Plant-fed CPB

The Colorado potato beetle, Leptinotarsa, decimlineata Say (Coleoptera: Chrysomelidae) colony, originally provided by the New Jersey Department of Agriculture, was reared on potato plants at room temperature under a light:dark regimen of L:D 16:8 in walk-in incubator chambers or in small cages at the Beltsville Agricultural Research Center. Each day, new plants were provided to the colony. Supplemental wild type CPB were obtained from potato field plots located at the research center. Insect colonies were maintained on Superior as well as the slower maturing Kennebec variety of potato. Seed potatoes were purchased in October-December and stored at 2–3°C to terminate dormancy and initiate uniform sprouting upon transfer to warm temperatures (15–20°C). Procedures for long term storage of seed potatoes (15–18 months) were obtained from Dan Palmer (New Jersey Department of Agriculture, personal communication).

For experimental tests, plant-reared CPB were fed on small bouquets of potato leaves, the stems of which had been placed in 20 ml vials of water. Appropriate-sized openings were cut into the bottoms of 6 oz plastic cups (WL Enterprises, Inc. Newark, NJ, USA, #026X clear 00090) so that the vial neck which had been fitted with a rubber ring would form a secure seal with the edges of the opening in the cup. The leaves protruded into the cup which was covered with a transparent plastic lid that had been punctured with pin-sized holes to allow for air flow. Thus, beetles were caged individually within the cups during the experiment. Cups were placed in controlled environmental chambers at 25 ± 1°C, RH of 70%, L:D 14:10 and a light intensity of 600 lux. Egg masses were collected and stored as described for diet-fed beetles.

Diet-fed beetles

Colony and experimental egg masses of the CPB were maintained on filter paper (Whatman #1) in 100 × 15 mm and Falcon 50 × 9 mm (PGC Scientific, Frederick, MD, USA, (#29-4285) plastic petri dishes, respectively. The dishes were held in incubators at 25 ± 1°C, 75–80% RH and L:D 16:8. Under these conditions, the eggs hatched in 4–5 days. Newly hatched to 18-h old larvae were placed onto small blocks of diet. Cubes of diet (approximately 18 × 12 × 6 mm for 1^st and 2^nd instars and 18 × 12 × 12 mm for 3^rd and 4^th instars and adults) were placed on small pieces of weighing paper (slightly larger than the diet piece) in 50 × 9 mm (1^st and 2^nd instars) and 60 × 20 mm (3^rd and 4^th instars and adults) petri dishes, the bottom of which had been covered with white filter paper (Whatman #1). The filter paper trapped the CPB wastes and the weighing paper prevented the filter paper from absorbing water from the diet. Beetles were placed on fresh diet in new petri dishes on Monday, Wednesday and Friday of each week. Prior to the weekend, a slightly larger diet cube was used. Petri dishes were maintained in incubators under the conditions described above. Until larvae became 2^nd instars, petri dishes were covered with a large piece of opaque paper so that larvae were encouraged to feed rather than to migrate toward the light. At the end of the 4^th (final) instar, larvae stop feeding, become light beige in color and leave the diet. These prepupae were transferred individually to 1 oz cups (Bioserve, Frenchtown, NJ, USA, #9052; matching lids = #9050) that were half full with slightly moistened potting soil (Promix, Meyer's Seed Co., Baltimore, MD) and then covered with transparent lids that had been punctured with 10–12 pin-sized holes. During pupal and pharate adult development, cups were maintained in incubators (25±1°C, 75–80% RH and L:D 16:8). Newly emerged adults were placed individually in petri dishes as described above.

