Madhya Pradesh is situated in the central part of India with an area of 308 thousand km² of which forest covers 76,429 km² (about 25% of the total land area). According to an estimate made in 1997, 60, 35 million population of Madhya Pradesh (M.P.), accounting for 6% of the total population of India (1,028 million), contribute to 8.6% of the total malaria cases. Epidemiological indices of malaria in Madhya Pradesh revealed a very dismal picture of malaria. An international team of experts reported a very high incidence of malaria in pregnancy. In Madhya Pradesh (rural) 183,000-1.5 million per year contract malaria in pregnancy, and result in 73,000-629,000 lost fetuses and 1,500 to 12,600 maternal deaths. Malaria is complex in Madhya Pradesh because of vast tracts of forest and high percentage of tribal settlement (20% of state population) (Singh et al., 2004). The magnitude of the problem can be accessed from an estimate made in 1987,  that 54 million individuals of various ethnic origins residing in forested areas of India and accounting for 8% of the total population contributed 30% of total malaria cases, 60% of total falciparum cases and 50% of malaria deaths in the country (Sharma, 1996).

 

Anopheles culicifacies s.l. is widely distributed in India and has been recorded in all mainland zones including Kashmir and high elevations in the Himalayas (up to 3000 meters) except islands of Andaman & Nicobar and Lakshadweep (Nagpal and Sharma, 1995; Subbarao, 1998; Rao, 1984). It is the most important vector in plains of rural India contributing 60-70% of reported cases annually (Sharma, 1996). Success stories in malaria control during 1950-1960, and malaria resurgence in the 1970s deal primarily with the control of An. culicifacies s.l. Biology and genetics of An. culicifacies has been extensively studied in India (Sharma, 2006; Barik et al., 2009), and presently characterized to be a species complex with five informally designated species A, B, C, D and E. These five sibling species are spread across India with distinct biological characteristics and role in malaria transmission.

 

Cytogenetical techniques’ involving karyotyping of polytene chromosome was one of the earliest tools for the study of anopheline genetics. Anopheline females in the semi-gravid stage have the best polytene chromosomes in ovarian nurse cells (Coluzzi, 1968). Larvae at the IV instar stage have polytene chromosomes in salivary glands. For those anopheline species which do not have good ovarian polytenes, larval salivary chromosomes can be used (but salivary gland polytene chromosomes are not very good in most anophelines). The advantage with adult females is that ovaries can be removed and fixed in modified Carnoy’s fluid (1:3 glacial acetic acid: methanol) and can be used at any time. Another advantage is that the same female can be studied for other parameters such as host preference, presence of sporozoites/sporozoite antigen, susceptibility to insecticides. Hunt and Coetzee (1986) describe storing of field collected mosquitoes in liquid nitrogen for correlated cytogenetic, electrophoretic and morphological studies. The preparation of polytene chromosomes from adult females is not difficult. Polytene chromosomes are the result of repeated replication of chromosomes at interphase without nuclear division, the process being known as endomitosis. Chromatids after division remain attached, causing thickening of chromosomes which results in the appearance of long ribbon-like structures with dark and light horizontal portions representing band and interband regions respectively. The dark and light regions represent differential condensation of chromosomes. The banding pattern of each chromosome is specific in a given species; thus, each species differs from others in characteristic banding pattern. Any changes in the pattern can be easily detected. In the polytene chromosome complement, only euchromatic regions are seen and the heterochromatic portions of the chromosomes which are under-replicated are not seen. This information will be helpful in better understanding of sibling species complex occurring in these areas and also the patterns of disease prevalence. Such information may also be helpful in developing of keys for sibling species identification and also to develop newer approaches for their control strategies.

 

An. culicifacies sibling species collected from different study sites i.e. Hoshangabad (Dhadav and Padav villages), Narsinghpur (Chinki, Bhalpani, Sherpipariya and Gotegaon villages) and Khandwa (Chighdhalia and Narmada Nagar villages) were processed for preparation of polytene chromosome. The ovaries of individual mosquito were processed for polytene chromosome examination and were identified using the paracentric inversion on the X-chromosome and chromosome arm 2.

 

X- Chromosome: Due to the presence of a very weak chromocenter, the X-chromosome is always seen isolated from the other autosomal arms in the complement (Figure 1); it is easily identified by its much smaller size. In sibling species A and D, 1 large dark band occur near centromere and 4 large dark band occur in the middle of X- chromosome are also important features of X+a+b (X-chromosome) (Figure 2) whereas in sibling species B and C of An. culicifacies, 1 large dark band far from centromere and a few light bands occurs in a small puff are important features of Xab (X- chromosome) (Figure 3). For identification of An. culicifacies sibling species based on X+a+b (X-chromosome) character definitive cytospecies identification was not possible because sibling species A or D have same patterns of X- chromosome and similarly sibling species B or C also have same patterns of Xab (X- chromosome).

