Journal of Vector Borne Diseases

RESEARCH ARTICLE
Year
: 2022  |  Volume : 59  |  Issue : 3  |  Page : 206--215

Salivary AsHPX12 influences pre-blood meal associated behavioral properties in Anopheles stephensi


Seena Kumari1, Tanwee Das De1, Charu Chauhan1, Jyoti Rani1, Sanjay Tevatiya1, Punita Sharma1, Veena Pande2, Rajnikant Dixit1,  
1 Laboratory of Host-Parasite Interaction Studies, ICMR-National Institute of Malaria Research, New Delhi, India
2 Department of Biotechnology, Kumaun University, Uttarakhand, India

Correspondence Address:
Dr Rajnikant Dixit
Laboratory of Host-Parasite Interaction Studies, ICMR-National Institute of Malaria Research, Dwarka, New Delhi
India

Abstract

Background & objectives: A successful blood meal acquisition process by an adult female mosquito is accomplished through salivary glands, which releases a cocktail of proteins to counteract the vertebrate host’s immune homeostasis. Here, we characterize a salivary-specific Heme peroxidase family member HPX12, originally identified from Plasmodium vivax infected salivary RNAseq data of the mosquito Anopheles stephensi. Methods: To demonstrate we utilized a comprehensive in silico and functional genomics approach. Results: Our dsRNA-mediated silencing experiments demonstrate that salivary AsHPX12 may regulate pre-blood meal-associated behavioral properties such as probing time, probing propensity, and host attraction. Altered expression of the salivary secretory and antennal proteins expression may have accounted for salivary homeostasis disruption resulting in the unusual fast release of salivary cocktail proteins and delayed acquisition of blood meal in the AsHPX12 knockdown mosquitoes. We also observed a significant parallel transcriptional modulation in response to blood feeding and P. vivax infection. Interpretation & conclusion: With this work, we establish a possible functional correlation of AsHPX12 role in the maintenance of salivary physiological-homeostasis, and Plasmodium sporozoites survival/transmission, though the mechanism is yet to unravel.



How to cite this article:
Kumari S, De TD, Chauhan C, Rani J, Tevatiya S, Sharma P, Pande V, Dixit R. Salivary AsHPX12 influences pre-blood meal associated behavioral properties in Anopheles stephensi.J Vector Borne Dis 2022;59:206-215


How to cite this URL:
Kumari S, De TD, Chauhan C, Rani J, Tevatiya S, Sharma P, Pande V, Dixit R. Salivary AsHPX12 influences pre-blood meal associated behavioral properties in Anopheles stephensi. J Vector Borne Dis [serial online] 2022 [cited 2023 Feb 6 ];59:206-215
Available from: http://www.jvbd.org//text.asp?2022/59/3/206/328814


Full Text

 INTRODUCTION



Adult mosquitoes of both sexes relies on nectar sugar for their regular metabolic energy production[1],[2]. However, an evolutionary adaptation of host-seeking and blood-feeding behavior in adult female mosquitoes is imperative for their reproductive success. The harmonious actions of the neuro-olfactory system drive the mosquito’s successful navigation towards a vertebrate host[3], but it is the salivary gland that facilitates rapid blood meal uptake from the host[4]. The mosquito’s salivary cocktail contains crucial bioactive molecules having anti-homeostatic[5],[6], anti-inflammatory, and immuno-modulatory properties[7],[8],[9], which counteract the host defense for rapid blood meal acquisition, usually in less than two minutes.

For the past two decades, several salivary glands encoded factors have been identified from different mosquito species[10],[11]. But the nature and function of the salivary cocktail remains largely unknown, especially when mosquitoes’ active physiological status changes from sugar-to-blood feeding. Substantial evidence shows that salivary gland secretory proteins are rapidly depleted upon blood-feeding[12],[13]. Our recent study also demonstrates that mosquito salivary glands gene expression switching ability is key to manage meal-specific responses. Additionally, we observed that the first blood meal not only modulates the molecular and cellular responses, it causes persistent changes in the salivary gland morphology[14]. Though we correlate that the pre-and post-blood meal-associated changes are pivotal to maintain salivary gland homeostasis, but the regulatory mechanism remains unexplored.

