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Research Article

Possible Cytogenetic Effect of Capsicum frutescens (Solanaceae) Extracts on Meiosis in the Grasshopper Taphronota thaelephora Stal. (Orthoptera: Pyrgomorphidae)

Seino RA1,2*, Dongmo TI2, Chifon RN2, and Shambo DN2

1Department of the Biological Sciences, Faculty of Science, The University of Bamenda, Cameroon


2Applied Biology and Ecology Laboratory (LABEA), Department of Animal Biology, Faculty of Science, University of Dschang, Cameroon


Corresponding author


Seino RA, Department of The Biological Sciences, Faculty of Science, The University of Bamenda, P.O. Box 39, Bambili-Bamenda, Cameroon; Tel. +237-77311434; E-mail: raseino@yahoo.co.uk


Received Date: 02 Feb 2014

Accepted Date: 23 July 2014

Published Date: 25 July 2014


Citation


Seino RA, Dongmo TI, Chifon RN, Shambo DN (2014) Possible Cytogenetic Effect of Capsicum frutescens (Solanaceae) Extracts on Meiosis in the Grasshopper Taphronota thaelephora Stal. (Orthoptera: Pyrgomorphidae). Enliven: J Genet Mol Cell Biol 1(1): 002.

Copyright


@ 2014 Dr. Seino RA. This is an Open Access article published and distributed under the terms of the Creative Commons Attribution License, that permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Abstract


Pepper, Capsicum frutescens is an important Solanaceae species used as a common spice for food in many countries and in particular Cameroonian households. The aim of this study was to investigate the cytogenotoxic effect of the aqueous extract from C. frutescens fruits on the pest grasshopper Taphronota thaelephora (Orthoptera: Pyrgomorphidae) germ line cells by cytogenetic studies that have never been done before for pepper extracts. For this, adult individuals of T. thaelephora were treated for 72 hours with different concentrations of pepper extracts (0%, 10%, 25%, 40%, 50% & 60%). Chromosome number and the meiotic behaviour of chromosomes were analyzed. The results revealed that the extract did not induce chromosome breaks but could induce laggards and bridges in meiotic anaphases 1 &2 and metaphase -2 in the male germ cells of T. thaelephora. Increasing the concentration of the extract also significantly increased (P<0.05) meiotic abnormalities in the germ line of T. thaelephora. Chiasma frequency significantly increased when the concentration of the aqueous extract of C. Frutescens was increased.


Keywords:


Capsicum frutescens; Aqueous extract; Cytogenetic effect; Taphronota thaelephora


Introduction


The cytogenetic effects of many plants extracts on grasshopper pest species are still unevaluated even though cytogenetic assays have been widely used in genotoxicity assessments to test compounds under both in vitro and in vivo conditions. A review of available literature revealed that studies have been carried out regarding the cytogenetic effects of plant extracts on other plants [1], fungi [2-4], on maize stem borers [5] and one on grasshoppers [6]. These investigations revealed that plant extracts induced marked decrease in mitotic index accompanied by considerable percentage chromosomal aberrations, meiotic abnormalities such as stickiness, disturbed chromosomes, bridges and lagging chromosomes [1,7-9]. The degree of cytological aberrations in either mitosis or meiosis is regarded as one of the dependable criteria for estimating the effect of a mutagen [10]. Mutagen induced chromosome abnormalities are the primary basis of genetic change therefore investigation of chromosome breakage, type of aberrations and their genetic consequences form an integral part of most of the mutation studies [11]. Capsicum frutescens (Solanaceae) commonly referred as pepper in Cameroon is widely distributed in the tropics and subtropics. It is a common spice for food in many Cameroonian households. It is eaten cooked or uncooked. Peppers (Chillies) originated in South America where they have been cultivated since ancient times. They were introduced to the rest of the world by Spaniards and the Portuguese. The ripe fruit has insecticidal properties and the effective substances are highest in the skin and in the seeds [12,13]. Active ingredients function as stomach poisons to target pests [13]. Extracts from C. frutescens have proofed good antifeedants for the African pest grasshopper Taphronota thaelephora [14].


Considering the potential insecticidal use of C. frutescens extract, and the lack of data about its cytogenotoxicity, this investigation was carried out to study the effect of C. frutescens on meiosis in the pest grasshopper T. thaelephora.


Materials and Methods


Preparation of Extract

Dried yellow pepper (Capsicum frutescens) fruits were obtained from the local market in Dschang. 100 grams of the finely ground dried fruits were stirred in 1 litre of distilled water and allowed to stand overnight. The filtrate (stock solution) was used to prepare five concentrations of 0%, 10%, 25%, 40%, 50% and 60% of pepper (Capsicum frutescens) (Table 1) and used for the study.


Administration of Extract

Sixty adult male grasshoppers of Taphronota thaelephora were separated into 6 groups of 10 grasshoppers each. The groups were labelled A, B, C, D, E & F and were respectively administered 0%, 10%, 25%, 40%, 50% & 60% of extract. 1 ml of the extract was injected into the peritoneum of each grasshopper and incubated for 72 hours. The grasshoppers were then dissected, the testes removed and fixed in Canoy’s solution (3:1 Ethanol – acetic acid) then stored in a refrigerator at 4°C until used.


Cytogenetic examination

Meiotic chromosomes were obtained using the classical lactic-propionic-orcein squash technique [15]. Slides were prepared for individuals treated with the six concentrations under study and examined for chromosome number, precocious movements of chromosomes in meiotic Anaphases (Laggards) and formation of bridges in first and second meiotic Anaphases and second meiotic Metaphases. The chiasma frequencies in 25 Diplotene cells for each of 10 individuals per treatment were recorded.


