Impact of alkaloids extract of Moringa olievera Lam. leaves on the development, fertility and demography of the southern cowpea beetle insect Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

The Southern Cowpea Beetle Callosobruchus maculatus (F.) is one of the most widespread insect pests of stored legumes, causing a considerable loss during storage, decreasing the net weight of the crops


Introduction
Cowpea (Vigna unguiculata (L.) Walp.) is an important legume in tropical and subtropical regions that provides nutritious grains with high concentrations of proteins, carbohydrates, lipids, minerals (Fe, Zn and P), vitamins, thiamine, and riboflavin for human consumption 1,2 .So, it is an important crop economically and nutritionally 3 .After harvest, cowpea beans are stored and used until the following harvest.However, the beans may include storage insects, primarily C. maculatus 4 .
The beetle C. maculatus (F.) (Coleoptera: Chrysomelidae) is the most harmful storage pest of cowpea (V.unguiculata (L.)) seeds.The quality of the stored seeds is quickly reduced when the insect is present 5 , which causes a lot of money to be lost 6 , and it causes huge losses, up to about 9% in developed countries and 20% or more in developing countries 7,8 .
Many scientific studies have been done on fumigating seeds with different concentrations of organic pesticides to mortality C. maculatus while the seeds are in storage.But because some species have become resistant to these chemical materials, they are not enough to get rid of this pest.Also, these pesticides were very expensive, and the peasants had trouble using them because of how they were made.Also, these chemicals could be harmful to the environment if they end up in food waste, and they can mortality other insects that are friendly with the environment 9, 10 so, researchers recently ended up using Inorganic salts and antioxidants can be used instead of pesticides because they work just as well against insects that don't live long, are safe, and cost less.Careful preparation of this material leads to eliminate of insects and achieving the goal in small quantities and in safe substances that lead to eliminate insects in a safe way and do not harm human health 11,12 .Insect pests have been controlled in ways that are better for the environment by using traditional extracts or products made from plants instead of conventional pesticides made from chemicals.Studies have shown that many plants have materials that are poisonous, repel, or attract insects 13 .M. oleifera (Mo), which is in the family Moringaceae, is one of these plants.It has a lot of glucosinolates, which kill insect pests by acting like a biological fumigant 14 .M. oleifera grows in the tropics.It is called a "miracle tree" or "wonder tree" because it has a lot of social and economic benefits because it is used in pharmaceutical, nutritional, antihypertension, and industrial applications 15 .Studies have shown that the plant's leaves have an effect on the pests that live in warehouses 16 .Also, the alcoholic extract of M. oleifera.Leaves have useful biological properties that depend on the type of solvent used to get the active ingredients out of the leaves 17 .The phytochemical analysis of Moringa leaves showed the presence of alkaloids, tannins, saponins and phenols as major secondary metabolites present in the leaves of the plant 18 .Alkaloids are toxic secondary metabolites that block ion channels, inhibit enzymes or interfere with neurotransmission leading to the death of insect pests 19 .So, the goal of this study was to find out how well alcoholic extracts of M. oleifera leaves protect cowpea V. unguiculata (L.) from being infected by southern cowpea beetle as a safe alternative for chemical insecticides, by using agestage two sex, life table analysis.

Insect culture
Cowpea seeds infested with this insect were gathered from Baghdad's local marketplaces.The seeds were cleansed of contaminants and purified by Prof. Imad Ahmed Mahmoud of the entomology laboratory of the College of Science for women at the University of Baghdad identified this insect as the southern cowpea beetle (C.maculatus).In order to create a permanent stock culture, 250 g of uninfected cowpea seeds were placed in the sterilized jar 16 × 8 × 8 Cm 3 for 24 hours to verify that they were free of beetle infestation.Males and females of the southern cowpea beetle were then introduced, wrapped with organza fabric, and secured with an elastic band to prevent insects from escaping.The bottles were placed in the incubator at 28± 2 °C and 60± 5% relative humidity, and the colony was continuously replenished 20 .

