Host-based life cycle traits and detoxification enzymes of major looper pests (Lepidoptera: Geometridae) of tea from Darjeeling Terai, India Soma Das & Ananda Mukhopadhyay
Phytoparasitica ISSN 0334-2123 Volume 42 Number 2 Phytoparasitica (2014) 42:275-283 DOI 10.1007/s12600-013-0358-1
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Author's personal copy Phytoparasitica (2014) 42:275–283 DOI 10.1007/s12600-013-0358-1
Host-based life cycle traits and detoxification enzymes of major looper pests (Lepidoptera: Geometridae) of tea from Darjeeling Terai, India Soma Das & Ananda Mukhopadhyay
Received: 6 June 2013 / Accepted: 8 October 2013 / Published online: 24 November 2013 # Springer Science+Business Media Dordrecht 2013
Abstract Three polyphagous looper caterpillars, Buzura suppressaria Guenée, Hyposidra talaca Walker and Hyposidra infixaria Walker, have established themselves as severe pests of tea [Camellia sinensis (L.) Kuntze] in the plantations of sub Himalayan West Bengal (Terai region). Schima wallichii (DC.) Korth. is a naturally occurring alternative host of all these looper species. To gain an insight into looper and host plant relationships, the present work contemplates studies on host preference, host-based life cycle traits and levels of detoxification enzymes, such as general esterases (GEs) and glutathione S-transferases (GSTs). From the study, host-induction of feeding preference was evident in all the three looper species. Hyposidra spp. exhibited similar post-embryonic development periods both on tea and S. wallichii, whereas B. suppressaria reared on tea needed a longer development period than on S. wallichii. Tea-reared caterpillars of Hyposidra spp. were significantly heavier, having higher quantities of GEs and GSTs than S. wallichii-reared ones. B. suppressaria, however, exhibited similar body weights on tea and S. wallichii. While GST level was higher in tea-reared B. suppressaria, its GE quantity was higher on S. wallichii. Although tea was found to be a more suitable host for Hyposidra spp., the host S. wallichii proved marginally better than tea for supporting B. suppressaria. However, S. Das (*) : A. Mukhopadhyay Entomology Research Unit, Department of Zoology, University of North Bengal, Darjeeling, West Bengal 734013, India e-mail:
[email protected] A. Mukhopadhyay e-mail:
[email protected]
all the three looper species could utilize the foliage of tea and S. wallichii successfully. So S. wallichii trees can act as a sylvan reservoir of the looper species, prompting their possible invasion of tea plantations and thus making management of looper pests in tea more difficult. Keywords Buzura suppressaria Guenée . Camellia sinensis (L.) Kuntze . Defense enzymes . Fitness traits . Host preference . Hyposidra infixaria Walker . Hyposidra talaca Walker . Tea loopers
Introduction Commercial plantations of tea (Camellia sinensis) in the sub Himalayan Terai region of West Bengal (26°30’N 26°48’N, 88°10’E - 88°51’E), located in northeast (NE) India, were started in the middle of the 19th Century. The extensive tea plantation in hill slopes, foothills and their adjoining plains offer food, shelter and breeding ground to a number of arthropods, resulting in creation of about 167 pests in NE India (Das 1965). These pests cause 11–55% annual loss in yield if left unchecked (Hazarika et al. 2009). Several geometrid species are recorded as major defoliating insect pests of tea in South Asia, including that of NE India (Bigger 2009). The looper caterpillar Buzura suppressaria (Lepidoptera: Geometridae) has been known as the major defoliator of tea for several decades from this region (Anon. 1994). However, recently two other species of geometrid loopers, Hyposidra talaca and H. infixaria, have become dominant (in place of B. suppressaria) and are causing extensive damage to the high yielding tea clones
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of the Terai-Dooars region of sub Himalaya (Das et al. 2010b). All the three looper species are polyphagous (Robinson et al. 2010). Many of them have likely migrated from the forest to tea ecosystem, as tea in this region has been planted largely by replacing forest and natural vegetations. Moreover, many shade trees of the plantations that harbor the loopers are of sylvan origin. There are many examples of host shifts or switching over to a new host by insects driven by factors such as: (i) possible abundance of food provided by the new host, (ii) change in quality of food which consequently may affect larval growth, survival, and fitness, and (iii) involvement of changed physiological costs for using a new host plant (Scriber & Slansky 1981; Tabashnik 1983; Weiss & Berenbaum 1989). Different species of host plants with varied physical characteristics and nutritional contents can influence the survival, growth, development period, reproduction and host choice behavior of herbivorous insects (Awmack & Leather 2002; Scriber & Slansky 1981). Moreover, as herbivores are confronted with a large amount of noxious chemicals in their plant food, they need to detoxify in order to make the food plant more acceptable (Schoonhoven et al. 2005) and for better utilization of the resource. Production of a higher level of detoxifying enzyme enables the insects to detoxify xenobiotics including plant secondary metabolites (Li et al. 2007). The process of induction of glutathione Stransferases (GSTs) and general esterases (GEs) in lepidopteran insects by host plants has been reported by many authors (Dominguez-Gil & McPheron 2000; Sintim et al. 2009; Yu 1982). Some information on host range expansion of the looper species B. suppressaria, H. talaca and H. infixaria in NE India is available from the recent literature (Anon. 1994; Antony et al. 2012; Basu Majumdar & Ghosh 2004; Mukhopadhyay & Roy 2009). Besides tea, H. talaca, H. infixaria and B. suppressaria are found to occur naturally on the forest host Schima wallichii (DC.) Korth. (common English name: needle wood; local name: Chimal, Chilaune) (Das et al. 2010b). S. wallichii is a commonly occurring tree of the sub-Himalayan wet mixed forest distributed in Terai and the Dooars regions of West Bengal (Champion & Seth 1968) along with Sal (Shankar 2001). The plant species is known for its medicinal properties and is reported to be present in the Medicinal Plant arboreta in the Conservation Areas in Terai and the Dooars regions of West Bengal (Das et al. 2010a).
