Published in Crop Sci 39:1896-1899 (1999)
© 1999 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Crop Science 39:1896-1899 (1999)
© 1999 Crop Science Society of America
NOTES
Insect clip cages rapidly alter photosynthetic traits of leaves
Steven J. Crafts-Brandnera and
Chang-chi Chua
a USDA-ARS Western Cotton Research Laboratory, 4135 E. Broadway Rd., Phoenix, AZ 85040-8803 USA
crafts{at}ix.netcom.com
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ABSTRACT
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In plant-insect interaction experiments, a clip cage is frequently used to isolate insects or other small pests on a leaf. Clip cage effects on the physiology of the leaf could possibly confound experimental results. Our objective was to quantitate the effects of an insect clip cage of the type typically used for small pests such as whiteflies (Bemisia sp.), aphids (Aphis sp.) and mites (Urticae sp.) on the photosynthetic traits of cotton (Gossypium hirsutum L. cv. Coker 100A-glandless) and muskmelon (Cucumis melo L. cv. Imperial 45) leaves. Clip cages that enclosed 11.3 cm2 of both the abaxial and adaxial sides of a leaf were attached to young fully expanded leaves. For the leaf tissue within the clip cage, incident radiation was decreased and leaf temperature was increased. After 24 h, chlorophyll content of tissue within the clip cage was significantly increased compared with non-caged-control samples taken from the opposite half of the same leaf. Three days after clip cages were attached to leaves, compared with controls, the tissue within the cage had a lower light-saturated, steady-state CO2 exchange rate (CER) and leaf soluble protein content. The cage effect on CER and soluble protein could be explained, at least in part, by decreased light-saturated initial Rubisco activity for leaf tissue within the clip cage. We conclude that the clip cages caused physiological and biochemical alterations of leaves that could alter insect nutrition. Thus, it is suggested that clip cage effects on leaf physiology and microenvironment must be considered when interpreting results of plant-insect interaction experiments.
Abbreviations: CER, CO2 exchange rate • Rubisco, ribulose-1,5-bisphosphate carboxylase–oxygenase
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INTRODUCTION
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THE USE OF CAGES
to contain insects or other small pests on plants is common and necessary to facilitate many types of experiments conducted by entomologists and crop scientists. Many types of cages have been designed, ranging from large cages for whole plants to small clip cages that can be used to isolate small pests on individual leaves (Simmons and Yeargan, 1990; Mowry, 1993; McAuslane, 1996). Although the potential for confounding effects of insect cages on experimental results has been recognized (Womack, 1984; Simmons and Yeargan, 1990; Heinz and Perrella, 1994), there is little documentation of specific effects on leaf physiology. Our objective was to determine the effect of small clip cages, commonly used for whiteflies (Bemisia sp.), aphids (Aphis sp.) and mites (Urticae sp.), on photosynthesis and nitrogen metabolism of cotton (Gossypium hirsutum L. cv. Coker 100A-glandless) and muskmelon (Cucumis melo L. cv. Imperial 45) leaves.
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Materials and Methods
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Seeds of cotton (cv. Coker 100A-glandless) and muskmelon (cv. Imperial 45) were germinated in a growth chamber held at 28°C in pans containing commercial potting mixture (Grow More, Inc., Gardena, CA1)
or on filter paper in petri dishes, respectively. After 1 wk, uniform seedlings were transplanted into 15- by 15-cm pots containing potting mixture. Plants were cultivated in a greenhouse under natural sunlight. Maximum daily irradiance averaged 1900 µmol m-2 s-1 PAR during the experiments. Greenhouse temperature was maintained at 28°C during a 15-h day and 24°C during a 9-h night. There were two cotton plants or one muskmelon plant in a pot. Beginning 10 d after transplanting, the plants were fertilized twice a week with 750 mL of a solution containing 2 g L-1 of Grow More 20-20-20 (percent as N-P2O5-K2O equivalent) fertilizer (Grow More, Inc., Gardena, CA). The nutrient solution was supplemented with 0.5 mL L-1 of a micronutrient solution containing 2 mM MnCl2, 10 mM H3BO3, 0.4 mM ZnSO4, 0.2 mM CuSO4, 0.4 mM Na2MoO4, and 0.1 mM NiCl2. Clip cage treatments were started at approximately four weeks after planting.
The fourth and third leaf above the cotyledon for cotton and muskmelon, respectively, was used as experimental material. The leaves had recently reached full expansion when experiments were initiated on 11 May and 11 June 1998, for cotton and muskmelon, respectively. At this time one clip cage, similar in design to those described in previous reports (Cohen and Harpaz, 1964; Melamed-Madjar et al., 1984; Mowry, 1993) was attached to the leaf. The clip cages, which enclosed both the adaxial and abaxial sides of the leaf, were made from small petri dishes that were 3.8 cm in diameter and 1.8 cm high (Falcon 3001, Becton Dickinson Labware, Lincoln Park, NJ). The bottom of the petri dish, used for the bottom of the clip cage (abaxial side of the leaf where insects are placed), was replaced with 52-mesh nylon net for air circulation. The top of the petri dish, used for the top of the clip cage (adaxial side of the leaf), had six 3-mm-diameter holes that facilitated air circulation. Foam rubber was glued to the rim of each cylinder that contacted the leaf surface. A large hinged hair clip was glued to each half of the cage and hinge tension was adjusted to apply minimal pressure to the leaf surface. The clip cages weighed an average of 7 g and were supported with a metal wire extending from the soil to the leaf such that normal leaf orientation was maintained and the pressure of the cage on the leaf was minimal.
