Tri-trophic interaction involving host plants, black legume aphid, Aphis craccivora (Hemiptera: Aphididae) and the predator, Cheilomenes sexmaculata (Coleoptera: Coccinellidae)

Interactions involving host plants (cowpea, groundnut, cotton, sunfl ower, greengram, blackgram) an insect herbivore, black legume aphid Aphis craccivora Koch (Hemiptera: Aphididae) and a predator, the zigzag beetle Cheilomenes sexmaculata (Fabricius) (Coleoptera: Coccinellidae) were investigated during 2014–2015. The preference of the aphid A. craccivora for host plants measured in terms of growth and multiplication was cowpea > groundnut > greengram > blackgram > sunfl ower > cotton (most preferred to least preferred). Cowpea was the most preferred host plant for growth and multiplication of A. craccivora. Aphid’s oviposition period was longest, fecundity greatest; adult longevity longest (188.40 ± 28.87 h; 52.00 ± 10.92; 231.60 ± 40.41 h), and nymphal mortality was lowest (0%) when reared on cowpea followed by groundnut, greengram and blackgram. Aphids had highest nymphal mortality (100%), with very few or no live adults produced, when reared on sunfl ower and cotton, the least preferred host of A. craccivora in this study. At the third trophic level, both the larvae and the adults of the coccinellid, Cheilomenes sexmaculata, consumed more of the aphids reared on groundnut than of those reared on blackgram, greengram, cowpea, sunfl ower and cotton. The biochemical constituents (phenols) present in cotton and sunfl ower, which contributed to the aphid’s nymphal mortality, also affected the feeding behaviour of the coccinellid. * Corresponding author; e-mail: kvhp@hotmail.com INTRODUCTION Plants interact in complex ways with the herbivores and pathogens that feed on them and also with the natural enemies of the herbivores and pathogens (Price et al., 1980). The role of plant chemistry (primary and secondary metabolites) in tri-trophic interactions is central to several aspects of trophic phenomena including, top-down versus bottom-up control of herbivores, enemy-free space and host choice and theories of plant defence. The effects of plant defence on natural enemies i.e. parasitoids and predators, depends on the specifi c attributes of the plant trait and the details of the physical, biochemical, and behavioural interactions between the natural enemies, their hosts (prey) and the host plant (Kennedy, 2003). Plant defence traits viz., primary and secondary metabolites and their chemistry are important in assessing the degree of compatibility between biological control and plant resistance approaches to pest control (Ode, 2006). Aphids are major insect pests in both tropical and temperate regions (Francis et al., 2000) of the world causing direct and indirect (as vectors) damage to cultivated crops. Eur. J. Entomol. 113: 551–557, 2016 doi: 10.14411/eje.2016.075


INTRODUCTION
Plants interact in complex ways with the herbivores and pathogens that feed on them and also with the natural enemies of the herbivores and pathogens (Price et al., 1980).The role of plant chemistry (primary and secondary metabolites) in tri-trophic interactions is central to several aspects of trophic phenomena including, top-down versus bottom-up control of herbivores, enemy-free space and host choice and theories of plant defence.The effects of plant defence on natural enemies i.e. parasitoids and predators, depends on the specifi c attributes of the plant trait and the details of the physical, biochemical, and behavioural interactions between the natural enemies, their hosts (prey) and the host plant (Kennedy, 2003).Plant defence traits viz., primary and secondary metabolites and their chemistry are important in assessing the degree of compatibility between biological control and plant resistance approaches to pest control (Ode, 2006).
Aphids are major insect pests in both tropical and temperate regions (Francis et al., 2000) of the world causing direct and indirect (as vectors) damage to cultivated crops.(Malick & Singh, 1980) and total reducing sugars (Somogyi, 1952) were estimated from fresh leaves of the six host plants as per the above protocols.There were fi ve replicates for each host plant.Both the biophysical and biochemical constituents of host plants were correlated with the aphid's performance on these plants.