Preparation and Separation of Extraction Leaves M. oleifera Alkaloids
Moringa oleifera leaves were collected from the garden of the College of Science Woman -University of Baghdad and diagnosed in the same college.Then the leaves were cleaned, washed from dust with distilled water and dried at room temperature.After the Moringa leaves were well dried, they were crushed by an electric grinder (Monilex Australia Pvt. Ltd.) for 45 s.Samples were stored in airtight glass containers at room temperature until use.Alkaloids were extracted as The effect of three concentrations of the alcoholic extract of the leaves of the M. oleifera plant was tested on the indicators of the growth of the insect population, as the fertility and life tables were adopted to test this effect.
The life table and fertility were regulated by rearing the beetle under standard conditions.The earlyemerging insect 24 hour's age was isolated from a small test colony taken from the original colony.After the emergence of the insect, the males were separated from the females, by distinguishing them based on their size, as the females are larger than the males, in addition to the presence of two dark bands at the end of the female's abdomen.10 adults (5 males, 5 females) were placed in a plastic petri dish (6 cm diameter) with 3 replications for each concentration in addition to the control treatment.Then the adults were treated with concentrations 1000, 2000, and 3000 ppm by spraying them with a 100 ml hand sprayer from a distance of about 10 cm to ensure that the insect was exposed well, then left to dry.Then 15 healthy cowpea seeds were added to each dish and incubated under standard incubation conditions.The beetle was followed daily, and the number of eggs laid daily and the survival rates of adults were recorded.30 eggs were isolated for each replicate and left until emergence.After emergence, a newly emerging pair (male and female) was taken and placed in a plastic petri dish, and healthy and infested cowpea was added to it by 14 replicates for each concentration.Then follow up and record, the number of eggs laid/ day and the number of dead adult/ day.

Life table analysis of C. maculatus (L.)
Individual C.maculatus life history raw data were analyzed using the TWO-SEX-MSChart program12, which is based on the age-stage, twosex life table theory 23 and the method described by Chi. 24,25.As follows: Survival rate (sxj) (x = age, j = stage), which is the probability that a newly laid egg will survive to age x and stage j, and fecundity fxj, which is the number of hatched eggs produced by a female adult at age x were calculated.

Results
Phytochemical screening of alkaloids 1-Mayer reagent: The result was the appearance of a white precipitate, evidence of positive detection 26 .2-Wagner reagent: This resulted in the appearance of a brown precipitate as evidence of positive detection 26 .