Results and Discussion


Chromosome Number

Diplotene and Metaphase 1 cells obtained from individuals subjected to the different treatments revealed 9 bivalents and one univalent giving a chromosome complement of 2n=19. The univalent was the X- chromosome because it exhibited the reversal type of heteropycnosis a characteristic of Orthoptera grasshoppers [16]. In all the cells examined, chromosomes were acrocentric in morphology. No telocentric, metacentric or sub-metacentric chromosomes were present. Chromosomes have been described as acrocentric in T. Thaelephora [17]. Since telocentrics, metacentric and submetacentrics were absent, this indicated that treatment of T. thaelephora did not result in chromosome breakages (structural aberrations) and hence mutation. Since these were absent in treated and control individuals, it is conclusive that the extract of C. frutescent was not mutagenic.


Meiotic Studies

Chromosome aberrations are important cytogenetic endpoints that are routinely used in cytotoxicity and genotoxicity evaluations [18-21]. In the present studies, Diplotene (Prohase-1), Metaphase-1, Anaphase-1, Metaphase -2 and Anaphase-2 stages were recorded in both treated and control individuals of T. thaelephora. Treatment with aqueous extract of C. frutescens did not affect the sequence of the meiotic process in the individuals of T. thaelephora. However, the extract induced some aberrations that included laggards and bridges in Anaphase-1, Metaphase -2 and anaphase -2. Table 2 revealed that 0 and 10% of extract did not induce any laggards and bridges (meiotic abnormalities) in this grasshopper. Meiotic abnormalities were recorded with treatment of concentrations of the extract that were above 25%. Laggards were more commonly observed than bridges in Anaphase-1, Metaphase- 2 and Anaphase -2 cells. The highest percentage of laggards (14.29%) was recorded with treatment of 60% of C. frutescens extract. The highest percentage of bridges (7.79%) was recorded with a treatment of 60% of extract. More abnormalities were observed in Anaphase -1 cells than in the other meiotic stages considered. Therefore the meiotic abnormalities increased progressively with increase in concentration of extract. Different types of mutagenic alterations detected by cytogenetic techniques are thought to have aneugenic (chromosome lagging and effects on spindle) effects while chromosome aberrations are thought to arise from chromosome breakage and exchange [1,20,21]. We observed aneugenic effects in our study mainly when we applied concentrations higher than 25%of the extract.


Chiasma Frequency

A few studies have been carried out to determine the effect of plant extracts on the meiotic process in animals and in particular pest grasshoppers. One of such studies revealed that the aqueous extract of the seeds of Annona muricata affected chiasma formation in the pest grasshopper Zonocerus variegatus L. by changing the intracellular environment of germ cells [6]. During this study, mean chiasma frequency was observed to increase progressively with increase in concentration of aqueous extract of C. frutescens (Table 3). Mean chiasma frequencies were subjected to the Student-Newman – Keulsone wayvariance analysis. This analysis revealed mean chiasma frequencies recorded for all test concentrations were significantly higher (p < 0.5) than for the control. However, there was no significant change in mean chiasma frequencies with subsequent increase in concentration of C. frustescens extract (Table 4). These results indicated that 10% of the aqueous extract of C. frutescens sufficiently modified the internal environment of the germ cells in T. thaelephora as to induce a change in chiasma formation. Therefore, extracts of C. frutescens possess some genotoxic properties which could produce meiotic aberrations in the pest grasshopper T. thaelephora.


Concentration (W/V) of dry pepper (C. frutescens) Weight of dry pepper (C. frutescens) powder (grams) Volume of distilled water (litre)

0%

00.0

1.00

10%

100

1.00

25%

250

1.00

40%

400

1.00

50%

500

1.00

60%

600

1.00

Table 1: Preparation of aqueous solutions of pepper (C. frutescens) used in the study



Treatment Dose Total number of cells scored Anaphase - 1 Metaphase - 2 Anaphase - 2 Total abnormality %
Laggards Bridges Laggards Bridges Laggards Bridges

Control

150

-

-

-

-

-

-

-

10%

200

-

-

-

-

-

-

-

25%

170

-

-

-

-

-

0.10

5.88

40%

188

0.12

0.13

0.13

0.10

0.12

0.13

38.83

50%

175

0.12

0.13

0.13

0.10

0.12

0.13

38.83

60%

154

0.12

0.13

0.13

0.10

0.12

0.13

47.40


Table 2: Percentage (%) frequency of male meiotic aberrations in Capsicum frutescens treated T. thaelephora


Treatment Total number of cells scored Individual Mean

1

2

3

4

5

6

7

8

9

10

Total

Control

250

11

11

10

11

15

15

10

11

11

11

116

11.60±1.74a

10%

250

12

12

14

11

12

12

12

14

11

12

122

12.20±0.98ab

25%

250

15

13

12

11

13

13

12

11

13

15

128

12.80±1.33ab

40%

250

14

12

11

14

12

11

12

14

14

12

126

12.60±1.20ab

50%

250

12

14

12

13

14

12

12

13

14

14

130

13.00±0.89ab

60%

250

15

13

14

13

13

13

13

13

15

14

132

13.60±0.80b

Total

1500

-

-

-

-

-

-

-

-

-

-

754


Table 3:Mean chiasma frequency in Capsicum frutescens treated T. thaelephora Means with the same letter are not significantly different at P ≤ 0.05 (Student-Newman-Keuls Test) Means were compared with analysis of variance (ANOVA) using Student-Newman-Keuls Test at P ≤ 0.05 SPSS 15 for Window


Control Sum of squares df Mean Square F Significance

Between Groups Within Groups Total

23.533
86.400
109.933

5
54
59

4.707
1.600

2.942

0.020


Table 4: ANOVA for male mean chiasma frequencies in Capsicum frutescens treated T. thaelephora ONEWAY ANOVA

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