Feeding and Development of Cydia pomonella (Lepidoptera: Tortricidae) Larvae on Apple Leaves

Abstract

We present leaf consumption, maximum body weight, head capsule width, and testicular ultrastructure of four sequential instars in codling moth reared solely on apple leaves of Honeycrisp variety. The developmental parameters for Cydia pomonella (L.) larvae fed on leaves are compared with those reared on artificial diet. Head capsules in the second, third, and fourth instars reared on leaves were significantly smaller than those in larvae fed artificial diet. Maximum body weights were significantly lower in all instars fed leaves compared with larvae fed artificial diet. More than 60% of larvae fed leaves molted to the third instar, and ≈3% entered the fifth instar, but none pupated, demonstrating an inability to fully compensate for the reduced nutritive value of a leaf-based diet. A study of testicular ultrastructure revealed that feeding codling moth larvae exclusively on apple leaves allowed formation of testes, but also triggered an intense, abnormal autophagocytosis that caused premature senescence of this tissue.

Neonate larvae of an important cosmopolitan pest of apples, the codling moth, Cydia pomonella (L.), bore into fruit and feed until their larval development is complete. However, codling moth larvae are known to be polyphagous and can feed not only on apples, but also pears (Howell et al. 1992), cherries (Moffitt et al.1992), nectarines (Curtis et al. 1991), prunes (Yokoyama and Miller 1999), and even walnuts (Vail et al. 1993). Moreover, >80% of codling moth eggs are deposited on leaves (Jackson 1979), which suggests that larvae may also feed on leaves.

Whether or not codling moth larvae feeding on leaves could also pupate and reproduce is unknown. We did not find any report on how starvation or poor nutritive value of food may influence development of insect testes. Of particular interest is the question of whether the gonads of leaf-fed codling moth larvae are capable of developing to maturity.

Data on the performance of C. pomonella larvae on leaves would contribute to our basic knowledge about this species, indicating potential survival rates of the larvae in the field, before fruit infestation, and may identify new targets for codling moth control. It may also delineate possible limits of codling moth polyphagy. Here we present feeding dynamics of four sequential codling moth instars reared solely on apple leaves of Honeycrisp variety. The developmental and reproductive parameters (head capsule width, maximum body weight, and ultrastructure of the testes) for C. pomonella larvae fed on leaves are compared with those reared on artificial diet.

Materials and Methods

Codling moths originating from Yakima, WA, were kept in cylindrical cages lined with wax paper as an oviposition surface. The moths were given saturated sucrose solution and held in a growth chamber at 25°C, 70–80% RH, under a photoperiod of 16:8 (L:D) h. Neonates (0–3 h old) were placed on 2 by 2-cm pieces of apple leaves (Honeycrisp, U.S. patent No. 7197) with a fine brush, and individually held in transparent, 50 by 9 mm, Falcon Tight-Fit Lid Dishes (Benton Dickinson Labware, Franklin Lakes, NJ). In total, 200 larvae in three replications were used in this experiment. The pieces of apple leaves were replaced with fresh ones every 2–3 d throughout the duration of the experiment. To prevent dehydration, wet filter paper was placed on the bottom of each dish. Plastic grating (1 by 1-cm mesh) separated the wet paper from leaves and larvae, protecting them from drowning.

For comparison, 100 larvae, in three replications of 33–34 neonates each, were individually reared in plastic, transparent 1-oz cups, on artificial diet according to Howell (1971). Each cup contained one diet cube of approximate volume 15 cm ³, and was infested by one neonate. Diet-fed larvae were transferred and handled in the same manner as leaf-fed individuals.

Randomly chosen larvae were weighed every day using an ultra-sensitive Cahn Electrobalance, (Cahn Instrument Company, Paramount, CA). All larvae were examined daily for molting and mortality. Shed head capsules were measured using an ocular micrometer, the day after ecdysis to the next instar. For leaf-fed larvae, amounts of leaf tissue consumed were calculated for each larva, at the end of each instar, as follows: the surface area of consumed leaf was determined in micrometer units using a stereo microscope equipped with an ocular square-mesh reference grid. Separately, surface areas of 33 randomly chosen fragments of leaves were determined by the same method, these leaf fragments were dried, weighed on the electrobalance, and micrometer units were calculated and converted to milligrams of leaf dry weight. Based on this conversion, leaf surface areas consumed by larvae during our experiments were expressed in milligrams of leaf dry weight.

GraphPad InStat (GraphPad Software, San Diego, CA) was used for statistical analysis. All sets of data on maximum body weight and head capsule width passed tests for normality (P < 0.05). Regression analyses were used to test relationships among larval maximum body weights or head capsule widths, respectively. Maximum body weights were converted to logarithms of maximum body weights before being analyzed by means of linear regression. Additionally, mean maximum body weights and mean head capsule widths were analyzed with analysis of variance (ANOVA) followed by Bonferroni comparison, among larvae fed leaves and those fed Howell’s artificial diet. Regardless of the test used, the results were regarded as significantly different at P < 0.05.