Adults–mating, oviposition and egg harvest

Adult beetles typically have an obligatory pre-oviposition period of 5 days during which time feeding, egg maturation and mating take place. The sex of adult beetles was determined by examining the ventral tip of the abdomen; males have a slight depression on the posterior ventral tip of the abdomen just anterior to the anal opening. On the 4^th–7^th day post-emergence, adult beetles were sexed and, on the 9^th–11^th day, mated; each pair was placed in a 60 × 20 mm petri dish which contained a diet cube. After mating, pieces of brown paper towel were stretched over the top of the petri dish under the lid. The circular piece of paper towel was cut large enough so that it protruded approximately 9 mm beyond the edge of the lid of the covered petri dish. The paper towel served as a medium for egg laying. Since beetles eat their own eggs, eggs were collected one or two times each day. A circular area was cut around the egg mass and the small circles of paper, each containing an egg mass, were stored individually in petri dishes (50 × 9 mm) lined with filter paper. Conditions for egg storage were the same as for beetle rearing. When excessive fungal or bacterial growth was observed eggs, beetle larvae and/or adults were surfaced sterilized prior to being stored or transferred to fresh diet. Surface sterilization was performed by placing beetles in weak Chlorox bleach (3 ml household chlorox in 97 ml of distilled water) for 2–3 minutes, rinsing them in distilled water and blotting them dry with paper toweling.

Preparation of potato leaf, lettuce leaf and tomato fruit powders

Potato leaf, lettuce leaf and tomato fruit powders are not commercially available and are prepared by the user. Our potato preparation was usually obtained from field- or greenhouse-grown “Superior” or “Kennebec” potatoes, although any common potato variety would probably be suitable. Leaves and petioles were removed 3–4 weeks after the plants had sprouted and before flowering. The fresh weight of the 6–8 leaves obtained from a typical plant had a wet weight of 100–200 grams, and a final dry weight of 10–20 grams. Fresh leaves were washed, placed in a convection oven at 60°C, dried for 24 hours and ground to a fine powder in a 250 ml Waring stainless steel blender cup. The powder was sifted sequentially through 20 (850 microns) and 40 (420 microns) mesh strainers (Fisher Scientific,  www.fishersci.com), and the process was repeated until almost all of the powder had been ground sufficiently to pass through the 40 mesh strainer. Lettuce leaf powder was prepared from store-bought Romaine lettuce. Leaves were washed, dried, ground and strained through mesh as described above. It was of paramount importance that all the water be removed from the potato and lettuce leaves prior to grinding. The tomato fruit powder was prepared from fresh tomatoes either collected from field plots or purchased locally. Tomatoes cut into small cubes were homogenized in the same blender that was used to prepare the potato powder. The homogenate was poured into pyrex baking dishes to a depth of 10 mm and dried for 24 – 48 hours in a convection oven (64°C). The dried tomato was placed in the blender and ground to a fine powder. Passage through mesh strainers was not necessary. The potato and lettuce leaf powders were stored in containers at room temperature until needed. The tomato fruit powder must be stored in air-tight jars as it has a greater tendency to absorb water than does the potato or lettuce leaf powder. The shelf life of the potato leaf and tomato fruit powder is at least 6 months. We have successfully used potato leaf powder that was stored for 12 months.

Potato, Lettuce+Potato and Lettuce diets

Four artificial diets were prepared as described in Table 1, all of which were based on a diet developed by Forrester (APHIS-USDA, unpublished report, personal communication to R. A. Bell). The Potato diet contained potato leaf powder, the Lettuce diet contained lettuce leaf powder, and the Lettuce+Potato diet contained both leaf powders. All three also contained tomato fruit powder. The Lettuce diet and the Lettuce+Potato diet both contained the same amount of lettuce leaf powder, but the latter diet contained an additional 3.75 grams of potato leaf powder and 1.25 grams less of tomato fruit powder. The modified Forrester diet differed from the original Forrester diet in its tomato fruit powder and potato leaf powder content. The original diet contained 5 and 10 gms of these powders, respectively.

All dry ingredients, except for the agar, were weighed and put in a common container. The combined ingredients, called the “premix,” were mixed in the blender at low speed for three 30 sec intervals so that ingredients were evenly distributed. The premix could be stored in sealed plastic bags for several weeks to exclude moisture. A 500 ml batch of diet was prepared in a 1-liter Waring stainless steel blender cup. Five hundred ml of water was poured into a beaker, placed in a microwave oven and brought to a full boil. It is very important to preheat the blender cup with very hot (preferably 180°C) water prior to use. The hot water was quickly emptied from the blender and replaced with 384 ml of boiling water used to prepare the diet. With the blender operating at low speed (a rheostat can be used to control speed), the agar was added to the water. The agar was blended at very low RPM so that the water/agar was held at a high temperature (85–87°C. An oven-type thermometer can be used to insure that the desired temperature is obtained. The blended agar was then returned to a beaker and heated to boiling in a microwave oven (about 10 seconds at the “High” setting). With the aid of a pipette, the oil was dispensed into the diet and blended thoroughly. Finally, while gradually increasing the speed control, the pre-mix ingredients were added and blended for 1 min at high speed. Diet was poured into containers to a depth of about 12 mm, containers were covered with 2-ply soft paper toweling and allowed to cool for 40 – 60 minutes. For holding and storage of the diet, various sizes of freezer containers (manufactured by Rubbermaid Co.) designed for food storage were used. Diet was covered tightly and stored in the refrigerator until needed. Freshly made diet provided the best results. The shelf (refrigerator) life of the diet was only 2 days. With longer storage times, the beetles did not feed as well (unpublished results).