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Host shift mechanism followed by establishment of the looper species as tea pests in the Terai – Dooars region of NE India needs to be understood from the ecological and physiological point of view. As literature on these aspects is very scanty, the present investigation has been undertaken to add to the bioecological knowledge on these looper pests. The findings may help in answering the following questions: (i) How suitable is tea as a host to the polyphagous looper species in this region as compared with other natural/wild hosts? (ii) Does any feeding preference operate in host shift mechanism and consequent continuation of feeding on a host? (iii) What is the status of detoxifying enzymes in looper caterpillars when feeding on different host plants reflecting their adaptive traits on a host? The present study on food preference and life cycle performance of the three looper species on the host plants C. sinensis and S. wallichii was undertaken along with quantification of GEs and GSTs to gain an insight into the host plant–looper caterpillar relationship and the underlying process leading to establishment of these loopers as severe pests of tea.
Materials and Methods Specimen collection and rearing of insects Gravid female moths of B. suppressaria, H. talaca and H. infixaria were collected in nature and brought to the laboratory. They were kept separately in 9 cm (ht) x 6 cm (diam) plastic containers with paper toweling for laying eggs. Once laid, batches of eggs were transferred to 12 cm (ht) x 13 cm (diam) plastic containers with the opening covered with a piece of fine cloth and the base toweled with tissue paper. Twigs of host plants were provided as food, with their cut ends immersed in water-filled small plastic tubes so that neonates could obtain ready food immediately after hatching. Twigs of leaves were arranged in such a way that enough space in between leaves was present for free movement of the tiny caterpillars. After emergence, the neonates were found to move towards light and showed a tendency to aggregate on the more illuminated side of the container, form webs and eventually die. To avoid this mortality, side walls of containers were covered with opaque black paper. Neonates hatching in mass were kept in these emergence containers for 1–2 days and then transferred individually to culture containers. Side by side mass cultures on different hosts were
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maintained in larger containers (28 cm ht x 28 cm diam) under the laboratory conditions. Host plant material Tea twigs (C. sinensis) of the clonal variety TV 26 were used for rearing, as this is the high- yielding clone commonly planted in Darjeeling Terai. A plantation of this clone (about 20 years old) is organically maintained in the experimental plot of the Department of Zoology, North Bengal University (NBU) located in Terai region. S. wallichii leaves were collected from a forest patch adjacent to a tea plantation ca 5 km away from the NBU campus. Food preference test A food preference test was conducted using dual choice method after Schoonhoven et al. (2005) with minor modifications. One-day-old final instar larvae (5th or 6th instar) were used after 3 h of starvation for the food preference test. Leaf area was measured graphically before and after feeding. Petioles of the leaves were immersed in water-filled plastic tubes to avoid wilting. A set of such leaves representing two different hosts, C. sinensis and S. wallichii, were placed in a plastic container (20 × 12 × 4 cm) with tissue paper toweling and the test caterpillar was released at the center. A reading was taken after 12 h. Total consumption in each set of leaves and the area consumed of each host leaf was calculated in percent of total consumption to determine the feeding preference. If X and Y sq. cm. area were fed on from the two host leaves (A and B), respectively, then percent consumption of leaf A = {X/(X + Y)} x 100. “Induction index”, which expresses the degree of food imprinting, was calculated after de Boer & Hanson (1984) for each plant pair, on which the species were reared, as follows: The difference in feeding preferences for two plant species is expressed in a choice index (range: –100 to +100) which measures the mean consumption of plant A minus that of plant B. The degree to which preferences are induced is expressed as an induction index (range: 0 to 200), which is the absolute difference between the choice indices for plant pair A, B obtained for two groups of larvae reared on either plant A or plant B. Besides leaf area consumed, consumption was also calculated based on leaf dry weight to see the correlation between leaf area fed and leaf mass fed. After the experimental period, the remaining pieces of leaves were dried at 60 °C to a constant weight and then compared to the dry weight of the equivalent original
