Effect of Aphis odinae (Hemiptera: Aphididae) infestation on sugars and amino acid content in mango 1 1 2 3 D. Lokeshwari *, A. Verghese , S. Shivashankar , N.K. Krishna Kumar , 4 5 H. Manjunatha & R. Venugopalan 1
Division of Entomology and Nematology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bengaluru-560 089, India 2 Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bengaluru-560 089, India 3 Division of Horticultural Science, Indian Council of Agricultural Research, Krishi Anusandhan Bhawan – II, New Delhi-110 012, India 4 Department of Biotechnology and Bioinformatics, Kuvempu University, Jnanasahyadri, Shankaraghatta, Shimoga-577 451, India 5 Division of Economics and Statistics, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bengaluru-560 089, India The mango aphid, Aphis odinae (Toxoptera odinae) (Van der Goot) (Hemiptera: Aphididae), is an occasional, phytophagous sucking pest of mango. These aphids are very often associated with natural enemies and an ecological understanding, especially on insect–crop relationships, is needed in management. This will be manifested in altered sugar and amino acids contents of the plant. In light of this, the effect of aphid feeding on the content of total soluble sugars (TSS) and free amino acids (FAA) in mango shoots was studied. The amount of TSS and FAA in infested and uninfested shoots with varying levels of aphid infestation, namely low, medium and high, were quantified. Results indicated a significant reduction in the amount of TSS and FAA in infested shoots due to aphid feeding (P < 0.05). At maximal aphid abundance (251–300 aphids/shoot), TSS declined by 32 % and FAA by 47.5 %. Further, regression analysis between aphid numbers and the quantity of TSS/FAA yielded simple linear equations with R2 = 0.99, which can be used as an alternative way to estimate aphids numbers. The present study discusses the influence of aphid herbivory in mango shoots, a critical factor in understanding aphid–plant interactions and their damage to the host. It can be possibly extended to similar other aphid infestations. Key words: Aphis odinae, free amino acids, infestation, mango aphids, total soluble sugars.
INTRODUCTION The mango aphid, Toxoptera odinae (Van der Goot) (Hemiptera: Aphididae), a phloem-feeding pest is assuming importance in south India. Their damage is characterized by honeydew excretion which covers the leaves resulting in photosynthetic inhibition and transmission of plant pathogenic viruses (Van Emden & Harrington 2007). The mango aphid has one of the broadest host ranges of at least 45 plant families (Blackman & Eastop 2000); however, the major host plant is mango (Mangifera indica L., Anacardiaceae). It forms dense colonies on young shoots or on the under surface of young leaves along the main veins and petioles attended by many species of ants. Both adult and immature stages cause serious damage *Author for correspondence. E-mail:
[email protected]
to leaves, tender shoots and inflorescences. The aphids affect the crop by feeding on the phloem sap. The plant sap is a limiting factor to aphid growth, development and survival (Wilkinson & Douglas 2003). It is a main source of nutrients with high concentrations of sugars (especially sucrose) and free amino acids providing an abundant source of carbon and nitrogen (Douglas 2006). Consequently, aphids collect high quantities of phloem sap to extract amino acids (Doorsher 1988) and unassimilated excess sugars are voided in their honeydew (Klingauf 1987). Therefore, the amount of total soluble sugars (TSS) and free amino acids (FAA) decreases within tissues of different plant species infested by Aphis gossypii (85 % TSS and 33 % FAA) (El-Khawas & El-Khawas African Entomology 22(4): 823–827 (2014)
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2008), Brevicoryne brassicae (48.5 % TSS and 30 % FAA) (Khattab 2007) and Lipaphis erysimi (16 % TSS) (Singh & Sinhal 2011). However, there has been no available information so far on how aphids influence the sugar and amino acid content within tissues of a tree. Further, knowledge of how this aphid species affects the nutritional quality of affected parts in a specific insect–host relationship can be of great significance to assess pest status and to devise methods of minimizing the effect of infestation on yield. In this regard, the present study focuses on the influence of mango aphid, A. odinae, feeding on the amount of major nutritional constituents, namely TSS and FAA in infested and uninfested mango shoots. MATERIAL AND METHODS The present study was carried out from August– October 