Heme-containing peroxidase enzymes, having conserved function throughout vertebrates and invertebrates’ taxa, play a crucial role in the maintenance of cellular and physiological homeostasis by scavenging free radicals (NOS/ROS)[15],[16]. Out of total 889 putative insect heme peroxidases indexed in the National Centre for Biotechnology Information (NCBI) database, at least 39 heme peroxidases have been predicted from blood-feeding mosquitoes, which are distributed to six highly conserved HPX lineages[16]. Though salivary peroxidase constitutes important bioactive vasodilator molecules, their functional role has not been established[17].

Here, we demonstrate that AsHPX12, a heme peroxidase homolog, abundantly expresses in the salivary glands, and influences the blood-feeding associated behavioral properties in the mosquito Anopheles stephensi. Using dsRNA mediated gene silencing experiments, we show that salivary AsHPX12 dysregulation significantly delays in probing time, and blood meal acquisition propensity of the mosquitoes. Possibly, this is caused by salivary physiological homeostasis disruption and altered expression of olfactory and salivary proteins. Furthermore, a significant transcriptional modulation in response to blood feeding and P. vivax infection, suggests that salivary AsHPX12 may have a unique role in the maintenance of salivary gland physiology, and Plasmodium sporozoite survival and transmission.

 Material & Methods



Sequence identification and in silico analysis

We have recently analyzed tissue-specific RNAseq analysis of P. vivax infected mosquito, and among the top 35 salivary genes encoding salivary secretory proteins, we noticed HPX12 also differentially up-regulated in response to Plasmodium vivax infection[18]. To further verify whether other HPX members also express in salivary glands, we made the catalog from salivary RNAseq data and compared read count values of at least six transcripts encoding heme-peroxidases [Table 1]. A high read count both in naïve blood-fed and P. vivax infected salivary RNAseq database allowed us to select HPX12 as the target gene for functional study in the current investigation. The RNAseq database has been submitted to the NCBI data repository with accession no. SRR8476334.{Table 1}

A multiple BLAST (www.ncbi.nlm.nih.gov/BLAST/search) analysis was carried out against either at NCBI (NR database) and/or mosquito species-specific database available at VectorBase (www.vectorbase.org) to dig out complete molecular information such as homolog sequence selection; gene-structure, ORF finding, domain, and motifs-prediction (Fig. S1–S4). The phylogenetic trees were prepared with selected 14 peroxidase protein sequences by the neighbor-joining method in MEGA X program as described previously[19]. We aligned all shortlisted peroxidase protein sequences by the ClustalX algorithm (www.clustal.org) where the reliability of the branching was tested by 1000 bootstrap values of the replicates, showing associated taxas information next to the branches. The tree was drawn to a scale of 0.050, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method shown in the units of the number of amino acid differences per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). The processed phylogenetic tree was examined based on clusters and nodes formed.

Mosquito rearing and maintenance

Indian strain of An. stephensi mosquito was reared and maintained in the insectary at the temperature of 28 ± 2°C, relative humidity of ~80% with 12 h light-dark cycle as mentioned previously[20]. The adult mosquitoes were fed daily on sterile sugar solution (10%) using a cotton swab throughout the experiment.

