DEMOGRAPHIC AND PHYLOGENETIC EVOLUTION OF SITOPHILUS ZEAMAIS SUBSERVIENT TO MILLET IN SENEGAL (WEST AFRICA).

1. Faculty of sciences and Department of Animal Biology Technology BIOPASS Laboratory UMR 022 IRD-CBGP University Cheikh Anta DIOP Dakar, Senegal. 2. Faculty of sciences and Department of Animal Biology Technology BIOPASS Laboratory UMR 022 IRD-CBGP University Cheikh Anta DIOP Dakar, Senegal. 3. University of sine saloum ELHADJ IBRAHIMA NIASS sing-sing, BP 55, Kaolack, Senegal. 4. Faculty of sciences and Department of Animal Biology Technology BIOPASS Laboratory UMR 022 IRD-CBGP University Cheikh Anta DIOP Dakar, Senegal. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 04 August 2019 Final Accepted: 06 September 2019 Published: October 2019

Food security remains a major challenge in Senegal. The millet that occupies the most important place in cereal production can play a fundamental role in taking up this challenge. But it is strongly ravaged by a beetle of the Curculionidae, Sitophilus Zeamais, in 4 agroecological zones (AEZ) where it is substantially exploited. The solution required to eliminate this pest is the use of pesticides, despite the horrible consequences that accompany it. It is possible to find healthy solutions. This article aims to identify the type of selection (positive or negative) that specifies each population and the degree of kinship of their individuals. The importance of detecting the demographic signal of AEZ populations is to be able to identify agroecological zones that are likely to favor survival or extinction of the insect. Positive selection militates more in favor of the adaptation of individuals than negative selection. Thus, insects of S. Zeamais subservient to millet were sampled in each AEZ. The exploitation of Cyt.B gene sequences led to the conclusion that the NBA population has undergone a positive selection, while the BMC population was negatively selected. SBA and SOHC populations have not been selected. Thus, the agroecological zone of the NBA is likely to favor the survival of the insect whereas the AEZ of the BMC is likely to favor a bottleneck. Phylogenetic trees have highlighted a close relationship of individuals in each AEZ.
Copy Right, IJAR, 2019,. All rights reserved. …………………………………………………………………………………………………….... Introduction:-85 the use of pesticides, chemicals whose use has a negative impact on living beings and the environment. Otherwise, genetic studies on S. Zeamais so far have focused on its phylogeny and its distribution in Africa in general. No study on the genetic effect of the environment and its consequences on the insect targeting the specific case of Senegal has been made.
To avoid the enormous losses caused by the insect, natural solutions can be studied. This article fits into this perspective. It aims on the one hand to identify the agroecological zones naturally likely to favor the survival or the extinction of S. Zeamais, by the detection of the demographic signal which characterizes each one of them and on the other hand the degree of kinship of their populations. Indeed, agroecological zones where genetic drift and natural selection exert a positive selection are likely to favor the survival of the insect in contrast to those undergoing negative selection.
Insects of S. Zeamais were sampled for this purpose in each of the four AEZs.
The 43 sequences of the Cytochrome B gene corresponding to these individuals were exploited by population genetics software (Bioédit, DNAsp, Mega, Harlequin ...), in relation to demographic and phylogenetic parameters, in relation to the aforementioned objectives.

Harvesting individuals
The collection of infested Millet samples in the different AEZs made it possible to isolate individuals of S. zeamais for each zone. It has been done in the fields, in storage facilities where grain is highly vulnerable to infestation, but 86 also in marketing places where there is a high chance of encountering infested millet from different AEZs. After isolation, individuals from each AEZ are placed in tubes containing 96% alcohol.
To code individuals for their host plant, we capitalized the first letter of the insect's genus name and then specified the type of host plant of the individual using the first two letters of the plant (The first letter in upper case and the second in lower case), we have specified the locality of origin (the first letter in capital letters and the second in lowercase), then specify the serial number. Example a Sitophilus zeamais individual who was harvested in Bambey on Mil with the order number 12 is coded as: SMiBa12. if it was on corn from maize, the code would be SMaBa12.  Table 1 summarizes the localities of the AEZs where the harvests took place, the number of individuals sampled for each AEZ, the geographical coordinates of the localities and the codes of the individuals.