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leaves for the calculation of the amount of leaf mass ingested by the larvae (Santana & Zucoloto 2011). The data concerning food preferences were analyzed statistically by Wilcoxon non-parametric tests (signed rank test for P-choice and rank-sum test for P-induction) since the data distributions were similar but generally not normal. Life cycle parameters Life cycle traits, i.e., duration of different developmental stages of the caterpillars and the total development period from egg to adult, were recorded on different hosts. Length of each larval instar and body weight from 3rd instar onwards were recorded for the freshly molted caterpillars. Length and weight of female and male pupae and adults were recorded. A fine electronic balance (BT 124 S, d = 0.1 mg, Sartorius) was used. Host-based life cycle parameters of B. suppressaria and the two species of Hyposidra were studied during spring time (March – April 2011) at a mean maximum temperature of 32.2 ± 2.8 °C and mean minimum temperature of 19.0 ± 3.5 °C. The data related to life cycle traits were analyzed by Student’s t-test. Quantification of detoxification enzymes Chemicals used: Bovine serum albumin (BSA), α-naphthyl acetate (α-NA), α-naphthol, 1-chloro-2,4-dinitrobenzene (CDNB), reduced glutathione (GSH), and Fast Blue BB salt (Sisco Research Laboratory (SRL), Mumbai, India) were used. Solutions of α-NA (30 mM), GSH (50 mM) and CDNB (50 mM) were prepared fresh just before use. Enzyme preparation: Activity of detoxification enzymes, general esterases (abbreviated as GEs) and glutathione S-transferases (abbreviated as GSTs) was measured in midgut tissues of final larval instars. Caterpillars were dissected in ice-cold phosphate buffer (0.1 M, pH 7.0). The food bolus was removed completely and the tissue was used for homogenization. Individual midgut was homogenized in ice cold 0.1 M sodium phosphate buffer (pH 7.0) and centrifuged at 10,000 g for 15 min at 4 °C in a high speed refrigerated centrifuge (Sigma 3 K30). The resultant postmitochondrial supernatant was divided into 100 μl aliquots and stored at −80 °C to be used as the enzyme source for GEs and GSTs activity assay and to estimate the amount of total protein. General esterase activity: General esterase activity was measured using α-naphthyl acetate (α-NA) as
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substrate according to the method of van Asperen (1962) with minor modifications. Ten μl of supernatant was taken in each well in triplicate in a 96-well microplate. The microplate reader used was of Opsys MR (Dynex Technologies, Chantilly, VA, USA). Two hundred microliters of 30 mM α-NA was added to each well for the reaction to occur. The reaction was stopped after 10 min by adding 50 μl of staining solution containing 0.1 % Fast Blue BB salt and 5 % SDS (3:5).The plate was left for 5 min for equilibration and absorbance was recorded at 450 nm. The change in absorbance was converted to end product (α-naphthol) using the standard curve of α-naphthol (5–500 mM). Blanks were set at the same time using a reaction mixture without protein extracts. Glutathione S-transferase (GST) activity: GST activity was estimated using the method of Habig et al. (1974) with minor modifications. Fifty μl of 50 mM CDNB and 150 μl of 50 mM GSH were added to 2.78 ml of sodium phosphate buffer (100 mM, pH 6.5). Then 20 μl of enzyme stock was added. The contents were shaken gently, incubated 2–3 min at 20 °C and then transferred to a quartz cuvette in the sample cuvette slot of UV-Visual Spectrophotometer (Rayleigh UV-2601, China). The reaction was carried out in duplicate. The reaction mixture (3 ml) without enzyme was placed in the reference slot for zeroing. Absorbance at 340 nm was recorded for 10–12 min employing kinetics (time scan) menu. The GST activity was calculated using the formula CDNB-GSH conjugate (μM mg protein-1 min-1) = (Absorbance increase in 5 min × 3 × 1000)/ (9.6* × 5 × mg of protein) (*9.6 mM cm-1 is the extinction coefficient for CDNB-GSH conjugate at 340 nm). Protein quantification: Enzyme activities were corrected for protein concentration. The total protein content of the homogenate was determined by the Folin – Lowry method (Lowry et al. 1951) using BSA as standard. Statistical analysis of the enzyme quantification was done by Student’s t-test. Statistical software Graph pad Instat version 3.1 was used for all the analyses.