dsRNA mediated gene knockdown assays

To knock down AsHPX12 the expression of dsRNA mediated gene silencing protocol was used to amplify the target cDNA sequence through PCR. While for control dsrLacZ pre-cloned bacterial gene segment in Plasmid vector was amplified by PCR using LacZ perimers. The amplified PCR product was examined by agarose gel electrophoresis, purified (Thermo Scientific Gene JET PCR Purification Kit #K0701), quantified, by Nano-Drop (Thermo Scientific, USA). For PCR based protocol, the designed dsRNA primers were tagged with T7 overhang of 20bp sequences and therefore the purified PCR product was subjected to double-stranded RNA synthesis using Transcript Aid T7 high-yield transcription kit (Cat# K044, Ambion, USA). The bacterial specificity for the designed LacZ primers was determined by searching against An. stephensi genomic as well as transcript databases. A low quality or no-match, especially to transcript database ensured that designed LacZ primers have negligible chances of cross-match to any mosquito gene (Fig. S5 & S6). The details of all the qPCR, as well as dsRNA primers used in this study, are mentioned in the supplemental data sheet (ST-1). About ~69nl (3 μg/ul) of purified dsRNA product was injected into the thorax of cold anesthetized 1–2 day old female mosquito using a nano- injector (Drummond Scientific, CA, USA). The knockdown of the respective gene was confirmed by quantitative RT-PCR after 3–4 days of dsRNA injection.

Blood-feeding assay

To track the possible role of AsHPX12 in blood-feeding both control (LacZ injected) and test (AsHPX12 injected) mosquito groups (50 each) were offered rabbit blood meal after 4 days of the dsRNA injection. The mosquitoes were allowed to feed for 20 min, after which we scored the number of mosquitoes that had fed. For the statistical analysis of blood-feeding propensity (percentage of mosquitoes that probed within a fixed period), the feeding phenotype of knockdown mosquitoes was compared with the respective control group mosquitoes[21]. For probing assay, the control and AsHPX12 knockdown mosquitoes were starved for 4–5 h before exposure to vertebrate host, and calculated probing time (the initial insertion of the proboscis into the skin to the initial engorgement of blood) as described earlier[22]. Briefly, LacZ and HPX-12 knock-down mosquitoes (25 in each batch) were released into the modified Olfactometer which comprised two arms (6 cm diameter), each ending in two independent tests and control chambers (length 44 cm, width 36 cm). Before the initialization of the experiment, both groups of mosquitoes were allowed to acclimatize for 30 minutes within the chamber. After acclimatization two rabbits were kept into the arms of the Olfactometer and probing time was calculated.

Sample collection and RNA extraction

Experimentally required tissues were dissected and pooled from the cold anesthetized adult female mosquitoes under different physiological conditions. To examine the tissue-specific expression of target genes, selected tissues such as hemocyte, spermatheca, olfactory system were dissected from 3–4 day old naïve sugar-fed mosquitoes. Salivary glands were dissected from 25 blood-fed (Rabbit) mosquitoes and collected at different time series (3h, 24h, 48h, 72h, 10 days and 14 days). For the collection of salivary glands infected with P. vivax sporozoites, 3–4 day old An. stephensi mosquitoes were fed on the P. vivax infected patient’s blood (~2% gametocytaemia) through a pre-optimized artificial membrane feeding assay. The confirmation of the P. vivax infection was done by staining the midgut with 5% mercurochrome to visualize the oocysts after 4 days post-infection (DPI), as described earlier[18]. After confirmation of positive infection, ~20-25 mosquitos’ salivary glands were dissected at 9-12DPI and 12-14DPI, respectively. Total RNA from the salivary gland, midgut, and other tissues was isolated using the standard Trizol method as described previously[14].

cDNA preparation and gene expression analysis

~1μg total RNA was utilized for the synthesis of First-strand cDNA using a mixture of oligo-dT, random hexamer primers and Superscript II reverse transcriptase, as per the described protocol (Verso cDNA synthesis Kit, Cat#AB-1453/A, EU, Lithuania)[14]. For differential gene expression analysis, routine RT-PCR and agarose gel electrophoresis protocols were used. The relative abundance was assessed by SYBR green qPCR master mix (Thermo Scientific), using Illumina Eco-Real Time or Bio-Rad CFX96 PCR machine. PCR cycle parameters involved an initial denaturation at 95°C for 15 min, 40 cycles of 10 sec at 95°C, 15 sec at 52°C, and 22 sec at 72°C. After the final extension, both melting, as well as amplification curves, were examined for quality assurance. Each experiment was performed in three independent biological replicates. The relative quantification results were normalized with an internal control (Actin), analyzed by 2-ΔΔCt method, and statistical analysis was performed using Origin 8.1. Differences between test samples and their respective controls were evaluated by paired Student’s t-test and one-way ANOVA test.

