) tool search analysis predicted the presence of a similar but varying number of domain containing protein homologs in diverse organisms [Table 2], supporting their evolutionary conserved role.
Figure 1: Molecular analysis of An. culicifacies attractin (Ac-atrn) gene: (a) Domain architechture of Ac-atrn gene; (b) Schematic representation of the genomic architecture of the Ac-atrn gene. Five green coloured boxes indicates the exons, blue line denotes the introns and +1 mark the transcription initiation site. The size of the exons and introns correspond to the size of the boxes and lines; (c) Multiple sequence alignment of a segment of An. culicifacies attractin with other mosquitoes, flies, invertebrates and vertebrates homologs showing high degree of conservation in the amino acid sequence. One of the CUB domain is highlighted with horizontal red line; and (d) Phylogenetic relationship of Ac-attractin indicating that An. culicifacies attractin is clustered within the mosquito domain and have much greater similarity with mosquito attractin than flies and other insects. Attractin sequences of human, Mus musculus and invertebrate Aplysia californica were also considered in this analysis for out-group clustering.

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Table 1: Domain architecture of Ac-atrn gene

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Table 2: Identity match of Ac-atrn with other mosquitoes, insects and vertebrate homologs

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In silico analysis of the full-length transcript, revealed that Ac-atrn is a single copy gene having six exons and five introns with 50 bp flanking sequences at the 5’ upstream region and 50 bp overhang at the 3’ downstream region [Figure 1]b. Multiple sequence alignment analysis of the Ac-atrn gene showed a high degree of conservation in the predicted domains namely, EGF, a cysteine rich repeat and CUB domain [Figure 1]c. Furthermore, a comprehensive phylogenetic analysis of Ac-atrn gene revealed a conserved relationship among blood feeding as well as non-blood feeding insects, and animals.

Food source stimulates attractin response in early larval development

To unravel whether Ac-atrn has any role during the aquatic development of the mosquito, a relative gene expression analysis was performed at different developmental stages of mosquito. A real-time PCR analysis showed relatively higher expression of Ac-atrn in the young L1 larvae when compared to egg and other developmental stages [Figure 2]a. Starvation of 10 h did not alter Ac-atrn expression, when compared to freshly emerged un-starved larvae of the same age. However, surprisingly, a two-fold (p < 0.01) up-regulation of Ac-atrn was observed in the naïve as well as the starved larvae, when given fresh food supply prior and after starvation, respectively [Figure 2]b.
Figure 2: Transcriptional profiling of attractin gene in An. culicifacies developmental stages—(a) Real-time PCR mediated developmental expression of Ac-atrn in An. culicifacies; and (b) Relative expression analysis of Ac-atrn under food stressed conditions in I instar larvae; L1–L4: Larval stage 1–4.

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Cold stress arrests attractin expression

In order, to test whether external/environmental stress influence the Ac-atrn expression, the young larvae were exposed to an overnight cold stress, and their expressions were compared with unstressed larvae. Though, cold stress did not affect the survival of the larvae, depletion of Ac-atrn to a negligible level (p < 0.002) was noticed [Figure 3]. It was also observed that cold treatment temporarily arrested the motility of the larvae, which was recovered to the normal active stage when kept back at room temperature for 3–4 h. Along with the recovery of larval movement, Ac-atrn expression also reached to normal level after 3–4 h of the recovery phase. However, 4–5 h of heat exposure to the larvae at 42 °C did not alter Ac-atrn expression significantly (p < 0.1).
Figure 3: Differential gene expression analysis of Ac-atrn gene in the I instar larvae under temperature stressed conditions.

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Nutritional stress may influence attractin response in the adult mosquito brain

A tissue specific relative expression analysis indicated that Ac-atrn constitutively expresses in the olfactory tissues, central nervous system and the reproductive organs of both male and female mosquitoes [Figure 4]a. But, ~2.5-fold higher level of expression was observed in the neuro-olfactory system than the reproductive tissues for both the sexes of naïve adult mosquitoes [Figure 4]a. An initial gene expression analysis of sugar fed versus blood fed olfactory system did not show any significant alteration in the Ac-atrn expression [Figure 4]b. Furthermore, Ac-atrn expression level in the sugar fed and starved (24 h) mosquito's olfactory system remains unaltered [Figure 4]c.
Figure 4: Tissue specific transcriptional behaviour of Ac-atrn—(a) Tissue specific relative expression analysis of Ac-atrn; MOLF: Male olfactory tissue (Antennae, maxillary palp and proboscis); FOLF: Female olfactory tissue; MBr: Male brain tissue; FBr: Female brain tissue; FRO: Female reproductive organ; MRO: Male reproductive organ; (b) Ac-atrn expression pattern in sugar fed and blood fed olfactory tissues; FOLF_SF: Sugar fed female olfactory tissue; FOLF_BF: Blood fed female olfactory tissue; (c) Transcriptional response of Ac-atrn in the olfactory tissues of both male and female mosquitoes under food deprived condition; MOLF_SF: Sugar fed male olfactory tissue; MOLF_Starve: 24h starved male olfactory tissues (Same for the females); (d) A time dependent transcriptional profiling of Ac-atrn in the brain tissues of male An. culicifacies mosquitoes under food deprived condition; MBr_SF: Male brain dissected from sugar fed mosquitoes; MBr_Starve_6h: Male brain dissected after 6 h of starvation (Same in case of other time points); (e) A time dependent transcriptional profiling of Ac-atrn in the brain tissues of female mosquitoes under food deprived condition; FBr_SF: Female brain dissected from sugar fed mosquitoes; FBr_Starve_6h: Female brain dissected after 6 h of starvation (Same in case of other time points); and (f) Survival curve of 3–4 days-old adult male and female mosquitoes under food deprived conditions.