Molecular method of analysis DNA extraction
The extraction is the DNA release technique of the cell. It includes the individualization of cells (digestion) and the destruction of their plasma and nuclear membranes (lysis).
The digestion of the cells consisted of placing their paws and prothorax into tubes containing ATL buffer and K proteinases. After incubation, the tubes were centrifuged to separate the supernatant from cell debris.
To destroy the cell membranes, first cell lysis buffer (AL) was added, then some ethanol (96%) after incubation into the tubes. Then the tubes are transverse in silica membrane columns. Finally, the centrifugation of the tubes allowed to retain the DNA on the siliceous membranes of the columns because negatively charged.

DNA purification
The tubes DNA was purified by adding 2 buffers AW1 and AW2 in each column. After Centrifugation of the tubes and precipitation of the DNA at the bottom, the buffers and contaminants are discarded. The columns are then replaced in other tubes in which buffer AE has been added to unhook the DNA. The DNA is thus removed and stored at -20 ° C.

PCR of the mitochondrial gene Cytochrome B
The PCR of the mitochondrial gene Cyt.B was carried out by two primers CB1 (5'TATGTACTACCATGAGGACAAATATC-3') and CB2 (ATTACACCTCCTAATTTATTAGGAAT-3'). For each sample (tube), the amplification was made from a total volume of 25 μl, of which a mixed volume of 23 μl and a volume of 2 μl of DNA extract. The mixed volume was constituted by: 18.3 μl of milli water, 2.5 μl of 10 × buffer, 1 μl of additional MgCl 2, 0.5 μl of Dntp, 0.25 μl of each primer and 0.2 μl of Taq polymerase.
The conditions under which the PCR was performed are as follows: 1. The DNA strands were first separated with a temperature of 94 ° C for 3 minutes. This first denaturation was followed by 35 denaturation cycles of 1 minute at the same temperature. 2. The synthesis of complementary strands (elongation) was made at 72 ° C. for 10 minutes. After amplification, the fragments are sent to a South Korean company for sequencing. 87

Bioinformatics Analyzes
The sequences were corrected and aligned by the Clustal software implemented in the Bioédit version 7.2.5 programs (Hall, 1999).
The demographic history of the populations sampled in the different agroecological zones was apprehended from a "mismatch distribution"    Thus, the NBA AEZ with positive selection favors the survival of the insect while that of the BMC which has been negatively selected is conducive to a bottleneck. The other 2 AEZs, namely the SBA and the SOHC, whose populations are characterized by Tajima's D and H values of Fay and Wu null, have no effect on the adaptability of the insect.

Mismatch distribution
The phylogenetic trees (Maximum Likelihood and Neighbor Joining) revealed 3 clades: 2 clades strongly supported by posterior probabilities superior to 90%, formed individually of insects subservient to SBA and SOHC and another weakly supported clade (16%), consisting of both BMC and NBA insects. Insects, those of SOHC and BMC are phylogenetically closer.

Conclusion:-
The study that we made in Senegal on 43 individuals of Sitophilus Zeamais subscribed to millet and identified in four (4) agroecological zones revealed on the one hand a demographic expansion of the population of the NBA and the BMC which is respectively the result of a positive selection and negative one, and a complete lack of polymorphism of the SBA population and that of SOHC. The agroecological zone of the NBA thus gives the insect a strong adaptability while that of the BMC which can be at the origin of a bottleneck through the genetic drift and the natural selection, is hostile to the insect. NBA and SOHC AEZs have no effect on the suitability of the insect.