Volume 3, December 2017, pages 12-18

Infection induced polypeptides in the hemolymph of female mosquitoes of species A  and B of Anopheles culicifacies complex  

Anil Sharma1,2*, Hardev Parashar3, Baseerat Hamza1 ,Tridibes Adak3

1International Center for Genetic Engineering and Biotechnology, New Delhi, India 
2 Biological Innovations Research Development Society, New Delhi, India 
3National Institute of Malaria Research, New Delhi, India 

* Corresponding Author Email: anil.mrc@gmail.com | Tel: +919990963163

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All anophelines are not vectors of malaria. Many factors involved in determining the vectorial status of a mosquito are still unknown. Anopheles culicifacies, the most important malaria vector of India, exists as a sibling species complex. The members of the species complex differ in their capacity to transmit malaria. A.culicifacies A and C are efficient malaria vectors whereas species B is a poor vector. The susceptibility of mosquitoes has been correlated with their ability to pose immune responses against infections. In this context, immune-responsive proteins of two differentially susceptible A. culicifacies mosquitoes were identified when challenged with the Gram-Positive bacteria, Micrococcus leutus and rodent malaria parasite, P. vinckei petteri. The female mosquitoes belonging to species A and species B of A. culicifacies complex were challenged with sterile injury or with M. leutus and the proteins were resolved. Similarly, the mosquitoes were also experimentally infected with P. vinckei petteri and their protein profile was studied.  The midguts of the infected mosquitoes were dissected on the 5th day and their blood feeding, the oocyst rate and mean oocyst number per midgut were recorded. A. culicifacies mosquitoes responded promptly to bacteria infection by secreting polypeptides in the hemolymph within one hour of infection. In the hemolymph of the female mosquitoes of species A and B, septic and sterile injury induced totally different sets of proteins. A 65 kDa polypeptide was induced only in infected hemolymph samples of species B but not in species A hemolymph.


Anopheles culicifacies , Micrococcus leutus , P. vinckei petteri, Hemolymph


Mosquitoes have developed innate immune mechanisms during the course of evolution. The understanding of the mosquito immune response against their natural and unnatural parasites is increasing rapidly with the studies involving pattern recognition peptides, signaling pathways and antimicrobial peptides (Barillas et al. 2000; Dimopoulos et al. 2001). In parallel with these studies are genetic approaches that are beginning to identify the genes involved in the immune response (Dimopoulos et al. 2000).  
Malaria control in India is largely the story of the control of A. culicifacies (Subbarao 1988). This vector is responsible for 60-70% of total malaria cases per year in the Indian subcontinent. A. culicifacies exists as a complex of five sibling species. Member species of this taxon differ in their distribution pattern, larval and adult biology, responses to insecticides, blood feeding preferences and capacity to transmit disease. Among all the biological variations, the differential disease transmission potential of different members of the complex is probably of greatest epidemiological importance.  
Among members of the Culicifacies complex, only species A, B, and C has been colonized so far. Various host-parasite interaction studies involving Culicifacies A, B and C and different Plasmodium species unequivocally demonstrated that species A is the most susceptible while species B is the least susceptible to both human (Adak et al. 1999; 2006) and rodent Plasmodium infection (Kaur et al. 2000). The natural variability in malaria susceptibility because of differential genetic factors of mosquitoes is not yet been clearly understood. However, studies involving A. culicifacies have shown the correlation between different types of mosquito immune responses with differential mosquito susceptibility to various parasitic infections (Rodrigues et al. 2007; Sharma et al. 2014). To combat malaria further understanding of the immunity of A. culicifacies to natural and unnatural invaders is required. The susceptibility of mosquitoes to malaria parasites is found to be associated with their immunity to other infections. The gut biota of mosquitoes is found to be influenced by the gut biota composition. The gut biota negatively influences the vectorial competence of the mosquitoes, Anopheles stephensi (Sharma et al. 2013) and A. gambiae (Cirimotich et al. 2011).  
In the present study, the female mosquito of differentially susceptible member species of Culicifaices complex was experimentally infected with the gram-positive bacteria, Micrococcus leutus and rodent malaria parasite, P. vinckei petteri and the induced peptides were recorded and compared. 

Materials and Methods

2.1.Mosquito strains 

The cyclic colonies of A. culicifacies were established from wild indoor-resting mosquitoes collected from different geographical areas in India. Species A was collected from village Burari, North-East district of Delhi (280 37’N, 77012’E), and species B from village Ladpur, District Sonepat, Haryana (280 48’ N, 760 28’E). The strains were maintained as cyclic colonies in the laboratory. 

2.2.Use of animals

The animals i.e., New Zealand rabbits and Balb/c mice were maintained in the animal house of  National Institute of  Malaria Research as per the recommendations of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines and were used in the experiments as per the guidelines of the Ethical Committee of the Institute. 