2009 on 15-year-old unsprayed mango trees cv. Totapuri maintained at Indian Institute of Horticultural Research, Bengaluru, India (12°58’N 77°35’E). Survey of A. odinae infestation was carried out at fortnightly intervals. Trees were kept free from insecticidal sprays to encourage natural infestation of the aphids. Three infested trees were randomly chosen out of 10 and labelled. The infested tender mango shoots were labelled and the aphid numbers were monitored from August to September on a weekly basis. Data indicated that aphid numbers within the plant ranged from low, i.e. 100 aphids/shoot to high 300 aphids/shoot. Infestation was classified into three classes, namely low (100–150 aphids/ shoot), medium (151–250 aphids/shoot) and high (251–300 aphids/shoot). The sampling units constituted same-aged terminal tender shoots (7 cm length) on which aphids colonized selectively. Five terminal shoots of each class along with five uninfested control shoots, i.e. a total of 20 shoots were selected from each tree and carefully cut, inserted into individual polythene bags and sealed immediately to prevent the escape of aphids. The actual numbers of aphids infesting each shoot was counted using a stereo binocular microscope in the laboratory. A total of 60 shoots were subjected to biochemical analysis in October. Extraction of sugars and amino acids from samples was done by homogenizing the shoots in 20 ml of 80 % ethanol. The clear supernatants were collected and evaporated on a water bath maintained at 80 °C. The dry residue was dissolved in 10 ml of
distilled water and used for the estimation of sugars and amino acids. The amount of total soluble sugars was quantified using the dinitrosalicylic acid (DNS) method (Miller 1959). Sample extract (0.1 ml) was pipetted into test tubes and the volume was made up to 1 ml using distilled water. DNS reagent (0.5 ml) was added and contents were heated on a boiling water bath for 5 min. The contents of the tubes were cooled and diluted to 20 ml using distilled water. Absorbance was recorded at 540 nm using Beckman DU-64 UV-Vis spectrophotometer, U.S.A. The amount of total soluble sugars present in the samples was calculated using a calibration graph prepared using known concentrations of glucose. Free amino acids content in the extracts was estimated by ninhydrin method (Lee & Takahashi 1966). Sample extract (0.1 ml) was mixed with 1.9 ml of ninhydrin reagent at pH 6.0. The test tubes was covered with aluminium foil and heated on a water bath for 15 min. The tubes were cooled and the absorbance was recorded at 570 nm. The amount of free amino acids present in the samples was calculated using a calibration graph prepared using known concentrations of glycine. Data were subjected to a two-way analysis of variance (ANOVA) using PROC GLM of SAS V9.3 in order to determine the variability in the amount of sugars/amino acids in shoots sampled among trees. The significance was tested at the 5 % level of probability using least significant difference (LSD) (Gomez & Gomez 1984). In addition, data were subjected to linear regression analyses with number of aphids per shoot as ‘y’ and the amount of total soluble sugars/free amino acids as ‘x’. PROC REG of SAS V9.3 (SAS Institute 2010) was utilized for model building. The coefficient of determination (R2) was used to evaluate the goodness-of-fit. Normality test was performed using Shapiro-Wilk (W) statistic to determine whether or not model-generated residuals follow a normal distribution, a prerequisite for developing a holistic model. RESULTS A significant quantitative difference in the amount of TSS and FAA was observed between uninfested and infested mango shoots (Table 1). A strong negative relationship was evident with aphid infesta-
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Table 1. Total sugars and amino acids content at different infestation levels in mango shoots. Infestation group
Uninfested Low Medium High
Mean sugars ± S.E. (mg/g fresh weight)
Mean amino acids ± S.E. (mg/g fresh weight)
15.81 ± 0.02 a 13.48 ± 0.01 b 12.06 ± 0.04 c 10.75 ± 0.02 d
0.362 ± 0.001 a 0.282 ± 0.001 b 0.238 ± 0.001 c 0.190 ± 0.001 d
Means in a column followed by different letters show significant difference (P < 0.05).
Fig.1. Relationship between amount of sugars and aphid numbers in mango shoots.
tion and TSS/FAA content, which was highly significant (P < 0.05; F = 568, S.E.M. = 0.62) (Figs 1, 2). Approximately, 32 % decline in the amount of TSS was observed at maximal aphid abundance
Fig. 2. Relationship between amount of amino acids and aphid numbers in mango shoots.