Statistical analysis of the data

Statistical analysis was performed using Origin 8.1[23]. All these data were expressed as mean ± SD differences between test samples and their respective controls were evaluated by paired Student’s t-test, and the one-way ANOVA test was considered significant if the p-value was less than 0.05. Each experiment was performed at least thrice to validate the findings.

Ethical statement

All of the experimental procedures involving malaria-infected patient blood samples were approved by Institutional Ethics Committee, ICMR-NIMR, Delhi. All protocols for rearing and maintenance of the mosquito culture were approved by the Institute Animal Ethics Committee.

 RESULTS



Identification, annotation, and phylogenomics analysis of AsHPX-12

Our recent RNAseq study demonstrates that P. vivax sporozoites significantly modulates the molecular architecture of the mosquitoes’ salivary glands. To identify the transcripts having a crucial impact on salivary physiology and antioxidant defense responses, we selectively cataloged at least seven transcripts encoding heme-peroxidase homologous proteins [Table 1] from the salivary transcriptomic data. Relatively a high read count of HPX12 than other heme peroxidase family members prompted us to investigate its possible role in the mosquito salivary gland physiology. The BLASTX analysis of the selected 1806bp long HPX12 transcript showed 84% identity with the HPX12 homolog of Anopheles gambiae (AGAP029195). A homology search against An. stephensi database showing 100% identity to and predicts full-length AsHPX12 transcript (ASTE016356) is a 1874bp long single-copy gene with four exons and three introns [Figure 1]A. We noticed that AsHPX12 transcripts encodes a 587 amino-acid long peptide containing Animal heme peroxidase domain and seven motifs including Amidation, Casein kinase-II phosphorylation, protein kinase C and N-myristoylation site, cAMP and cGMP dependent protein kinase phosphorylation site [Figure 1]B.{Figure 1}

Multiple sequence alignment of putative HPX members from insects and mosquitoes indicated a high degree of sequence conservation among all aligned HPX12 homolog members from mosquito and insect species (Fig. S7). Phylogenetic analysis showed all fourteen aligned HPX12 homolog proteins clustered in two major clades; while upper clades showed higher conservation within Anopheles mosquitoes, however, lower clades separated into two sub-clades clustering one for mosquitoes (Aedes & Culex), and second for other non-blood-feeding insects [Figure 1]C.

AsHPX12 abundantly expresses in the salivary glands of adult mosquitoes

To predict the possible role of the identified salivary AsHPX12, first, we performed developmental expression analysis. Our initial RT-PCR-based analysis revealed AsHPX12 ubiquitously expresses throughout the developmental stages of the mosquitoes [Figure 2]A. A comparative tissue-specific transcriptional profiling by real-time PCR revealed that HPX12 abundantly expresses in the salivary gland than other tissues of in 3-4 day old naïve mosquitoes. Taken together we hypothesize that AsH-PX12 may have a salivary-specific role in the regulation of mosquito’s blood-feeding associated behavioral properties.{Figure 2}