Parameters for monitoring/comparing the effect of diet on beetle growth, development and fecundity

For each larval and pupal stage, the effects of a given diet on maximum weight (not determined for 1^st and 2^nd instars), stage duration and percent mortality were examined. For adults, the maximum weight, percent mortality and fecundity were also determined. The diet treatments were: plant leaves (control), Potato diet, Lettuce+Potato diet, and Lettuce diet. Results for parental and F₁ generations were combined (P-diet-I and PL-diet-I) and results for F₇ and F₈ (P-diet-II and PL-diet-II) generations were combined. For the diet containing only lettuce (Lettuce diet), only F₈ generation beetles were used; previous generations (P through F₇) had been reared on the Lettuce+Potato diet. Experimental beetles were examined daily through the 9^th day post-adult emergence. Instars were identified by head capsule width, the appearance of exuviae, and the black color of the pronotum and the anterior beige and posterior black stripe visible on the pronotum of the 3^rd and 4^th instars respectively (see Wardojo, 1969 for photographs). Prepupae were identified by the disappearance of black pigmentation (diet-fed beetles and leaf-fed beetles were light beige and reddish in color, respectively), their relative inactivity and the curved shape assumed by the beetle once it had entered the prepupal stage. Larvae [red with black markings and beige with black markings for plant and diet-reared beetles, respectively (Fig. 1A)] were weighed every other day, and since beetle development was not synchronous, there were sufficient beetles whose weights were determined the day before the molt to determine the mean maximum weight of each instar for each experimental treatment. For adults (also weighed every other day), the maximum weight observed for each beetle that had initiated feeding by day 7 post emergence was used in calculating the mean maximum weight. Beetles that had not initiated feeding by day 7 post emergence died sometime after day 10 post emergence. Adult beetles were sexed, and since the sex of the beetle influenced its weight, only those beetle larvae and pupae that reached the adult stage were included in the calculations for mean maximum weight of each of the stages. Mean duration and mean percent mortality were also determined. Since there was no significant difference in the durations for males and females of any given stage (determined for those that survived to adulthood), all larvae that had not died by the end of a given stage were used in the calculation of mean duration. For percent mortality, we could not determine the effect of sex, since the sex of beetle larvae and prepupae was not easily determined until they had molted to the adult.

The number of egg masses laid per day, number of eggs per mass and percent hatch served as measures of relative reproductive function of parental and F₁ generations and F₇ and F₈ generations of beetles reared on the various diets. Data were not collected until 5 days after beetles had mated. As mentioned previously, egg masses were collected twice daily on weekdays and once per day on weekend days.

Effect of diet on the initiation of adult feeding

Adult beetles that had not gained weight and had not defecated by day 9 post emergence were considered to be non-feeders. For each treatment group, the percent of males and the percent of females that had not initiated feeding was determined. After day 9, each non-feeding beetle was placed on Potato diet and was maintained on this diet until either feeding was initiated or the beetle died. The percentage of beetles that initiated feeding was determined.

Parasitization by Edovum puttleri

E. puttleri, originally obtained from the New Jersey Department of Agriculture, were reared as described in Hu et al. (1999b). For parasitization, CPB egg masses were placed in small plastic Petri dishes, irradiated to destroy developing beetle embryos as described by Hu et al. (1999b) and placed in a jar containing approximately 500 7–14 day-old E. puttleri adults. After 2 h, parasitoids were removed. Egg masses produced by CPBs reared on plant leaves, Potato diet, Lettuce+Potato diet and the modified Forrester diet were compared. For the three treatment groups reared on artificial diet, F₂ and F₃ generation CPBs were used. Mean percent parasitization and adult emergence as well as mean adult weight and longevity were determined for wasps in the four treatment groups.