Pots were arranged in a completely randomized design with eight replications sampled at 4 and 3 times for cotton and muskmelon, respectively. Analysis of variance was performed at each sampling time. For each replicate, the leaf tissue enclosed within the clip cage and comparable leaf tissue from the other half of the same leaf were sampled at 1000 h. All measurements for the clip cage treatment were made on leaf tissue that was enclosed within the clip cage. Clip cages were removed and light-saturated, steady-state CER was determined, after which a 0.8-cm2 leaf disc was removed and frozen in liquid N2 for subsequent enzyme assay. Immediately after leaf disc removal, the chlorophyll fluorescence yield was determined, and finally two additional leaf discs (each 0.4 cm2) were removed and kept on ice prior to soluble protein and chlorophyll extraction. Controls were sampled in the same manner as described for the clip cage treatment, with leaf discs and chlorophyll fluorescence yield being determined on the portion of leaf tissue that was used for steady state CER measurement.
Light-saturated, steady-state CER and stomatal conductance to CO2 were determined on 6 cm2 of leaf area using a LI-COR 6400 (LI-COR Inc., Lincoln, NE) portable photosynthesis system. The PAR within the sample chamber was maintained at 1800 µmol photons m-2 s-1 using a built-in LED light source and the partial pressure of CO2 within the sample chamber was maintained at 350 µL L-1. To estimate the effect of shading on CER, measurements were made at light intensities ranging from 300-to-1200 µmol m-1 s-1 phosynthetically active radiation. Chlorophyll fluorescence yield was determined on 0.8 cm2 of leaf area with a WALZ PAM-2000 fluorometer (Walz, Effeltrich, Germany) as described by Feller et al. (1998). Soluble protein was extracted in 0.5 mL of buffer containing 50 mM sodium phosphate, pH 7.5, 10 g L-1 polyvinylpolypyrrolidone, and 15 mM ß-mercaptoethanol and determined according to the method of Bradford (1976) after centrifugation at 13000 g for 30 seconds. After extraction overnight in the dark in 1 mL of methanol, the chlorophyll concentration was determined as described by Holden (1976). Rubisco activity was rapidly extracted and assayed from light-saturated leaf tissue in 1.5 mL of buffer as described by Feller et al. (1998). Leaf temperature of controls and of leaf tissue contained within a clip cage was measured with a small thermocouple pressed to the bottom of the leaf. The reduction in light intensity caused by the top of the clip cages was estimated with a LI-COR Model LI-189 light meter (LI-COR Inc., Lincoln, NE).
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Results
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It is reasonable to assume that the insect clip cages would alter the microenvironment of the leaf tissue contained within the cage. Indeed, non-uniform shading by the top of the clip cage reduced the light intensity intercepted by the leaf tissue within the cage by 30 to 70%, compared with non-caged leaf tissue. Additionally, depending on the exposure of the caged leaf tissue to the sun, the leaf temperature was increased by 0.0 to 3.5°C.
Attaching clip cages to cotton and muskmelon leaves led to a significant increase in chlorophyll content within 1 d (Fig. 1) . For cotton, treatment differences in chlorophyll content disappeared by 7 d as a result of increased chlorophyll content of the control leaf tissue. The increase in chlorophyll content of the control cotton leaf tissue at Day 7 of the time course was associated with increased shading of the sample leaf by new vegetative growth. For muskmelon, the leaf tissue enclosed within the clip cage maintained higher chlorophyll content compared to controls over a 7-d period. Throughout the experimental time course, the ring of tissue bounded by the edges of the cages became progressively more chlorotic for both plant species.
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Fig. 1 The effect of clip cages on chlorophyll content of cotton and muskmelon leaves. Data points represent the mean ±SE of eight replicate samples. Significant differences between treatments at a given time point determined by the LSD are indicated by *** for P = 0.001
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Contrary to what might be expected by chlorophyll content (Fig. 1), light-saturated, steady-state CER was decreased by the clip cage treatment (Fig. 2)
within 1 and 3 d for cotton and muskmelon, respectively, and remained significantly lower until the end of the experiment. Clip cages also led to decreased chlorophyll fluorescence yield relative to the controls (data not shown). This measurement, which is indicative of the quantum yield of photosynthesis (Schreiber et al., 1986), provided another indication of the inhibitory effect of the cages on photosynthetic capacity. Clip cage inhibition of light-saturated CER was not associated with stomatal closure as stomatal conductance to CO2 was either not affected or increased by the clip cage treatment, compared to controls, for cotton and melon, respectively (data not shown).