AsHPX-12 may regulates pre-blood meal associated behavioral responses

To test the above hypothesis, we first examined the knockdown effect of AsHPX12 on the mosquito’s preblood meal-associated behavioral properties influencing the blood meal acquisition process. While performing behavioral assays, unexpectedly, we observed an increased host attraction of AsHPX12 knockdown mosquitoes towards vertebrate host. However, interestingly, we also observed that the silencing of HPX12 ([Figure 3]A, p<0.002)), significantly increases propensity by 31% (p<0.0067) and probing time to ~180 seconds as compared to ~100 seconds (p<0.000692) of the control mosquitoes group ([Figure 3]C; ST-2). Together, these data suggested that salivary HPX12 disruption may impair salivary physiology, resulting in the altered functions during the blood meal acquisition process. It is well known that host-seeking behavioral property is regulated by the concerted actions of the odorant-binding proteins (OBPs) and odorant receptors (ORs). In a bid to check the correlation of salivary AsHPX12 with pre-blood meal-associated mosquitoes’ olfaction, we evaluated and compared the expression of selected OBPs/ORs in the naive and HPX12 knockdown mosquito’s salivary gland and olfactory tissues [Figure 3]C). Among all the tested OBPs exceptionally, OBP10 showed a significant (p<0.001) up-regulation in both the salivary gland and olfactory tissues in AsHPX12 knockdown mosquitoes [Figure 3]C & [Figure 3]D, while expression of odorant receptor remains unchanged in the olfactory as well as salivary glands [Figure 3]D. We noticed that knockdown of HPX12 also alters the expression of salivary secretory enzyme apyrase which inhibits ADP-dependent platelet aggregation [Figure 3]E, and other salivary-specific proteins such as 53.7kDa, 37.3kDa and Anophelin also play an important role to facilitate blood meal uptake [Figure 3]F.{Figure 3}

Blood meal and P. vivax sporozoite boost salivary AsH-PX-12 expression

Next, to evaluate how blood meal affects the expression of heme-peroxidase family proteins, we profiled and compared time-dependent transcriptional responses of all seven HPX family members in the salivary gland of blood-fed mosquitoes. Compared to other members, exceptionally HPX12 showed a gradual induction within 6 h of blood-feeding, which was further increased by ~16 fold (p<0.0025) within 24 h of blood-feeding, and gradually cease to basal level after 72 h of blood-feeding [Figure 4]A. Since, earlier studies demonstrate that a gut-specific AsHPX15, may favor Plasmodium development by the formation of a crosslinking mucin layer at the luminal side of the gut epithelium, we also tested whether Plasmodium infection also modulates HPX12 responses in the mosquito salivary glands. Detailed transcriptional profiling showed a gradual elevation of the AsHPXH level in response to salivary invaded P. vivax sporozoite infection [Figure 4]B.{Figure 4}

 DISCUSSION



The evolution of conserved antioxidant defense system enzymes is necessary to maintain physiological homeostasis, but its role in the salivary physiology of hematophagous insects remains unknown[16]. Here, we identify and characterize a salivary-specific AsHPX12 transcript encoding heme-peroxidase enzyme, from malaria vector An. stephensi. We demonstrate AsHPX12 is key to regulate salivary homeostasis, and mRNA depletion by dsRNA silencing significantly impairs pre-blood meal-associated host-seeking abilities influencing the blood meal acquisition process of the adult female mosquitoes.

Host-seeking and blood-feeding behavioral adaptation are unique to adult female mosquitoes for their reproductive success. The navigation trajectory during active host-seeking is achieved by coordinated neuro-olfactory actions to find and locate a suitable vertebrate host[4], and once located the desired host, the salivary actions facilitate a rapid blood meal acquisition process, usually, in less than two minutes. Several studies suggest that during blood feeding the salivary cocktail composition is rapidly depleted[13], but how the host-odor activated salivary glands manage ‘prior and post’ blood-meal associated changing physiologies is largely unknown[14]. We aimed to screen and test whether the salivary Anti-oxidant enzyme system plays important role in the regulation of changing physiological homeostasis maintenance. Our recent observation of salivary HPX12 transcript enrichment in response to P. vivax infection[18], prompted us to test its possible role in salivary physiology. Sequence conservation of AsHPX12 with other mosquito species suggested its conserved functions in the blood-feeding behavior of hematophagous insects. We found AsHPX12 is constitutively expressed in all the developmental stages of An. stephensi mosquitoes.