Number of aphids consumed by C. sexmaculata
The stock culture of the coccinellid, C. sexmaculata, was kept in a laboratory.Initially pupal stages were collected en masse from nearby agricultural fi elds.These pupae were kept in plastic boxes, 9 cm × 14 cm, till adult emergence.The adults were provided with aphids reared on cowpea.The eggs laid on the walls of the plastic boxes were collected and the larvae and adult beetles that developed from them were used to determine the number of aphids they consume.
Newly hatched fi rst instar larvae were placed in small plastic containers (7 cm × 4 cm) and provided with third and fourth instar nymphs of A. craccivora.The larvae were provided with known numbers of aphids and after 24 h, the numbers of live aphids were counted and the total number of aphids consumed by the larvae recorded.The values were expressed as total no. of aphids consumed by each larva.The aphids were provided with a small piece of leaf of the respective host plant.The same procedure was followed for determining the number of aphids consumed by the adult beetles.

Statistical analysis
All the data were subjected to ANOVA and DMRT analysis using the SPSS 13.0 software package (SPSS Inc., 2004).
On cotton and sunfl ower, none of the aphids completed its nymphal development and died before reproducing.The nymphal duration of each instar was recorded for those survived but did not moult into the next instar..Of all the host plants tested, aphids had the shortest developmental times (104.40 ± 8.09 h), longest oviposition period (188.40 ± 28.87 h) and highest fecundity (52.00 ± 10.92), when reared on cowpea, followed by those reared on groundnut (fec.21.12 ± 2.64), greengram (fec.12.62 ± 8.38) and blackgram (fec.9.87 ± 8.67) (Table 1 and  2).herbivore, A. craccivora, and an insect predator, C. sexmaculata.The substrate used for growing plants was red soil : compost in a 3 : 1 ratio in plastic pots 23 cm × 20 cm.The seeds of the test plants were treated with fungicides (Mancozeb @ 2gKg -1 ) to prevent fungal diseases.

Studies on the biology of A. craccivora
Biology of the aphid A. craccivora was studied in the laboratory at 28 ± 2°C and 75-80 per cent RH.The second or third freshly opened leaves of the host plants at the peak vegetative stage were used for studying the biology of the aphid.The leaves were excised and placed singly in 9 cm diameter glass Petri dishes.To keep the leaves fresh, the Petri dishes were lined with moist blotting paper.One fi rst instar nymph of the aphid was placed on each of the excised leaves.Development of the aphids in terms of number of moults, duration of each instar, nymphal mortality, adult longevity and adult fecundity was recorded every 12 h.There was a total of 20 replications per host plant.Body length, body width, head capsule width, antennal length, cornicle length and caudal length of all the nymphal instars and adult aphids, reared on the six host plants were measured using an ocular micrometer.There was a total of ten replicates per host plant and the data were expressed in millimeters.

Trichome density
Trichome density was measured on the abaxial surfaces of the leaves of the six host plants.Number of trichomes on a 0.25 cm 2 area of leaf was counted and expressed as number of trichomes per 0.25 cm 2 leaf area.
The same trend in size was also recorded for adult aphids.Maximum adult body size was greater when they were reared on cowpea (2.33 ± 0.14 mm × 1.18 ± 0.02 mm) than on greengram; blackgram and groundnut (signifi cantly different).Similarly, the maximum head capsule width of the adults reared on cowpea (0.44 ± 0.02 mm) was greater than that of aphids reared on greengram and blackgram (signifi cantly different).The smallest head capsule widths of adults was recorded for aphids reared on groundnut (0.32 ± 0.00 mm) (signifi cantly different from those of aphids reared on the other host plants) (Table 4).
Of all the host plants the highest protein content was recorded in the leaves of cowpea (335.21 ± 29.58 mg/g) followed by cotton (306.06 ± 71.92 mg/g) and blackgram (275.01 ± 76.73 mg/g) (not signifi cantly different).The lowest protein content was recorded in leaves of sunfl ower (139.53 ± 26.13 mg/g), which is signifi cantly lower than that recorded in the leaves of all the other host plants.The leaf protein content of greengram and groundnut are intermediate between those of cowpea and sunfl ower.
Total phenol content was highest in the leaves of cotton (260.46 ± 31.43 mg/g) followed by that in the rest of the host plants (signifi cantly different).Least total phenol content was recorded in leaves of cowpea (49.29 ± 17.04 mg/g).
Correlations between the aphid's performance (in terms of biological parameters) and plant physical and biochemical constituents of the various host plants, revealed that the density of trichomes on the leaves of the different host plants were signifi cantly negatively correlated with adult longevity (r = -0.752,fecundity (r = -0.799),total life cycle (r = -0.762)and a signifi cantly positively correlated with percentage nymphal mortality (r = 0.682) (Table 6).
The number of aphids consumed by adult male beetles was highest when they were provided with aphids reared on groundnut (588.5 ± 27.57 aphids), which is statistically signifi cantly different from that consumed when provided with aphids reared on the other host plants.The least numbers of aphids consumed by adult male beetles were recorded when they were provided with aphids reared on cotton (425.00 ± 17.89 aphids) and sunfl ower (469.11± 28.46 aphids) (not signifi cantly different).The numbers of aphids consumed by adult male beetles when they were provided with aphids reared on greengram (539.23 ± 32.13 aphids), cowpea (519.5 ± 14.84 aphids) and blackgram (509.00 ± 37.87 aphids) did not differ signifi cantly and were intermediate between the number aphids reared on groundnut and cotton consumed (Table 7).
Adult female beetles consumed the highest numbers of aphids when they were provided with aphids reared on groundnut (903.00 ± 39.59 aphids), which is statistically different from when they were provided with aphids reared on the other host plants.Adult female beetles consumed the lowest number of aphids when they were provided with aphids reared on cotton (528.00 ± 12.72 aphids) followed by sunfl ower (547.00 ± 9.89 aphids) (not statistically different) (Table 7).