 

Identification of Anopheles culicifacies sibling species using polytene chromosomes is very useful in the confirmation of various facts of evolutionary genetics and inter and intra specific chromosomal homologies. The preparation of a standard map is the first important step which acts as a reference for comparative studies. For example, the banding pattern specificity has been utilized in distinguishing different species of the same genus or subgenus and different populations of the same species (Dubinin and Tiniakov, 1945; Carson and Stalker, 1947). One of the essential requirements of cytogenetic investigation of mosquitoes is the preparation of "standard" polytene chromosome maps. Such type of polytene chromosomal mapping have been done in An. subpictus, An. vagus, An. hyrcanus nigerrimus and An. barbirostris, An. albimanus, An. aconitus, An. culicifacies, An. quadrimaculatus. The difference in the banding pattern and the presence of chromosomal polymorphism or inversion was observed among and within population of An. sundaicus might be useful as a diagnostic character in distinguishing cryptic species and chromosomal variability. In our study the collected mosquitoes were subjected to polytene chromosome preparation for viewing the difference in chromosomal patterns of An. culicifacies sibling species. Sibling species C of An. culicifacies was identified from Hoshangabad (Dhadav and Padav villages). The diagnostic inversion genotypes for sibling species C was Xab; 2+ g1h1 and species D of An. culicifacies was identified from Narsinghpur (Chinki, Bhalpani, Sherpipariya and Gotegaon villages) and Khandwa (Chighdhalia and Narnada Nagar villages), The diagnostic inversion genotypes for sibling species D was X+a+b; 2i1+h1. In other study the examination of polytene chromosomes of An. culicifacies from villages around Delhi and in the states of Gujarat and Madhya Pradesh, India, revealed the presence of two fixed inversions on chromosome arm 2. Species B was fixed for the g1 inversion in addition to a and b on the X-chromosome; thus, species B had Xab; 2g1 arrangement (Subbarao et al., 1983). In Gujarat and Madhya Pradesh, two chromosome 2 arrangements, g1+h1 and +g1h1, within Xab populations were seen. In a large sample examined, only four double-inversion heterozygotes (2g1+h1 /+g1h1) were observed; therefore, Xab; 2g1+h1 and Xab; 2+g1h1 were considered as two reproductively isolated populations. The new population with Xab; 2+ g1h1 was designated as species C (Subbarao et al., 1983). Similarly in a few populations in northern India, the i1 inversion on chromosome arm 2 was found polymorphic in species A, and it was found fixed in the southern Indian populations of An. culicifacies species A (Subbarao, 1984). The latter population with the X+a+b; 2i1 arrangement was found sympatric with species B. The evidence for reproductive isolation between species A (X+a+b; 2+g1+h1) and the population with the X+a+b; 2i1 h1 arrangement came from two locations (inversion i1 includes the g1 inversion region and the distal breakpoint is the same for both the inversions). In northern and central India, a deficiency of heterozygotes was found for i1 inversion in the X+a+b populations (Subbarao et al 1988; Vasantha et al., 1991) while in southern India a total absence of heterozygotes was found between species A (X+a+b; 2+g1+h1) and the population with the X+a+b; 2i1+h1 arrangement (Suguna et al., 1989). The X+a+b population with the new chromosome 2 arrangement 2i1+h1 were designated as species D. The explanation for the heterozygotes (+i1/ i1) of i1 inversion observed in northern India is that this inversion is floating in species A with varied frequencies in different populations and is fixed in species D (Vasantha et al., 1991). However it is evident from the literature that species E of An. culicifacies cannot be differentiated from species B because they have homosequential polytene chromosome arrangements. Species E requires mitotic chromosome examination of male progeny and/or vectorial potential needs to be established for distinction from species B (Kar et al., 1999). In the absence of either of these, identification of species E may not be accurate. Mitotic chromosome examination of male progeny and/or vectorial potential studies for sibling species B and E has not been done in present study due to availability of small number of samples. Cytogenetics involving the karyotyping of polytene chromosome was one of the earliest tools for the study of anopheline genetics. Among the disadvantages of this technique is that the polytene chromosome preparations must be made from ovarian tissues or brain cells of fourth instar larvae. This limits the samples to either adult blood fed female mosquitoes or late 4th instar larvae. In addition to the paucity of experienced personnel trained to read polytene chromosome preparations paracentric inversions are not abundant and the number of specimens that can be processed in a given time is also limited. Despite these limitations, this method remains integral for much of the contemporary work. Nevertheless, this standard photomap of polytene chromosome of An. culicifacies provides a reference point for further studies.