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But surprisingly, a time dependent starvation significantly modulated Ac-atrn expression in the brain tissue of adult mosquitoes of both sexes [Figure 4] d and [Figure 4]e. The male mosquitoes brain showed an early transcriptional response (6 h after starvation) of Ac-atrn gene (p < 0.0001) under nutritional stressed condition [Figure 4]d, whereas, female mosquitoes brain showed a delayed elevation of Ac-atrn at 30 h of starvation (p < 0.0001) [Figure 4]e and the mortality rate of male mosquitoes was much higher than their female counterpart [Figure 4]f.

Age and sex specific olfactory response of attractin may influence mating behaviour

Given the multi-functional properties of atrn, it was further tested whether age dependent maturation affects the Ac-atrn response in mating behaviour of the mosquitoes. To examine this relationship, an age and sex specific relative expression analysis of Ac-atrn gene was performed in the olfactory and brain tissue of An. culicifacies mosquito. A significant and continuous increase (~6 fold for female OLF and ~3.5 fold for male OLF) in Ac-atrn expression was observed till the Day 7 in the olfactory tissue of both virgin male and female mosquitoes [Figure 5] a and [Figure 5] b. However, as compared to the olfactory tissue brain did not show any significant modulation, except an initial increase of Ac-atrn level in the aging adult female mosquitoes [Figure 5]c
Figure 5: Age and circadian clock dependent transcriptional response of Ac-atrn transcript in male and female An. culicifacies mosquitoes—(a) Age dependent relative transcriptional regulation of Ac-atrn in female mosquito olfactory system. FOLF-1D: Female mosquito of 1 Day-old (Similar pattern for others); (b) Transcriptional response of Ac-atrn in male mosquito according to their age; MOLF_1D: Male mosquito of 1 Day-old (Similar pattern for others); (c) Age dependent relative transcriptional profiling of Ac-atrn in female mosquitoes' brain. FBr_1D: Female mosquitoes' brain of 1 Day-old (Similar pattern for others); (d) Circadian time dependent and the mating status dependent expression pattern of Ac-atrn in the reproductive organ of both male and female mosquitoes; Virg_12Pm: Virgin mosquitoes dissected at 2400 hrs; Mated_5Pm: Mated mosquito dissected at 1700 hrs; MAG: Male accessory gland; Virg_O/N and Mated_O/N: Virgin and mated mosquito dissected after overnight exposure to each other respectively; and (e) Circadian time dependent and the mating status dependent expression pattern of Ac-atrn in the olfactory tissue of both male and female mosquitoes.

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A circadian dependent transcriptional profiling indicated that mating status did not alter the Ac-atrn expression in the reproductive tissue of both the sexes [Figure 5]d. However, a significant (>2.5-fold) change in the Ac-atrn expression in the mated mosquito's olfactory system provides an evidence that attractin may facilitate pheromone guided male-female courtship behavior [Figure 5]e.

  Discussion Top

The functional characterization of novel genes regulating different physiological responses in mosquitoes, is very important for targeted control of mosquito population. To unravel the smell detection mechanism, we have recently carried out a comprehensive RNA-seq analysis of the olfactory system in the mosquito An. culicifacies[19]. From the RNA-seq data, identification of a unique gene encoding attractin protein, prompted us to investigate its role in the mosquito An. culicifacies. Existing literature search demonstrated that atrn gene plays crucial role in immunity, neurophysiology and reproduction in vertebrates like human and mice[1],[2], [5],[6]. Aplysia californica is the only invertebrate species where atrn gene guides sperm motility towards distantly located eggs for successful fertilization[10],[11]. However, its role has not been investigated in any of the mosquito species. To resolve its possible evolutionary functions a detailed in silico analysis was performed. A comprehensive phylogenetic analysis showed a conserved relationship among all these organisms predicting that atrn gene may have similar multi-physiological role in the mosquito An. culicifacies.