DISCUSSION
This study revealed that A. craccivora did signifi cantly best when reared on cowpea (shortest developmental time and lowest nymphal mortality) followed by groundnut, greengram, blackgram, sunfl ower and cotton [highest nymphal mortality (Table 1)].Based on the size (body length and width) of the different instars and adults this aphid did best when reared on cowpea and worst on groundnut.On cotton and sunfl ower, the nymphs did not survive to the adult stage (Table 1).
It is generally believed that aphids fed on poor quality host plants are smaller and lighter, take longer to develop and are more vulnerable to attack by natural enemies than those fed on high quality plants (Dhaliwal & Dilwari, 1993).Partial plant resistance coupled with the action of predators is a more effective way of reducing the number of aphids infesting faba bean than either predator or plant resistance alone (Shannag & Obeidat, 2008).In the present study, the poor quality of groundnut might account for the small size of the aphids reared on this plant and the high quality of cowpea for the large size of the aphids reared on this plant.The low nymphal mortality of aphids when reared on cowpea could be due to its high protein content, low density of trichomes and low phenol content (Table 5).
The high phenol content and high trichome density of cotton and sunfl ower might account for the high aphid nymphal mortality recorded on these plants.This aphid failed to complete its development on these two plants (Table 1).
It is well documented that plant trichomes (glandular and non glandular) act as barriers (antixenosis resistance) to movement and reduce the survival of aphids (Farrar & Kennedy, 1991;Simmons et al., 2003;van Emden, 2007).This is also the case for biochemical constituents such as plant secondary metabolites.A. gossypii has a shorter adult life and lower fecundity when reared on varieties of cotton with a high polyphenol gossypol content (Du et al., 2004).Wheat cultivars with a high Hydroxamic acid content are more resistant to attack by Sitobion avenae and its growth is negatively correlated with Hx levels (Fuentes-Contreras & Niemeyer, 1998).
Because of the low nutritional quality of groundnut, aphids reared on this plant are small and both larvae and adults of C. sexmaculata need to consume more aphids when on groundnut (Table 7).In contrast, because of the high nutritional quality of cowpea, aphids reared on this plant are large (Table 3 and 4) and both the larvae and adults of C. sexmaculata need to consume fewer aphids when on cowpea (Table 7).Similar fi ndings are reported by Barkhordar et al. (2013).They studied the effect of host plant resistance in the tri-trophic interaction between wheat genotypes, the aphid, Schizaphis graminum, and the coccinellid beetle, Coccinella septempunctata.They report that the low weight and small size of the aphids reared on resistant genotypes resulted in the ladybird eating more aphids per unit time on these plants.Messina & Sorenson (2001), studying tri-tropic interactions involving larvae of the lacewing Chrysoperla plorabunda (Fitch) and aphids reared on resistant and susceptible wheat cultivars, found that lacewing larvae caused a greater reduction in density of Russian wheat aphid, Diuraphis noxia (Mordvilko) on a tolerant-resistant line (carrying the Dn4 gene) than on its near-isogenic susceptible parent.Similarly, Giles et al. (2002) report an increase in survival rate, decrease in developmental time and larger Coccinella septempunctata, when fed on the aphid Aphis pisum reared on Medicago sativa, than when fed aphids reared on Vicia faba, They report that the tritrophic interactions appear to be modulated by the response of A. pisum to the biochemical constituents in the host plants.The aphid, Myzus persicae fed on sweet pepper is a more suitable food for the lady beetle, Adalia bipunctata due its better nutritive value; whereas the same aphid fed on tabacco is less suitable because it is toxic (Jalali & Michaud, 2012).
The study on the effect of the biophysical and biochemical constituents of the host plants (Table 5), revealed that both cotton and sunfl ower had signifi cantly higher numbers of trichomes and higher quantities of phenols.Both trichomes and phenols had a signifi cant negative effect on the aphid's biological characters and positive effect on aphid nymphal mortality (Table 6).It is concluded that the high quantity of phenols and high trichome density of cotton and sunfl ower resulted in the poor performance of the  aphids reared on these plants (Table 1 and 2).Consequently, when both larvae and adults of C. sexmaculata were provided with aphids reared on cotton and sunfl ower they consumed far fewer of these aphids than of those reared on the other host plants.The biochemical constituents (phenols) in cotton and sunfl ower that resulted in the high aphid nymphal mortality on these plants (Table 1), might also have affected the feeding behaviour of the coccinellid (third trophic level) (Table 7).
Several workers have hypothesised that the harmfulness of certain aphid prey for their aphidophagous predators results from the presence of toxic plant biochemical constituents (Malcom, 1990;Hodek & Honek, 1996;Francis et al., 2000).The present results provide an insight into the negative effect host plants (based on their secondary metabolites) can have on the third trophic level.However, contrary to the present fi ndings, Du et al. (2004) report that high levels of gossypol in cotton had an antibiotic effect on Aphis gossypii but a positive effect on the growth and development of Propylea japonica in the third trophic level.The toxic aphid Aphis sambuci is consumed by larvae and adults of Coccinella septempunctata at twice the rate as that of two essential species of prey Acyrthosiphum pisum and Aphis philadelphi (Nedved & Salvucci, 2008).The result may be due to the fact that the period the prey were exposed to the predator was only four hours and the predators were starved prior to the onset of the experiment.It is believed that starved predators generally do not distinguish between toxic and non toxic prey.However in the present study the predators were allowed to feed on insect prey (reared on different host plants) throughout their larval and adult development and hence the differences are apparent.
It can be argued that plant secondary metabolites have a negative effect on herbivores in reducing their fi tness and making them more vulnerable to both natural enemies and abiotic conditions.However, this is not always the case as when the host plant is favourable for the herbivore it is also likely to be favourable prey for its natural enemies as seen in the case of groundnut in this study.However, if the secondary metabolites are toxic, as in the case of cotton and sunfl ower in this study, the negative effect on the herbivore may also have a negative effect on its natural enemies.