(251–300 aphids/shoot) in comparison to uninfested shoots. Similarly, about 47.5 % reduction in FAA content was observed at maximum aphid abundance. The linear regression equations generated in this study strongly suggested their utility in estimating aphid numbers using different infestation classes. Since estimation of aphid numbers by in situ visual counting is rendered difficult as these herbivores have high reproductive rates and unit density, these models when tested for their reliability would be useful in numerous ecological experiments. In this regard, the models obtained were tested using statistical analysis and various statistical measures, namely R2, W. Linear regression equations and various statistic values are presented in Figs 1 and 2. Higher value for R2 statistic revealed better fit of a model to data. The normality test statistic value (W) was within the critical region (P < 0.05) confirming the statistical adequacy of the fitted model. DISCUSSION Interactions between plants and herbivorous insects have provided numerous insights into a range of important ecological and evolutionary processes (Lewinsohn et al. 2005). The relationship between aphid infestation and its effect on the plant growth has been investigated in different aphid-host plant combinations and various changes in plant growth rates have been ascribed (Hawkins et al. 1985). However, plant response to aphid feeding has been reported to be specific to particular aphid system investigated (Hawkins et al. 1985). Reports indicate that phloem-mobile sugars and free amino acids are the principal source of carbon and nitrogen for aphid respiration and growth (Brodbeck & Stong 1987; Van Emden & Harrington 2007). They ingest large quantities of vital cell sap which contains sugars and nitrogenous compounds to extract the necessary amino acids (Montllor
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1989; Dixon 1998). Therefore, sucking large volume of plant sap by aphids leads to the impoverishing effect on the level of sugars and free amino acids within infested parts. Aphid feeding has been correlated with a decrease in TSS and FAA content in various herbaceous plants (Singh & Sinhal 2011). The amount of TSS in aphid (Brevicoryne brassicae)-infested cabbage leaves was 64.21 mg/g in contrast 124.74 mg/g in uninfested leaves and FAA in infested cabbage leaves was 3.5 mg/g in contrast to 5.01 mg/g in uninfested control (Khattab 2007). In mustard, the aphid (Lipaphis erysimi)-infested mustard stem had 84 mg/g of TSS in contrast to 100 mg/g in uninfested stem (Singh & Sinhal 2011). In squash and cabbage, the amount of TSS in aphid (A. gossypii and B. brassicae)-infested leaves reduced significantly from 7.35 mg/g to 1.09 mg/g and 7.95 mg/g to 1.23 mg/g, respectively (El-Khawas & El-Khawas 2008). Correspondingly, the FAA reduced significantly from 0.24 mg/g to 0.16 mg/g and 18 mg/g to 16 mg/g, respectively, due to aphid feeding (El-Khawas & El-Khawas 2008). All of these results indicated that the mango aphid efficiently utilizes the photosynthates for its growth and reproduction, disturbing the activities of host plant. The results obtained in the present study are consistent with earlier reports on impact of aphid feeding on various herbaceous plants. In addition, the study generated linear regression equations with R2 = 0.99. Similar linear regression analysis was reported between sugarbeet root aphid Pemphigus betae and P. fliscicornis population density vs sugar content in infested sugarbeet roots with R2 = 0.97 and R2 = 0.94, respectively (Hutchinson & Campbell 1994; Zarrabi 2007). The study suggests that growing mango shoots rich in sugars and amino acids, provided good nutritional conditions to the aphids for their growth and development resulting in higher rates of aphid multiplication. Sugars and FAA in phloem sap play a vital role in insect physiology. The dietary sucrose in aphids is hydrolyzed to its constituent monosaccharides (glucose and fructose) and are assimilated (Wilkinson et al. 1997) while, the FAA participate in protein biosynthesis and used in various biogenetic pathways. Further, they determine the quality of honeydew excreted by sap feeders (Volkl et al. 1999) which plays a role in attracting ants that safeguards aphids from predation, parasitism and fungal infection (Stadler & Dixon 2005, 2008; Nielsen et al. 2010). Therefore,
the quality of honeydew affects the selection of ants species and intensity of ant attendance, a critical factor in devising effective management strategies especially biological control. For example ants respond most intensively to honeydew containing high amounts (70 %) of the trisaccharide melezitose (Volkl et al. 1999; Fischer et al. 2001, 2002). In addition, linear regression models generated in this study, aids in counting aphids in a much easier way. However, this requires further investigation before implementing in commercial orchards. Thus the basic understanding of aphid abundance in relation to the changes in biochemical constituents of the host plant is essential to understand the fundamentals of aphid physiology, honeydew production, interactions with ants and natural enemies, which further aids in developing resistant varieties and integrated pest management systems. The present study serves to be the starting point with reference to the biochemical changes induced by mango aphid, A. odinae feeding on mango shoots. CONCLUSION The present study focuses on the effect of Aphis odinae feeding on sugars and amino acids content in mango thus providing a basic understanding of aphid abundance in relation to the changes in biochemical constituents of the host plant. This aids in understanding the fundamentals of aphid physiology, honeydew production and tritrophic interactions. This further aids in developing resistant varieties and integrated pest management systems. The study also provides a relationship between aphid numbers and sugars/amino acids content. The present study discusses the influence of aphid herbivory in mango shoots, a critical factor in understanding aphid–plant interactions. Further, it is expected that this study would open up similar exploratory studies in other minor herbivores like cicadellids, pseudococcids, coccids, psyllids, etc. for development of effective pest management strategies. ACKNOWLEDGEMENTS The authors thank the Director, Indian Institute of Horticultural Research, Bengaluru for encouragement and provision of facilities to carry out the research. Thanks are also due to Indian Council of Agricultural Research (ICAR), New Delhi for financial support through the Out Reach Programme
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