Previously, a heme-containing salivary secreted peroxidase in the mosquito An. albimanus has been suggested to act as a vasodilator through hydrogen peroxide-dependent destruction of serotonin and noradrenalin, but the functional role in the regulation of physiological homeostasis remains uncertain[24]. To establish a functional correlation of AsHPX12 we performed dsRNA mediated knockdown and evaluated the altered behavior properties in An. stephensi mosquitoes.

Surprisingly, an enhanced eagerness of AsHPX12 knockdown mosquitoes towards a rabbit host, but a significant delay in probing time, suggested that HPX12 is key to regulate salivary physiological homeostasis and oxidative stress responses, as described recently[28]. A preliminary analysis with trypan blue, which is a dye used to stain dead cells, the HPX12 silenced mosquito salivary glands showed an abnormal morphological disruption than control mosquito salivary glands (Fig. S8).

Additionally, we also noticed that HPX12 knockdown causes a significant enrichment of odorant-binding protein expression in both the salivary gland and olfactory system, but in parallel down-regulate some of the salivary cocktail protein expression such as Apyrase which is key to regulate pre-blood meal associated host-seeking properties [Figure 3]E and [Figure 3]F. Recently, we have demonstrated that rapid blood meal acquisition not only alters the molecular architecture, also impairs the salivary glands’ morphological and cellular architecture in the adult female mosquito An. culicifacies[16]. Here, our observation of altered expression of salivary proteins, caused by HPX12 dysregulation suggests that AsHPX12 may have a direct influence on mosquito’s host-seeking abilities affecting the blood meal acquisition process. Although observational evidence of host-seeking and biting behavioral manipulation by pathogens is increasing, the underlying mechanisms benefiting pathogen transmission remain elusive. Proteomic analysis suggests that malaria infection may modify mosquito behavior by modulating the expression of either neuro-regulatory synapse-associated protein or by changing ATP synthesis pathways, affecting the potential role of ATP as a neuromodulator[25]. A significant change in the salivary apyrase activity, an ADP-degrading enzyme that helps the mosquitoes to locate blood, has been correlated for altered host-seeking behavior[26]. The observation of increased probing time, coupled with altered expression of odorant-binding proteins in the AsHPX12 silenced mosquitoes further corroborate and support the idea that the down-regulation of salivary apyrase is crucial to enhances host attraction of the Plasmodium-infected than uninfected mosquitoes.

Earlier HPX15, the potent immune-modulator in the gut, was found to function as an agonist and favor endogenous bacterial population as well as Plasmodium parasite survival by the formation of a cross-linked mucin barrier on the luminal side of the midgut[27]. Elevation of AsHPX12 mRNA level after 24 h of blood-feeding, and consistent up-regulation in response to salivary invaded P. vivax sporozoites, we propose that AsHPX12 may have a dual role in the management of salivary gland homeostasis following blood-feeding and long term survival of stored sporozoites in the salivary glands. Further insights into the sporozoite storage mechanism within the salivary gland may allow us to confirm this hypothesis.

 CONCLUSION



In summary, we demonstrate that salivary-specific AsHPX12 plays an important role in the regulation of pre-blood-associated behavioral properties. A parallel transcriptional modulation in response to blood feeding and P. vivax infection, suggests salivary AsHPX12 may have a pivotal role in the management of physiological homeostasis, Plasmodium sporozoite survival, and transmission.

Conflict of interest: None

 Acknowledgements



We would like to thank all the technical staff members of the central insectary for mosquito rearing and Kunwarjeet Singh for lab assistance. We are grateful to the patients of the malaria clinic, who contributed to the study. Finally, we thank Xceleris Genomics, Ahmedabad for NGS sequencing. Work in the laboratory was supported by the Indian Council of Medical Research (ICMR) (Ref#5/87(301)v2011ECD-II), Government of India. SK is recipient of the CSIR Research Fellowship (09/905(0015)/2015-EMR-1).

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