CONCLUSIONS
It is concluded that it is necessary to carry out detailed and comprehensive trophic level studies before advocating the use of partial plant resistance (based solely on secondary metabolites) in insect pest management, as the secondary metabolites present in host plants could affect the efficacy of the predators in the third trophic level (van Emden, 1995).

Table 1 .
Duration (in h) and percentage mortality in the nymphal instars of A. craccivora reared on the six different host plants.

Table 2 .
Post nymphal developmental periods (in h), adult longevity and fecundity of A. craccivora reared on the different host plants.

Table 3 .
Morphometrics (mm) of fourth instar nymphs of A. craccivora reared on different host plants.
a ± 0.01 Values followed by same letter are not signifi cantly different at 0.05 level as per DMRT.

Table 4 .
Morphometrics (mm) of sdults of A. craccivora reared on different host plants.

Table 5 .
Biophysical and biochemical characteristics of the different host plants.
b ± 0.61 Values followed by same letter are not signifi cantly different at 0.05 level as per DMRT; TSS -Total Soluble Sugars; FAA -Free Amino Acids.

Table 6 .
Correlation (Pearson)("r" values)between the different growth parameters of the aphids and different host plant characters (biophysical and biochemical).

Table 7 .
The number of aphids reared on the different host plants consumed by the larvae and adults of the coccinellid beetle (C.sexmaculata Fab.).
a ± 12.72 Values followed by same letter are not signifi cantly different at 0.05% as per DMRT.