2.3.Mosquito rearing

Cyclic colonies of the above-mentioned mosquito strains were kept in an insectary maintained at a temperature of 28±10 C and 70-80% RH as described by Adak et al. (1999). The insectary was fitted with a simulated dusk and dawn machine with a photoperiod of 14 hour day and 10 hour night. Adult mosquitoes were offered water-soaked resins and 1% glucose soaked cotton pads as a source of energy. Female mosquitoes were allowed to feed on rabbit blood for their ovarian development. 

2.4.Maintenance of gram-positive bacteria

Micrococcus leutus bacteria were cultured in a medium containing 0.2% yeast extract + 0.1% Beef extract + 0.5% peptone + 0.5% NaCl at 30o C for 24 h at 250 rpm. Following incubation, the bacteria pellet was stored at 0oC until used.

2.5.Experimental infection of bacteria

Four to six day old adult female Culicifacies mosquitoes were briefly anesthetized at ~0oC for exactly half a minute. Unconscious mosquito was laterally placed on Whatman No. 1 filter paper under a dissecting microscope. An aseptic wound was made on the lateral side of the thorax of mosquito, using a fine entomological needle contaminated with Micrococcus leutus. Mosquitoes injured with sterile entomological needle served as control. Immediately after the injury mosquitoes were allowed to recover in a humid chamber for an hour at lower temperature i.e., at 24-25oC to decrease mortality.  

2.6.Maintenance of rodent Malaria parasite

Rodent malaria parasite, Plasmodium vinckei petteri 279BY used for infection of mosquitoes was originally supplied by Prof. Irene Landau, Museum National d’Historie Naturelle, Paris. P. vinckei petteri infected blood was mixed with equal volume of incomplete RPMI culture media and inoculated into healthy 4-5 weeks old inbred Balb/c mice to maintain the malaria parasite in the laboratory. Thin blood smears were prepared from the parasite inoculated mice and stained in JSB stain after 3 days of inoculation (Singh 1956). The parasite-positive mice were used for feeding Anopheles stephensi mosquitoes to maintain malaria cycle in the laboratory. Maintenance of mosquito-parasite cycle in laboratory involved regular monitoring of the parasitemia of infected mice for the presence of at least 0.05% mature gametocytes to ascertain the ideal time for feeding.  
The sporogonic development of the malaria parasite was monitored by observing mosquito midguts stained in a drop of 0.5% mercurochrome (Eyles 1950) after 5 days of infective feeding for the presence of oocysts. Salivary glands were observed on 14th day after feeding to assess sporozoite positivity. Sporozoite-positive mosquitoes were starved and were allowed to feed on four to five week old healthy Balb/c mice immobilized in molded wire mesh cages to infect the mice with P. vinckei petteri in order to maintain the malaria cycle in the laboratory. 

2.7.Estimation of parasite positivity

Mosquitoes fed on P. vinckei petteri infected mice were kept in the insectary maintained at 23-24oC and 70-80% RH to check the sporogonic development of the parasite. Midguts of the parasitized mosquitoes were examined on 5-6 days post-blood feeding and the number of midguts positive for oocyst out of total midgut dissected, i.e., oocyst rate, was recorded. A number of parasite challenge experiments were carried out during the course of the study; however, only the samples from the batches of mosquitoes where infectivity was detected were used in the present study. 

2.8.Experimental feeding to mosquitoes

Four to six day old adult female mosquitoes of Culicifacies A and B were fed on P. vinckei petteri infected BALB/c mice blood to study the response of A. culicifacies to P. vinckei petteri. About 200 female mosquitoes in one cage were allowed to feed on infected mouse having at least 0.05% gametocytemia. The mosquitoes fed on a strained healthy mouse constitute the control. Insectary was maintained at 23-24oC and 65 –70% relative humidity. Since feeding success was always better in the evening than in the daytime, all the feeding experiments were carried out in the evening. Mice were removed from the cages after one hour of feeding. Unfed and partially fed mosquitoes were discarded and only fully fed were kept in the insectary.  

2.9.Sample preparation

Hemolymph samples were prepared before or after 1, 4 and 8 hours of injury or before and after 12, 18 and 24 hours of from the mosquitoes. For hemolymph extraction, mosquitoes were knocked down by chilling at 0oC. Hemolymph was collected by amputating the legs and wings of 25 mosquitoes on a slide containing ice-cold sterile phosphate buffered saline (PBS) pH 7.4. Thorax of each mosquito was pressed using an entomological needle to expel the hemolymph droplet into the PBS solution. Every care was taken to collect a first clear droplet of the hemolymph to avoid contamination of fat bodies.


The proteins of A. culicifacies mosquitoes were resolved using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The quantity of the proteins in the hemolymph and crude extract of body tissue were measured by Bradford's method (Bradford 1976) using bovine serum albumin as a standard. Hemolymph samples were mixed with 4X sample buffer containing 40% glycerol, 250mM Tris Cl, pH 6.8, 0.8% SDS, 0.002% bromophenol blue and 2% of ß-mercaptoethanol. The qualitative analysis of soluble proteins was performed on 10% polyacrylamide gels essentially following the procedure of Laemmli (1970) at a constant current of 30 mA.  The gels were stained in 0.5% Coomassie brilliant blue G-250 solution and de-stained in 10% acetic acid. The gels were photographed on ‘Syngene Gel Documentation System’ using ‘Gene SnapTM’ software and analyzed in ‘Gene ToolTM’ software.  