Life table analysis of C. maculatus (F.)
The results in Table 1 give information on the population growth of C. maculatus after treatment with concentrations of 1000, 2000 and 3000 ppm of the alkaloids extract of M. oleifera.First, it can be seen in the table that the highest median survival was at 3000 ppm concentration which is 51%, while the lowest median survival at 1000 and 2000 ppm concentration was 49% respectively.In addition, the survival rates (lx) for the three concentrations decreased compared to the control of 77%.The results of the statistical analysis showed a significant difference in the survival rates between the three concentrations and the control treatment.This indicates that alkaloids extract concentrations have an effect on C. maculatus survival rate (lx).Moreover, it is clear from the Table 1 that the net reproduction rate (Ro) reaches its lowest level at the concentration of 1000ppm i.e. 4.76 females/female /generation, compared with the control which is 10.34 female/female/generation.Thus, it can be concluded from the results of the statistical analysis that there is a clear variation in the three concentrations.Also, the Table 1 gives information about the mean generation length (T) for 1000 ppm concentration which equals 24.58 days, while the (T) for 2000 ppm and 3000 ppm is 24.72 days and 24.6 days, respectively, compared to 24.62 days to control.However, statistical analysis shows that the extract does not cause any significant change in the mean generation length.Regarding the increased capacity (RC) female/female/day, it can be seen that it is equal to 0.063 for the concentration of 1000 ppm compared to 0.08 for the concentration of 2000 and 3000 ppm, while the control treatment is 0.09.Once again, the statistical analysis indicates that there is no significant difference between the concentrations on the one hand and between the concentrations and control on the other hand.
Looking at the intrinsic rate of increase (rm) female/female/day, it is 0.08 for each of 2000 and 3000 ppm compared to 0.06 for the concentration of 1000ppm and 0.09 for the control treatment.The statistical analysis shows that there are significant differences between the concentration of 1000ppm and all the concentrations, and there are no significant differences between the concentration of 2000 and 3000 ppm and also between control treatments.As for the finite rate of increase in female offspring per female per day it was found that it was 1.08 for each of 2000 and 3000 ppm compared to 1.06 for the concentration of 1000ppm and 1.09 for the control treatment, as well as the statistical analysis between the presence of a significant difference only in the concentration 1000 ppm from the rest of the concentrations, as well as between it and the control.Moreover, both the 2000 and 3000 ppm concentrations have the same weekly multiplication rate which is equal to 1.75, while it is 1.52 for the 1000 ppm concentration, and 1.88 for the control treatment.Statistical analysis showed that there was no significant difference between concentrations 2000 and 3000, while it was clear between concentrations 1000 and other concentrations and between the studied concentrations compared with the control treatment.The table 1 gives information about the corrected generation time (Tc) for the three concentrations 1000, 2000 and 3000 which are 26, 24.82 and 25.34 days, respectively, compared to 25.96 days for the control.However, there is not much difference between the concentrations.About the doubling time of the population, it reaches its highest level at a concentration of 1000 ppm, which is equivalent to 10.92 days, while it reaches its lowest level of 8.41 at a concentration of 3000 ppm, compared to the control treatment of 7.31 days.In fact, statistical analysis indicates significant difference between the 1000ppm concentration on one side, and the other and the control treatment on the other side.On the other hand, there was no significant change between the concentrations of 2000 and 3000 ppm or between them and the control treatment.The study showed that the fecundity rate or the number of eggs laid by a female reaches its highest level of 42.44 egg at a concentration of 3000ppm, while it becomes at its lowest level of 34.55 egg at a concentration of 1000ppm.It is worth noting that the fecundity rate of the control treatment is 32.75 egg .The statistical analysis showed that there was no significant change between the concentrations 1000 and 2000, but it was significant between the concentration of 3000 and the other two concentrations, as well as between all concentrations and the control.the results also showed that the fertility (percentage of hatching eggs) decreased gradually with increasing the concentration of the alkaloid extract, as the lowest percentage of hatching for eggs was recorded, which amounted to 62% at a concentration of 3000 ppm, while at a concentration of 1000 ppm it recorded 77%, while the highest percentage of fertility was recorded when the control treatment 88 %.The results of the statistical analysis indicated that there were significant differences between the fertility (hatching eggs) at the three concentrations, in addition to the control treatment.The results also showed in the Table1 that there was no effect of the different concentrations of the alkaloid extract on the longevity of the male and female the insect.No significant differences were recorded between the longevity when treated with different concentrations and with the control.As for the sex ratio (female: male), it was not affected much in the different treatments, as the ratio between males and females of the insect was close to (1:1) with a slight tendency to increase the proportions of males compared to females.Fig. 1 showed some information derived from life tables where the figure appears the relationship between the survival rates of the insect, its egg productivity and the sex ratio of the female during the life cycle of the C.maculatus after using three concentrations 1000, 2000, 3000 ppm of alkaloid extract of Moringa oleifera leaves It can be observed when using 1000 ppm of alkaloid extract, the first day of laying eggs was two days after the appearance of the beetle, which corresponds to the 24th day of its life cycle.In addition, the highest survival rate for when using this concentration was 49% and the highest number of eggs laid by each female was 14.35 egg and the number of females was 4.95 egg according to the sex ratio.On the contrary, on the sixth day of a female's life the lowest number of eggs being laid was on the 28th, which was 0 eggs when the survival rate was 0.02.As for the results, after using the concentration of 2000 ppm, the first laying of eggs was after two days from the emergence of the mature beetle, and the survival rate reached 49% and which corresponded to the 24th day of the life cycle.the highest number of eggs was 16.99 eggs for each female and noted that the number of females is 7.73 according to the sex ratio, while the lowest rate of laying eggs on the sixth day of female life was (0), and the survival rate was 0.07%.Regarding the results after using the concentration of 3000 ppm concentration, the first egg laying was after two days from the emergence of a mature female, and the highest survival rate was 51%, which occurred on day 24 of the life cycle.and the highest number of eggs laid by each female was 17egg.The number of females was 7.73 female, according to the sex ratio, It is worth noting that the lowest rate of laying eggs was on the sixth day of life, the female was zero and the survival rate was 0.07%.In addition, it has been noticed that control treatment, has begun laying eggs after two days from the emergence of the female.At the same time, the survival rate reached its highest level, which is 77%, and the highest number of eggs laid was 16.79 egg and the number of females was 7.47 female according to the sex ratio.However, the average of the lowest eggs laid on day 28 of the life cycle corresponding to the sixth day of a female's life was zero, meanwhile, the beetle's survival rate was 0.14%.