Developmental status of testes was compared between larvae fed leaves and those reared on artificial diet. The three experimental groups were as follows: (1) larvae reared solely on leaves, (2) larvae reared solely on Howell’s artificial diet, and (3) larvae that were reared on artificial diet for the first four instars, and starved for the first two days of the last (fifth) instar. Establishing this third experimental group was justified because in preliminary experiments larvae ceased leaf- feeding after entering the fifth instar and all died on day 3. Regardless of experimental variant, testes were excised on day 2 of the fifth instar, fixed in 3% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3), postfixed in 1% OsO₄, dehydrated, and embedded in Epon. Thin sections were cut and contrasted with alcoholic uranyl acetate and lead citrate. The sections were studied by transmission electron microscopy (Hitachi H-600, Hitachi High Technologies America, San Francisco, CA).

Results and Discussion

Codling moth larvae fed and developed on leaves throughout four consecutive instars. Upon entering the last (fifth) instar, feeding ceased and all larvae died on day 3. Cumulative leaf consumption, duration of particular instars and cumulative mortality are presented in Table 1. These findings may have an important practical value. Currently, in the U.S. Northwest, codling moth is controlled by sprays and mating disruption. Because codling moth larvae bore into the fruit and feed there until larval development is complete, contact pesticides are used to target the adults, eggs or neonates. Our recent study (Pszczolkowski and Brown 2002) showed that per os acting pesticides, such as Spinosad, may be successfully applied to target neonates fed leaves for 24–48 h after hatch. Also, the same report showed that leaf consumption may be stimulated by monosodium glutamate, and mortality from standard, field recommended concentration of pesticide increased by the addition of this feeding stimulant. Our present report extends those findings. Here, we show that at least 60% of neonates may continue feeding on leaves for as long as 7 d, where, in nature the majority of eggs are deposited. The amount of leaf tissue consumed over this period of time exceeded 2 mg of leaf dry weight per larva, well above 0.2 mg that needs to be ingested to assure almost 90% mortality in a population feeding on leaves treated with 2.85 ppm Spinosad (Pszczolkowski and Brown 2002). In the light of our present report, it seems that codling moth might be targeted not only as first instars, but also within the second, and maybe even in the third instar. How to arrest neonates on leaves, and prevent their boring into the fruit, remains an open question. However, our findings (Pszczolkowski and Brown 2002), with the report of Knight and Light (2001) identifying a new attractant for codling moth larvae, together suggest a new possible strategy for codling moth control. It seems that codling moth larvae might be targeted on leaves, with formulations containing a more environmentally friendly, noncontact pesticide (that would act if ingested), a feeding stimulant, and an attractant for neonate larvae.

Table 1.

Performance of codling moth, Cydia pomonella, larvae fed apple leaves

The parameters obtained in our regression analysis indicated that head capsule widths (r² = 0.9374, P < 0.05) and maximum body weights (r² = 0.9000, P < 0.05) of leaf-fed larvae increased significantly over four consecutive instars. Regression slopes for head capsule width differ significantly between leaf- and Howell’s diet-fed larvae; the slope parameter (95% CL) for leaf-fed larvae equaled 1.826 (1.718–1.935) and that for diet-fed larvae equaled 2.366 (2.191–2.541). Thus, the dynamics of head capsule growth were less in larvae fed leaves than in larvae fed artificial diet. There was a trend for larvae fed leaves to have smaller mean head capsule widths in all instars compared with larvae fed artificial diet (Fig. 1A), and these differences were statistically significant for the second, third, and fourth instars. Fifth-instar head capsules had characteristic chestnut-brown coloration both in leaf- and Howell’s diet-fed larvae. The parameters of head capsule growth in larvae reared on Howell’s diet, found in our study, corresponded with those reported previously for larvae growing on artificial medium (Williams and McDonald 1982), or on apples (Weitzner and Whalon 1987).

Fig. 1.

Growth of head capsule width (A) and maximum body weight (B) in larval populations of codling moth, Cydia pomonella. Solid circles represent larvae reared on artificial diet. Open circles represent larvae fed apple leaves. Statistically significant differences (ANOVA followed by Bonferroni comparison, P < 0.05) are indicated by asterisks. Numbers in brackets indicate numbers of larvae used

Leaf-based diet affected weight gain in codling moth larvae over all four instars. Regression slopes for maximum body weights did not differ between larvae fed leaves and those fed Howell’s diet; the slope parameter (95% CL) for leaf-fed larvae equaled 0.349 (0.322–0.376) and that for diet-fed larvae equaled 0.399 (0.371–0.426). Thus, the dynamics of weight gain were the same in both experimental groups. However, larvae cultured on leaves had significantly lower mean maximum body weights in all instars compared with larvae reared on artificial diet (Fig. 1B), which indicates that apple leaves have substantially less nutritive value than Howell’s artificial diet.

All larvae fed artificial diet pupated. In contrast, no larvae fed leaves pupated, demonstrating their inability to fully compensate for the reduced nutritive value of a leaf-based diet. This result is in accordance with the paradigm that lepidopteran larval body weight must exceed a species- specific threshold before the neurohormonal commitment to pupate (Nijhout 1981). Generally, in lepidopterans, the minimal weight for pupation is reached in the last instar, and varies between 25 and 50% of maximum body weight (Slansky and Scriber 1985), which would correspond to between 16 and 32 mg in the codling moth. But in our experiments no larva cultured on leaves exceeded 16 mg in weight.