Results Food choice test Choice tests indicated that caterpillars of B. suppressaria, H. talaca and H. infixaria consumed higher amounts of the leaves of the host on which they were experienced until final larval instar (5th instar stage
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for H. talaca and H. infixaria; 6th instar for B. suppressaria). The trends of entraining on a host were evident in all the three species (Fig. 1). Calculations based on leaf area and leaf dry weight consumed showed a strong correlation (r > 0.9) between preference based on leaf area and dry weight of the leaf. Based on the results of choice tests, ‘induction indices’ were calculated, which demonstrated the phenomenon of ‘induction of preference’. Higher induction in the final instar larvae of H. talaca and H. infixaria for the host plant pairs of tea and S. wallichii was evident. A lower but significant induction was observed in the final instar larvae of B. suppressaria, despite no clear preference for C. sinensis in the paired preference test when raised on the same host (Table 1). Host based life cycle parameters Generally H. talaca and H. infixaria completed their larval development through five instars, but in about 7 % of the cases there was an additional instar (6th). A reverse situation was observed in B. suppressaria, which normally passed through six larval stages to enter into pupation and in only a few cases (ca 6 %) it completed development in five instars, irrespective of the host plant. In H. talaca and H. infixaria, host-based difference in traits was noticed in the 5th instar, pupa and adult (Table 2). Body weights of these stages were significantly higher in both species when reared on C. sinensis than on S. wallichii. However, no significant difference in weights of the life stages, i.e., larva, pupa and adult, was evident when B. suppressaria was reared on C. sinensis and S. wallichii (Table 2). So, both the hosts equally supported growth and development of B. suppressaria. Development periods (egg – adult) were similar in H. talaca and H. infixaria when reared on C. sinensis and S. wallichii, but the period was approximately 2 days longer in B. suppressaria on C. sinensis in comparison with on S. wallichii (Table 2). Quantification of detoxification enzymes Midgut tissue extracts of the congeners of Hyposidra showed significantly higher amounts of detoxification enzymes, GEs and GSTs, when the larvae were reared on tea (C. sinensis) leaves than on jungle host, S. wallichii. In a similar trend, GST content of teareared B. suppressaria was higher than that of S. wallichii; however, in a reverse trend GE level was much enhanced in the same species on S. wallichii as compared with that on tea (Table 3).
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Fig. 1 Food choice exercised by loopers (Buzura suppressaria, Hyposidra talaca and Hyposidra infixaria) (mean±SE, n=20). C, Camellia sinensis; S, Schima wallichii. For each looper, columns labeled with different letters denote a significant difference in feeding preference in C:S host plant pair at P≤0.01 level
1968; Ting et al. 2002). Induction of feeding preference for the plant species experienced previously, was evident in H. talaca, H. infixaria and B. suppressaria. Induction of preference may contribute to the formation of biological races (Ting et al. 2002). At least, it reflects some adaptability in food choice and plasticity of preferences. The data on induction of preference showed the plasticity of the looper species to accept different host plants with more entraining and avidity, which in the pre-Jermy era was thought to be absent in insects. Many of the morphological features are considered as life history traits due to their contribution to the reproductive success of a species (Agnew et al. 2000, 2002). Morphological parameters such as maximum larval weight gain during final instar, pupal weight and adult weight were significantly higher in teareared H. talaca and H. infixaria than in the other host, S. wallichii. Body weight is an important fitness indicator in insect population dynamics (Liu et al. 2004). Egg production is usually maintained by nutrients
Discussion Chemical cues or plant volatiles play an important role in host plant selection by the insects (Schoonhoven et al. 2005).The forest tree S. wallichii and the cultivated tea bush C. sinensis provide different phenology and host environments for the looper species. As previous exposure to host odors is found to leave an effect on olfactory sensitivity of the insects (van Loon & Frentz 1991), the same may be instrumental for the loopers under study to identify a previously experienced host plant. Induction of food preference in lepidopterans through learning has been reported (Bernays & Wrubel 1985; Jermy et al. 1968). In many insect species the induction of preference usually develops after one or more instars remain on a particular host. A complex chemical stimulus participating in this process involves the central and peripheral nervous system (Hsiao 1985). In a number of lepidopteran insects, experience was found to induce their feeding preferences (de Boer & Hanson 1984; Jermy et al.
Table 1 Induction of preference of the loopers (Buzura suppressaria, Hyposidra talaca and Hyposidra infixaria) reared (experienced) and tested on pairs of host plants (C, Camellia sinensis; S, Schima wallichii) Species:
B. suppressaria
H. talaca
H. infixaria
Plant pair
C:S
Raised on
C
S
C
S
C
S
Choice index
17
−55
68
−54
74
−60
P choice
>0.5
0.002*
0.0007*
0.008*