Hemolymph ecdysteroid determinations

Hemolymph extraction was performed as described in Gelman and Woods (1983). Briefly, beetles (F₂ and F₃ generation) were anaesthetized by placing them in petri dishes containing small pieces of dry ice. Five to 10 microliters of hemolymph collected from a severed leg were placed in 1.5ml microcentrifuge tubes that contained 0.2 ml of ice-cold 75% aqueous methanol. Tubes were vortexed, kept on ice for at least 30 min and then centrifuged for 5 min at 4°C and 16,000 g. Supernatants or aliquots of supernatants were transferred to 6 × 50 mm borosilicate glass tubes and were stored in the freezer at −10°C. Prior to RIA, samples were dried in a Savant Speed Vac Concentrator (Forma Scientific, Marietta, OH, USA). RIA was performed as described in Gelman et al., 1997. Competitive studies showed that the ecdysone antibody had a high affinity for E, 20E, makisterone A, 26E, and 20,26E (Gelman et al., 1988). The radioactively labeled antigen was tritiated ecdysone (63.5 Ci per mmol). The concentration of ecdysteroids was determined from curves generated by 20E standards (50–4000 pg) and was therefore expressed as pg 20E equivalents/microliter hemolymph.

Statistical Analyses

To determine if the sex of the beetle significantly affected mean maximum weights, durations of various stages or percent non-feeding adults, Student's t-Tests (two-tailed, P< 0.05) were used. To determine the effects of diet on the various parameters selected to characterize beetle development, a one-way ANOVA was performed. When the variance of any one group was not equal to, or not slightly above 0, and when Bartlett's test of equal variance indicated that variances among groups were significantly different, a log transformation was performed prior to the one-way ANOVA. The Tukey (HSD) Comparison of Means Test (P< 0.05) was chosen as the post-hoc test to analyze for significant differences among the various treatment groups.

Duration of the four larval instars and the pupal stage of the CPB

For each larval instar, the length of the instar was significantly shorter when larvae were reared on potato plants than when they were reared on artificial diet (Fig. 2). However, with the exception of P-Diet I 2^nd instars, for any given instar, the durations for artificial diet-reared beetles were only between 0.8 and 1.5 days longer than the durations for leaf-reared beetles. When length of instar was compared for CPBs reared on the various artificial diets, in general the durations were slightly longer for P and F₁ generations than for F₇ and F₈ generations, excluding the F₈ beetles reared on the lettuce-only diet. For the pupal stage, the pattern was different. The time between prepupal burying into the soil and adult emergence was shortest for the P-Diet-I CPBs. Pupal durations for this group, the plant-reared beetles and the LP-Diet-I group were significantly shorter than for the P-Diet-II, LP-Diet-II and L-diet groups. As stated previously, for any given stage there was no significant difference in the stage durations for male and female beetles (results not shown).

Effect of diet on mean maximum weights of 3^rd and 4^th instars and of pupal and adult stages of the CPB

Maximum weights for 3^rd and 4^th instars, prepupae and newly emerged and older adults are provided in Fig. 3. Upon reaching the 4^th instar, except for F₇ and F₈ beetles reared on the Potato and Lettuce+Potato diets, the mean weight of female beetles was always significantly greater than that of male beetles. For both females (Fig. 3A) and males (Fig. 3B), leaf-reared beetles of all stages weighed significantly more than artificial diet-reared beetles. There were some significant differences in beetle weights associated with diet, but except for 5 – 9 day-old male adults reared on the Lettuce diet, for the most part, differences were small. Interestingly, for both males and females of all stages, CPBs fed on the Lettuce diet tended to weigh more than beetles fed on the other two diets, and the mean maximum weight achieved by male adults was not significantly different from the mean maximum adult weight of male beetles fed on potato leaves (Fig. 3).

