This is an Experimental analytical study conducted between Egypt and Sudan.

Study area:

Egypt: 500 samples were collected in the fourth larval stage. Samples were obtained from Egypt: Faiyoum Governate (Sinnuris Centre). Sinnuris is located between latitudes 29°15 and 30° 15 and longitudinal 30°0 and 30 °30.boundries: North-guaran Lake South-Western desert West Western desert. East-River Nile. It comprises an area of 1,778 km2 with an elevation of 30m from sea level.

Sudan: 500 samples were also collected in the fourth larval stage. Samples were collected from Gezira state (WadElshafie village), Sennar state and Khartoum state (Soba). Khartoum is located between 15 °26 and 15 °45 north and longitudes 32 °25 and 32° 40 east at an altitude of 405.6m above sea level.

Sample Collection:

500 samples were collected from Egypt and stored in a thermometer to be brought to Sudan for morphological identification. Also, 500 samples were collected from Sudan and bred in dishes containing normal water and placed in a room with an air conditioner. Larvae were fed with crushed bread (boxomat) for 3 days until changing to pupa.

Collection of Larvae:

Samples were collected via the dipping method according to O’Malley, (1989) was used [13]. The dipping technique is called Shallow skim. A WHO standard dipper was used to investigate the presence and/or absence of larval stages of Anopheles pharoensis species. Larvae were then collected by a Bilge pump or a Baster. They were emptied into a white enamel pan from which the larvae were examined and moved to a laboratory to be properly stored in Carnoy’s fixative and kept in a freezer pending the beginning of the experimental work.

Methodology:

a- Techniques for karyotyping:

Karyotyping was investigated using Polytene chromosomes.

1/Polytene Chromosomes:

Polytene chromosomes were obtained by the squash method.

Larval salivary glands were dissected and squashed using orecien stain method.

2/Cytotaxonomy and polytene chromosome map:

A reference system of chromosome mapping adopted by Sidky and Riad (1976) was used in this study[14].

b- Preparation of Orecien stain:

5 gm of orecien powder was added to 125 ml of acetic acid. The solution was shaken for 2-3 min at room temp. 125 ml of lactic acid was added to the solution and stirred for 2 minutes then it was filtered and stored in the laboratory.

c- Preparation of Carnoy’s fixative:

Carnoy’s fixative was prepared by adding 300 ml of ethanol to 150 ml of chloroform and 50ml of acetic acid [15].

d- Larval Salivary Polytene Chromosome Preparation:

Larval polytene chromosomes were prepared following Boakye et. al (1993)[16]. Fourth-stage larvae were removed from carnoy’s fixative and placed in a petri dish. Full-sized larvae were chosen. Each larva was dissected and its salivary glands were removed and converted to a clean slide. A drop of 60% acetic acid was put on the slide. The salivary glands were covered with a clean cover slip and squashed smoothly with the thumb. A drop of Orcein stain was added to the edge of the coverslip and the slide was placed on a bench to dry. Four hundred slides were prepared from An. Pharoensis species. The chromosomes were viewed under a LeitzDiaux 20 phase-contrast microscope with a camera attached to the microscope.

Mitosis in Anopheles pharoensis:

a-Polytene chromosomes:

Figure(1): shows a larval salivary gland polytene chromosome of Sudanese strains of Anopheles pharoensis. Polytene chromosomes have a characteristic clear banding pattern. No breakpoints and fragmentation were observed to occur in the Sudanese strain. The centromere is fan-shaped.

Figure (2): Clear bands appear in the Egyptian strain with two break points and fragmentation in the X -X-chromosome. The centromere is also fan-shaped.

b-X-chromosome Maps:

X-Chromosome Map of Sudanese strains:

Figure (3): Showing clear bands. No inversion was observed in the Sudanese stains of Anopheles pharoensis.

X-Chromosome Map of Egyptian strains:

Figure (4): Showing clear bands with inversions at junctions 1-C and 5-A on the X-Chromosome of Egyptian strains of Anopheles pharoensis. Inversions are indicated by arrows. This Map was drawn by Sidky and Riad (1976). [14]

X-Chromosome Map of South Africa (Zululand sample):

Figure (5): is a chromosome map adopted by Miles et al., [17]. This map is a comparative Polytene Chromosome map between the Sudanese and Egyptian Chromosome maps. Inversions also appear at junctions

1-C and 5-A similar to inversions of the Egyptian strain of Anopheles pharoensis.