The mean maximum body weights of larvae cultured on leaves correspond well with data of Markwick et al. (1995) on larval growth in codling moth reared on casein- deficient artificial medium. However, Markwick et al. (1995) reported that as much as 20% of codling moth larvae pupated despite very low protein content (0.06% of the wet weight) in the medium, suggesting that high protein content in the diet is not necessary for pupation in this species. However, our data on ultrastructure of spermatocytes in leaf-fed codling moth larvae suggest that sufficient dietary proteins may be necessary for appropriate testicular development.

In our experiments, transmission electron microscopy revealed a similar ultrastructure in larvae reared on artificial diet, regardless of whether the larvae were starved for the first 2 d of the fifth instar or not. In these experimental variants, diffuse stage spermatocytes were observed, and no cells in metaphase were found. Nuclei were of spherical shape, often surrounded by cisternae of endoplasmic reticulum (Fig. 2). Regularly shaped mitochondria with well developed cristae, and Golgi complexes were found around nuclei (Fig. 2). There were no obvious differences in shapes of spermatocytes, nuclei, mitochondria, and Golgi complexes between starved and nonstarved groups. However, we did not find moderately translucent particles, possibly fat deposits, in testes of starved larvae; whereas they were present in testes of nonstarved larvae (Fig. 3).

Figs. 2–6. (2) Testes of fifth instars reared on artificial diet. Nucleus (N) is surrounded by endoplasmic reticulum (R). Golgi apparatus (G), and mitochondria (M) are visible near the nucleus. Bar = 1 μm. (3) Moderately electron translucent bodies (L), probably fat deposits, were found in testes of fifth instars reared on artificial diet. Such structures were absent in testes of larvae fed leaves or reared on artificial diet, but starved for the first two days of the fifth instar. Bar = 1 μm. (4) Autophagy in testes of fifth instars reared on apple leaves. Disorganized nucleus (N) contains unidentified electron dense bodies (DB) and filamental structures (arrow). Mitochondria (M) are abnormally shaped. Arrowheads point to electron-translucent autophagic vacuoles. Bar = 1 μm. (5) Testes of fifth instars reared on apple leaves. Arrows indicate electron-dense bodies, remaining lysosomes located next to mitochondria. Bar = 1 μm. (6) Double-walled autophagic vacuole in testes of fifth instars reared on apple leaves. The arrowhead indicates double wall. Bar = 0. 1 μm

Testes of larvae fed leaves had dramatically different ultrastructure. Double-walled structures with electron-translucent areas were found in cytoplasm, often next to nuclei (Figs. 4 and 6). These structures are similar to macroautophagic vacuoles found in protein-deprived mouse hepatocytes (Mortimore et al. 1983). Nuclei were disorganized (Fig. 4) in a manner resembling that of human fibroblasts, which died from protein deprivation (Terman et al. 1999). There was no condensed chromatin found and some parts of the nuclei were electron-translucent. In some nuclei, specific electron-dense bodies and filamental structures were observed (Fig. 4). Mitochondria were abnormally shaped and few cristae were found (Fig. 4). We did not find normal Golgi complexes or endoplasmic reticulum in spermatocytes of leaf-fed larvae. Instead, numerous electron-dense bodies, possibly lysosomes, were found (Fig. 5); many of them next to mitochondria.

During starvation or protein deprivation, animal cells mobilize protein reserves by autophagocytosis, a vacuolar process that sequesters and degrades organelles or other constituents of cytoplasm as a source of free amino acids for metabolic use. In such a situation, demands on the lysosomal system are increased, and starving cells rely upon increased, reparative autophagocytosis (Brunk et al. 1995, Terman et al. 1999). However, if lysosomal reserves are limited, the autophagocytotic capacity may decrease to a level that is not sufficient for cell renewal even under normal conditions (Terman et al. 1999). In particular, starving or senescent tissue should not catabolize damaged mitochondria (Brunk and Terman 1999), which has been shown as a major contributor to aging (Ames et al. 1995).

We hypothesize that although feeding codling moth larvae apple leaves allowed formation of testes, it also triggered intense, abnormal autophagocytosis that caused premature senescence of this tissue. This process did not occur in testes of larvae reared on a more nutritious diet. Because 2-d starving of codling moth larvae resulted only in resorption of putative fat deposits, it seems that relatively long lasting exposure to poor nutrition is needed for induction of abnormal testicular autophagocytosis.

Apart from their practical aspects, our findings may provide an interesting experimental tool for studies on codling moth dietary requirements and mechanisms of food choice and taste perception. Supplementing the leaf-based diet with additional nutrients could establish what nutrients trigger pupation or allow proper testicular development. Measuring the intensity of feeding on leaves from various trees could delineate feeding preferences of polyphagous codling moth larvae.

Acknowledgments

This work was partially supported by Washington State Tree Fruit Research Commission, and by Washington State Commission on Pesticide Registration.