 

The adult Anopheline mosquito species were collected from different districts of Madhya Pradesh i.e., Hoshangabad (Dhadav and Padav villages), Narsinghpur (Chinki, Bhalpani, Sherpipariya and Gotegaon villages) and Khandwa (Chighdhalia and Narmada Nagar villages). These sites were selected on the basis as they represent the tribal belt along the streams of Narmada River and also show high incidence of malaria (Sharma, 2012; Singh, 2004; 2006; 2009). The locations and list of collection sites along with their longitude and latitude were given in Figure 7 and Table 1.

The adult mosquito and larval collection were made during the transmission period i.e. February-March and August-September in the morning period between 0600 hrs to 0800 hrs using mouth aspirator and battery operated torch. Only the fed anophelines were captured at various collection sites including human dwellings, cattle sheds, mixed dwelling and random collection sites.Larval samples were collected from selected breeding sites in the different localities. Larvae were collected with the help of dippers (cap. 250 ml).  Ten dips per sq m of breeding habitat surface area were taken.

 

All adult mosquitoes were brought to the laboratory and were identified using standard keys (Christopher, 1933; Wattal and Kalra, 1961; Das et al., 1990; Nagpal and Sharma, 1995). The prominent character that could be used to identify An. culicifacies from other Anopheles includes vein 3 mainly dark and inner costa interrupted other character apical pale band nearly equal to the pre-apical dark band. Each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection. From these collections the An. culicifacies female were separated and allowed for egg laying individually and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction (AS-PCR) (Singh et al., 2004a; Goswami et al., 2006; Sharma et al., 2014).

 

Fully engorged female Anopheles culicifacies mosquitoes were collected from field and were used for morphological identification and other subsequent studies. Eggs from the above field caught mosquitoes were collected and transferred to enamel bowls containing approximately two liters of dechlorinated water for further development into larval stages. The developing larvae were provided with yeast tablets as the source of food. After completion of the four larval stages (Eggs→ 1^st Instar Larvae→ 2^nd Instar Larvae→ 3^rd Instar Larvae→ 4^th Instar Larvae→ Pupae), the developed pupae were collected and kept in breeding cages for the emergence into adults. For rearing purpose each population, the adult mosquitoes of the An. culicifacies sibling species were released in separate 75 x 60 x 60 mm cages. The adult mosquitoes were fed with 10% sucrose solution soaked in cotton pads. For blood meal, a rabbit was made available as a source of blood on alternate days. Mosquitoes were reared in the laboratory until adult stage at 27^oC + 2^oC, relative humidity 80 to 90% and the light / dark cycle was controlled by Dawn and Dusk System (Figure 8).

3.5.1 Processing of field collected materials

Separate out semi-gravid females from collected materials and Etherize with anesthetic ether. Remove ovaries with the help of fine forceps and place them in modified Cornoy’s fixative (Methanol- acetic acid 3:1). Keep ovaries at least for 36 hours in Cornoy’s fluid at room temperature (22^oC-25^oC) before processing for the preparation of chromosome, if not to be used immediately, store them in a refrigerator. For long term storing, change the fixative in the vials after 1-2 months.

 

3.5.2 Preparation of polytene chromosome (For viewing under phase contrast optics)

Soften ovaries (removed from fixative after keeping for specified time) in a drop of 50% propionic acid for a minute after this remove propionic acid and add a few drops of diluted 2% lacto-aceto-orcein (1 part stock stain in 3 parts 50 % propionic acid). Keep in the stain for 10 minutes and add a few drops of 50% propionic acid and drop a clean cover slip over the material slowly. Tap on top of cover slip with a blunt ended tapper. Place the slide between two pieces of filter paper and using a circular motion rub hard with thumb on the cover slip to flatten and place the slide on a slide warmer 60^oC for a minute. For storing, place the slides in a horizontal coplin jar at the bottom with filter paper watted with 50% propionic acid.

 

Tyagi V: The main author of the paper and have made contribution to conception and design and also the analysis of data; Sharma A.K: This author also contribution to conception and involved in the acquisition of data by using various methods Yadav R.: Helped in the general supervision of the research group; Sukumaran D: Involved in drafting of Manuscript; Agrawal O. P.: Revising the MS critically for important intellectual contents; Veer V.: Have given final approval of the version to be published.

 

The authors are thankful to Dr. Lokendra Singh, Director, Defence Research & Development Establishment, Gwalior, Madhya Pradesh, India for providing all necessary facility to conduct this research work. Sincere thanks also to the scientists and supportive staff of Vector Management Division for their kind cooperation for carrying out the above work.