Initial developmental expression profiling of Ac-atrn gene indicated that an increase in atrn expression in emerging young larva may be important to taste, smell and move towards food sources. Although, nutritional stress do not influence the expression of Ac-atrn in the larvae, however, a food supply may accelerate its expression, possibly to regulate the larval movement towards the food source. This may be one of the unique features of atrn, having the water borne chemical communication (pheromone mediated) property, similar to sperm egg interaction in A. californica[10]. Furthermore, the exact role of Ac-atrn in the regulation of thermal stress is still not clear, however our study showed that cold stress may temporarily arrest atrn expression possibly to minimize the energy loss and hence facilitate its survival.

A ~2.5-fold higher level of expression was observed in the neuro-olfactory system than the reproductive tissues for both the sexes of naïve adult mosquitoes, suggesting its possible role in mosquito's behavioural biology and stress management. Though, a different food source and nutrient deprivation have only nominal effect in the Ac-atrn expression, but starvation causes significant modulation of Ac-atrn expression in the brain tissue of adult mosquitoes of both sexes. Furthermore, the time-course dependent relative expression data indicated that male brains are more susceptible to starvation induced neuronal damage as compared to female brains and thus consequently affect the mortality rate of male mosquitoes. Taken together, it is hypothesized that an early up-regulation of Ac-atrn in the male brain may be an attempt to protect the brain cells from fasting induced oxidative damage and consequently neuronal degeneration and death[21]. It is well known that human and other vertebrate's brain is a highly metabolic organ in the body which consumes a large amount of energy in the form of nutrition/food[22]. Although, brain is highly susceptible to oxidative damage due to the abundance of oxidizable material in the plasma membranes of neural cells, however, food deprivation has an added value which causes a failure in the oxidative stress management and thus leads to brain cells degeneration and death[21]. Further, a >10-fold elevation of Ac-atrn in the female brain during later stage of starvation suggested that female mosquitoes can survive for a longer period of time without any food source and thus are more adaptive to adverse environmental conditions which favour their evolution and existence. A continuous elevation of Ac-atrn gene was observed in the olfactory system, whereas brain showed poor modulation, except an initial increase, of atrn gene expression in aging mosquitoes. These data indicate that olfactory Ac-atrn may have an important role in the regulation of mosquito behavioural events and may regulate mating behaviour during early adulteration age. Thus, to test the possible role of Ac-atrn in mating behaviour a circadian dependent transcriptional profiling was performed. The data evidenced that Ac-atrn may facilitate pheromone guided male-female courtship behaviour as proposed in [Figure 6].
Figure 6: Proposed hypothesis for the possible functions of attractin gene in the mosquito An. culicifacies.

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For a successful mating, mosquitoes need to deal the complex events of swarm formation and courtship engagement, ending with successful insemination; a process possibly guided by natural dysregulation of quick-to-court protein in the olfactory system of the mosquitoes[23]. Though, it is yet to be clarified that how active swarm formation and courtship engagement is guided, but the present data suggested that Ac-atrn may have a key role to attract the couples during swarm formation, which is actively commenced on the onset of the sunset (1700 hrs).

  Conclusion Top

Among millions of insects, mosquitoes are evolved with extra specialization of sensory tissues that facilitate them to feed, mate, breed and adapt in diverse ecologies. In vertebrates and few invertebrates, a multi-domain proteins attractin, facilitate many physiological functions and thus have been regarded as a potential therapeutic target for many neuro-regulatory and sexual disorders. Under multiple innate physiological status of mosquito, the transcriptional response of attractin homolog Ac-atrn gene was evaluated, that was originally identified from the olfactory system of An. culicifacies. The comprehensive in silico analysis and transcriptional regulation studies indicates that Ac-atrn not only supports neuro-olfactory associated physiological functions but may also play a crucial role in courtship engagement behavioural responses. A functional characterization of Ac-attractin may help to design novel molecular strategy to disrupt the neuro-olfactory regulation and hence the malaria transmission.

Conflict of interest

The authors declare no conflict of interest.

  Acknowledgements Top

The authors thank the insectary staff members for mosquito rearing and Mr. Kunwarjeet Singh for technical assistance in laboratory. The financial support provided by the ICMR–National Institute of Malaria Research, Tata Education and Development Trust (Health-NIMR-2017-01-03/AP/db) and University Grant Commission is gratefully acknowledged. The authors are also thankful to the Xceleris Genomics, Ahmedabad, India for generating NGS sequencing data. Ms. Tanwee Das De is the recipient of UGC Research Fellowship (CSIR-UGC-JRF/20-06/2010/(i) EU-IV).

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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