Discussion
Understanding pest parameters is essential in Integration Pest Management (IPM).Our results showed that using the alcoholic extract of M, oleifera leaves in different concentrations 1000, 2000, and 3000 ppm reduced the survival rates (lx) of C. maculatus.This is due to the fact that the secondary compounds of plants, including alkaloids, may delay or speed up development or interfere with the life cycle of insects in other ways Earlier, 26 reported that alkaloids have a toxic and anti-nutritional effect on storage insects.This observation is consistent with the results of 27 , 28 it was reported that alkaloids, when increased in concentration, are better anti-appearances and more effective against immature stages of C. maculatus.
Hence alkaloids in increasing concentrations may have acted as ovulatory (active against eggs) and larvicides (active against larvae).This result is consistent with the work of the researcher 29,30 where he used extracts and powders of each of zanthoxyliodes, A. occidentale and M. oleifera, which contain alkaloids in preventing the Here, it turns out that the lower the survival rates, the less damage to cowpeas, whereas 30 indicated that the extent of damage during storage depends on the number of adults for each generation, the life span and the diversity of the host.Also, the net compensation rate (Ro ) decreased compared to the control treatment, an indication that the alkaloid extract reduced the number of females that replace a female in the generation a higher rate of population increase was observed in the populations of females treated with insecticides.It was noted that the three alkaloid extracts of Moringa leaves -compared to the comparison group -have a clear effect in reducing the egg hatching rate.The noticeable decrease in egg hatching can be linked to the fact that the extracts affected their general metabolic activities, including sexual activities, as indicated by 31,32 that plant extracts have the ability to pass through the chorion of eggs and thus prevent further development.Jaafar 33,34 mentioned that there was a significant effect of radish seeds and leaf extract Radish Raphanus sativus L containing alkaloids on the hatchability of cowpea beetle eggs for three generations, as the hatching rate of eggs decreased in the second and third generations than in the first generation.The results in the table also show the variation in the numbers of females and males that appeared between the treatments, and the concentration of 1000 shows the lowest percentage of females versus males, and this result is close to other results of 35 , 36 Where it was shown that extracts of Moringa oleifera, Ziziphus joazeiro reduced the appearance of male and female C. maculatus.In addition, the alkaloid extracts slightly shortened the longevity of the female at a concentration of 1000, and this contradicted the result of 37 , 38 where the methanol extract of leaves Anchomanes difformis (Blume) Engl and containing alkaloids was recorded the lowest mortality rate and longest lifespan for Callosobruchus maculatus 18.43-86.67%due to the presence of some bioactive compounds.However, the results showed an increase in the number of eggs laid by the female for the three concentrations compared to the control coefficient.However, the results show an adaptive reproductive strategy adopted by the females of the southern cowpea beetle, which seems to increase their daily fertility in line with the alkaloid extract that reduced their survival rates.

Conclusion
These results are of particular interest to those who seek to use biological insecticides such as alkaloid extract of M. oleifera leaves to control the pests of stored products.It is worth noting that the use of the lowest doses can have a significant and obvious effect on survival, hatchability and female rates.
Where does this help the laboratory study in evaluating the nature of the insect population treated with the alkaloid extract and with the completion of the study of life tables in the future and for several field generations can develop and evaluate appropriate control programs and develop integrated control programs for the insect economic importance.

Figure 1 .
Figure 1.Survival rate (lx) and fecundity (Mx) curves of Callosobruchus maculatus when treated by Alkaloids extract of Moringa oleifera insect and inhibiting its development.

Determination of the insect population growth indicators for C. maculatus by using age stage two sex life table Insects
22ecific predation or consumption rate, the agestage, two-sex life table offers a promising method for studies involving predator-prey relationships, biological control programs, economical injury levels in IPM, and determining the precise timing of various control measures22.
Mx female productivity of eggs at the age stage x, Fx total eggs laid at the age stage x, nx number of live females at the age stage x.mx = number of female from productivity of eggs at the age stage x, By knowing the survival rates and productivity rates, it was possible to extract the net reproductive rate(Ro) and average generation length(T) e generation Mean, The rate of internal increase in population(rm) Intrinsic rate of increase, specific rate of population increase(λ), (DT)h how long does it take for a population to double? as follows: Ro=∑〖lx.mx〗Where,Ro as the net Reproductive rate (number of productive females/female/generation), ∑〖lx.mx〗 the sum of the product of multiplying the life expectancy rates of females with the age productivity rates Tc=∑〖X.lx.mx〗/∑〖lx.mx〗Where,Tccohort generation time (in days) ∑〖X.lx.mxThe sum of the product of multiplication lx.mx with the age group X, ∑〖lx.mx〗 as the net compensation rate.T = ln Ro/rm T the corrected generation time rc = ln Ro/Tc rc innate capacity for increase Ln.Ro the inverse of the logarithm of the net compensation rate, Tc cohort generation time (in days) rm= ∑ e -rm.X lX mX = 1 Where, rm rate of intrinsic increase in population, the maximum population growth, the intrinsic rate of natural increase or the innate capacity for increase (females/female/day) λ=ermWhere, λ the finite rate of increase, the number of female offspring per female per day e represent antiloge Against the natural logarithm and its amount=2.718,rm rate of internal increase.DT=In2/rm DT doubling time, the number of days required by a population to double,