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Fig. 2 The effect of clip cages on light-saturated, steady-state CO2 exchange rate of cotton and muskmelon leaves. Data points represent the mean ± SE of eight replicate samples. Significant differences between treatments at a given time point determined by the LSD are indicated by * and *** for P = 0.05 and P = 0.001, respectively
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Similar to light-saturated CER (Fig. 2), the clip cage treatment led to decreased levels of soluble protein that were significantly different from controls within 3 d (Fig. 3)
. For cotton, the treatment differences became larger over time as soluble protein remained constant or declined for controls and treated leaf tissue, respectively. Temporal changes in soluble protein were markedly different for the two plant species, indicating that senescence of the controls was occurring during the experimental time course for muskmelon but not for cotton. Even though the soluble protein content declined markedly over the 7-d time course for the muskmelon controls, the decline was greater for the clip cage treatment.
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Fig. 3 The effect of clip cages on soluble protein content of cotton and muskmelon leaves. Data points represent the mean ± SE of eight replicate samples. Significant differences between treatments at a given time point determined by the LSD are indicated by * and *** for P = 0.05 and P = 0.001, respectively
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The initial activity of Rubisco extracted from light-saturated leaf tissue that was sampled immediately after measuring CER was decreased by the clip cage treatment (Table 1)
. The relative decrease in initial Rubisco activity for the clip cage treatment was in proportion to the decrease in light-saturated CER (Fig. 2) for both plant species. In addition to the clip cage effect on initial Rubisco activity, the results for controls corroborated the soluble protein data (Fig. 3), indicating that muskmelon, but not cotton, leaves were undergoing senescence during the experimental time course.
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Table 1 The effect of clip cages on the initial activity of Rubisco sampled from light-saturated leaf tissue. Numbers represent the mean ± SE of eight replicates
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Discussion
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The insect clip cages led to rapid and significant physiological and biochemical perturbations in both cotton and muskmelon leaves. The increased chlorophyll content of leaf tissue enclosed within the clip cage was most likely attributable to a direct effect of shading caused by the clip cage. For several plant species, including cotton, it has been shown that decreasing the photosynthetic photon flux density incident on a leaf leads to increased chlorophyll content (Anderson, 1986; Mae et al., 1993; Zhao and Oosterhuis, 1998).
Although chlorophyll content was increased by the clip cage treatment (Fig. 1), the assimilatory capacity of the leaf tissue was significantly reduced (Fig. 2 and 3). Indeed, the clip cages led to changes in leaf metabolism that were reminiscent of senescence symptoms, with characteristic declines in light-saturated CER, soluble protein and Rubisco activity (Feller and Fischer, 1994; Smart, 1994). Whether or not senescence was induced by the clip cages, these results provide additional documentation that chlorophyll content and declines in assimilatory capacity are not tightly linked (Smart, 1994). While the effect of clip cages on chlorophyll could be related to shading, it is possible that several microenvironment alterations, including shading, light quality, leaf temperature, relative humidity, and atmospheric gas composition, could have contributed to the observed decrease in photosynthetic capacity and soluble protein content.
Two observations relevant to insect-plant experiments can be made from our results. First, under our experimental conditions, the selected sample leaf differed in senescence characteristics in a species-dependent manner. The control cotton leaf did not display any senescence symptoms over an 11-d period, whereas the control muskmelon leaf was well into senescence within 7 d after beginning the experiment. Thus, the impact of leaf development on insect biology may be dependent on the plant species. The second and most important point from these experiments is that the clip cages significantly decreased light-saturated CER, soluble protein content, and Rubisco activity, which were indicative of impaired carbon and nitrogen metabolism. Furthermore, the CER measurements (Fig. 2) overestimated the actual rate of photosynthesis of the leaf tissue within the cage by 15 to 40% because the measurements were made under a light intensity that was saturating for photosynthesis. Therefore, the carbon and nitrogen nutrition available to insects would be markedly altered by the clip cage treatment, especially for insects caged for three or more days. On the basis of the senescence-like symptoms induced by the clip cages, it is possible that the contents of mineral nutrients other than nitrogen were altered. The negative effect of clip cages on mite and aphid survival (East et al., 1992; Kift et al., 1996) may be partially associated with nutritional deficiencies. In addition to the perturbations of leaf quality caused by the clip cages, altered microenvironment within the clip cage would also be expected to affect insect behavior and/or physiology directly.
In summary, leaf cages typical of the type used to isolate small pests such as whiteflies, aphids, and mites on individual leaves altered leaf physiological and biochemical processes, and thereby carbon and N nutrition available to the pest, in a manner that may confound experimental results. The cage effects were consistent for two plant species that exhibited differential leaf senescence characteristics. It is possible that cages could be designed more appropriately to lessen the detrimental effects on leaves. Nonetheless, the effects of clip cages on leaf physiological and biochemical processes should be accounted for by crop physiologists and entomologists when designing experiments that use clip cages to isolate small pests on plants for plant-insect interaction studies.
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ACKNOWLEDGMENTS
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The excellent technical assistance provided by Donald L. Brummett was greatly appreciated.
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NOTES
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1 Mention of a trade name does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval over other products that may also be suitable. 
Received for publication January 11, 1999.
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ABSTRACT
INTRODUCTION