Figures & Tables

Discussion

Anopheles pharoensis as a malaria vector is well established in Egypt [7] but there is a lack of certainty in the south Saharan Deseret’s of Africa even though a number of sporozoite-positive specimens were previously reported. Such conflicting views on the role of Anopheles pharoensis as a vector might be attributed to the existence of different sibling species deducted from Polytene chromosomes studied on materials from different African regions. In Egypt, Anopheles pharoensis vector populations are known by chromosomal Xa inversion [18].

The aim of this investigation was to study the genetics of Anopheles pharoensis in the Sudan and Egypt in-order to detect the presence of inversions in the X`-Chromosomes of Sudanese samples (the investigation also aimed to study the relation of this inversion with vectors in Sudan and Egypt).

The existence and apparent fixation of two different X-Chromosome arrangements in geographically separated populations of the taxon Anopheles pharoensis in nature represents either geographic variation within a single species, or at least two genetic species within the taxon [17].

A photo map of larval salivary gland Chromosomes of Anopheles annulipes (Cellia was presented by Booth et al., [19]. The standard Chromosome of Anopheles annulipes had one major puff with two prominent broken bands across it. The centromere end had a large puff. This agrees with the findings of this research where a large puff was observed at the centromeric end, also there are two broken bands, which indicate that an inversion in the X-Chromosome of the Anopheles pharoensis occurred in the Egyptian strain. The X-chromosome had also one major puff. In this research, Sudanese Anopheles pharoensis strains had no broken bands but had a large puff at the centromeric end and a major puff in the X-chromosome.

A photo map of the polytene chromosome of Anopheles pharoensis from Zululand was presented by Miles et al. (1983) inversions also appeared at junctions 1-C and 5-A [17], which agrees with the findings of this research similar to inversions observed in the polytene chromosome of the Egyptian strain of Anopheles pharoensis.

The Polytene Chromosome technique was used in this research to compare the X-chromosomes of the two strains (Egypt and Sudan) of Anopheles pharoensis and to give reasons why Sudanese strains cannot transmit Plasmodium parasites while Egyptian Anopheles pharoensis strains succeeded in transmitting the malaria parasite. Probably the absence of the inversion detected in the Sudanese strains of Anopheles pharoensis prevents Sudanese strains from acting as vectors of Malaria, while the presence of the inversion in the X-chromosome of the Egyptian strain increases its possibility of transmission of Malaria.

In Egypt, in general, and in Faiyoum Governate, in particular, Anopheles pharoensis is an important Malaria vector. In the Sudan, Anopheles pharoensis is not known to be causing malaria transmission anywhere in Sudan. The aim of this study was to answer the question of why Anopheles pharoensis is a major vector in Egypt and not a vector in the Sudan?

There were inversions detected in the X-chromosome of Egyptian strains at junctions 1-C and 5-A. From this finding one comes to the fact that Anopheles pharoensis in Egypt is capable of transmitting Plasmodium parasites due to the presence of inversions in the X-Chromosome while Anopheles pharoensis in the Sudan cannot transmit the Malaria parasite due to the absence of the inversions in the X-Chromosome.

It appears that Anopheles pharoensis is a Sudanese species that migrated to Egypt and on its migration it experienced different inversions in its X-chromosome when it reached Egypt. This changed its behavioural factors shifting it from a non–vector in Sudan to a vector of Malaria when reaching Egypt. Though further research is needed from the findings of this research, Anopheles pharoensis is probably a Sibling species.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Najlaa Siddig Nasir: Conceptualization, writing-original draft, supervision, methodology, data curation, writing-Review, and editing.
Fatima Fadul Ali: Conceptualization, writing-original draft.
Fatima M. Mohamed: Conceptualization, writing-original draft, supervision, methodology, data curation.
Mohamed Osman Elamin: writing-original draft, methodology, writing-Review, and editing.
Ali M. Alshehri: writing-Review, and editing.
Hatim M. Badri: writing-Review, and editing.
Wahaj.A. Khan: writing-Review, and editing.
Hatim A. Natto: writing-Review, and editing.
Mashael Alfaifi: writing-Review, and editing.
Ahmed A. Osman: writing-original draft, methodology, writing-Review, and editing.