Results and Discussions

Micrococcus leutus responsive polypeptides of two differentially susceptible member species viz., species A and species B of Culicifacies complex were studied in hemolymph after 1, 4 and 8 hours of the bacterial challenge taking corresponding aseptically injured tissue samples as control. Further naïve tissue samples were also used as a control for the sterile injury induced response. In addition, the Immune-responsive polypeptides were also studied in response to rodent Plasmodium vinckei petteri infection after 12, 18 and 24 hours of blood feeding and for comparison, the naive and uninfected group were taken correspondingly. Each experiment was conducted thrice each in three replicates. Only the reproducible polypeptides were recorded, analyzed and presented in the manuscript. Fold changes in the intensity of polypeptides were calculated using the raw volume of the polypeptides as digitalized by GeneToolTM software.
In the naïve hemolymph sample of Culicifacies, SDS-PAGE resolved twenty polypeptides ranging between 5 and 198 kDa molecular weight and between 1245.09 and 157035 pixels of raw volumes as calculated by the GeneToolTM software. In response to the sterile injury, three 46, 32, and 20 kDa polypeptides were induced; of which the 46 and 32 kDa were also induced upon sterile injury (Figure 1). In addition to the sterile injury inducible polypeptides, bacteria contaminated injury elicited an additional 45 kDa polypeptide. The sterile injury elicited two 37 and 20 kDa proteins in species B hemolymph while the bacteria contaminated injury elicited two 92 and 26.5 kDa polypeptides (Figure 2). It seems species B hemolymph poses a different response to sterile injury and septic  injury with no overlapping of the polypeptides. Lambrechts et al. (2004) have shown a positive correlation between bead melanization and clearance of injected bacteria in A. gambiae mosquito.  
Both the species of culicifacies complex responded promptly to the bacteria infection as evidenced by the early induction of polypeptides in their hemolymph. Mosquitoes have an efficient system of recognition of non-self (Gillespie et al. 1997). The invading bacteria are recognized and the fat bodies and hemocytes were signaled. Presence of a foreign substance in the body receives immediate attention and the clearance of the invader becomes the priority of the insect. In Anopheles albimanus, melanization and lysis of Gram-positive bacteria Micrococcus lysodeikticus was observed in the hemolymph within half an hour of the infection (Hernandez et al. 2002). 
Wound and microbial infection are known to activate various enzymes cascades such as pro-PO cascade (Ashida and Brey 1995; Lai et al 2002) and antimicrobial peptides (Boman and Hultmark 1987). Dimopoulos et al. (2002) reported upregulation of one cluster by injury but down-regulated in vitro in response to bacteria. Plasmodium-responsive prophenoloxidase encoding acppo6a gene of P. vivax-refractory A. culicifacies was also downregulated upon the bacteria infection, which was otherwise induced upon sterile injury (Sharma et al. 2014). The bacteria-inducible genes either play a role in the bacteria clearance (Dimopoulos et al.  2001) or in immune evasion (Juris et al. 2002). The immune evasion abilities of a parasite species are developed during the course of co-evolution with the host for the sake of their survival (Koella and Boete 2003). The refractory strains of A. gambiae are able to encapsulate allopatric strains of P. falciparum but not the sympatric strains of Africa (Collins et al. 1986).  
The healthy blood feeding induced five proteins of molecular weight 130, 45, 40, 32 and 26.5 kDa in the hemolymph samples of culicifacies A (Figure 3). The densitometric analysis revealed that the expression level of the 26.5 kDa was approximate ~2.7 fold as compared to the naive samples. In the Plasmodium infected samples, only three proteins, 45, 40 and 32 kDa protein was induced as compared with the naive hemolymph samples. Healthy blood feeding induced two proteins of molecular weight 32 and 26.5 kDa in the hemolymph samples of species B in first 24 hours of the blood feeding. Whereas, in the infected hemolymph samples a weak 65kDa protein band was noticed in addition to the 32 and 26.5 kDa polypeptides (Figure 4). Member culicifacies species are known to differ in a number of biological characteristics including their vectorial competence, which may have some inherent genetic basis (Adak et al. 1999; 2006). Species B is the least susceptible to malaria parasite infection among the members of the A. culicifacies complex and may possess inherent ability to respond to invading parasites over the other members of the complex (Adak et al. 2006). The midguts of Plasmodium infected mosquitoes were dissected on the 5th day of the blood feeding to score the comparative infection intensity in species A and species B of Anopheles culicifacies complex.  Species A was found to support more number of oocyst in its midgut as compared with the species B (Figure 5). The melanotic encapsulation of the parasites was also observed in the species B.  
In conclusion, the sibling members of the Anopheles culicifacies complex elicited different proteins in response to the bacterial or Plasmodial infection in their hemolymph.  


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