Effect of diet on percent mortality of the four larval instars and the pupal and adult stages of the CPB

Survival of 1^st instars was significantly better when beetles were reared on leaves as compared to artificial diet (Fig. 4). However, by the time the beetle became a 2^nd instar, a diet of potato leaves offered no significant advantage. Importantly, the mortality rate of pupae was four to five times higher when beetles were reared on plant leaves as compared to artificial diet.

Therefore, total (newly hatched to adult emergence) percent mortality was not significantly different among the treatment groups; the higher percent mortality observed in artificial diet-fed 1^st instar CPBs was balanced by the exceptionally high mortality observed for pupae that were leaf-fed during larval development.

It is noteworthy that P and F₁ generation CPBs fed on the Lettuce+Potato diet and F₈ beetles fed on the Lettuce diet exhibited a considerably, but not significantly, lower percent total mortality than did the other treatment groups (Fig. 4).

Adult mortality for the period of day 9 through day 30 was monitored in mated pairs. Mortality for all treatment groups ranged between 5 and 10%, and there was no significant difference among groups (results not shown).

Effect of diet on the initiation of adult feeding

Male CPBs of the parental and F₁ generations fed on the Lettuce+Potato diet and of the F₈ generation fed on the Lettuce diet had an abnormally high rate of non-feeding, approximately, 50% (Fig. 5). The percentage of non-feeding females for these two treatments was also somewhat higher than for females of the other two treatment groups (Fig. 5). By nine days post-adult emergence >90% of the beetles were alive and when provided with Potato diet, >86% of these beetles began to feed and survived.

After 5 to 7 days of feeding on the Potato diet, when switched to the Lettuce+Potato diet, these beetles continued to feed (unpublished results).

Effect of diet on hemolymph ecdysteroid titers of prepupae and pupae

For leaf-reared as well as artificial diet-reared CPBs, hemolymph ecdysteroid titers were very low just prior to prepupal formation (in late 4^th larvae) as well as in newly formed prepupae. By 24-h post-prepupation, titers had begun to rise to approximately 250 pg/microliter, and continued to rise sharply through 72-h post-prepupation, at which time they reached approximately 1000 pg/microliter (Fig. 6). Except for the prepupal stage, there were no significant differences in hemolymph ecdysteroid titers for the three treatment groups. For prepupae, ecdysteroid levels for potato diet-reared beetles were slightly but significantly higher than for the other two treatment groups.

Effect of diet on fecundity and percent hatch

The number of egg masses laid, the number of eggs per mass and percent hatch served as measures of adult reproductive function. For each mated pair, beginning on the 5^thday post-mating, these values were recorded for a period of 30 days. For each parameter, a comparison of means (one-way ANOVA, alpha = 0.05) for the 1^st, 2^nd 3^rd and 4^th 7–8 day period revealed that there were no significant differences among them (results not shown).

Number of masses/pair/day

CPBs reared on potato plants produced significantly more egg masses per day than beetles reared on artificial diets (Table 2). The F₇ and F₈ generations fed on the Potato diet laid significantly more egg masses than all other diet-reared beetles except for F₇ and F₈ generations reared on the Lettuce+Potato diet. For all diet-reared beetles, approximately 20% of the egg masses were destroyed by cannibalism, while for leaf-fed beetles, a smaller percentage of egg masses were cannibalized (results not shown). However, it was difficult to determine the exact number of egg masses that were eaten, and therefore, values were not corrected for cannibalism of egg masses.

Number of eggs/mass

Egg masses produced by beetles from all treatment groups except for the P-Diet-I and Lettuce groups contained similar numbers of eggs (Table 2).

Percent hatch

Percent hatch was highest for leaf-fed beetles and beetles fed on the Lettuce diet, and lowest for beetles fed on the Potato diet (P-Diet-I and P-Diet-II) (Table 2). Therefore, the addition of lettuce leaf powder to the diet appears to promote egg hatch.

Number of offspring/day/pair

For each individual pair, the mean number of offspring/pair/day for each treatment group is provided in the next to the last column of Table 2. Leaf-fed beetles produced between 8 and 10 times more offspring than did all other treatment groups except for the Pot-Diet-I group; the latter produced approximately 28 times fewer eggs than the beetles fed on potato plant leaves. The last column of Table 2 provides information concerning the percent of beetle pairs that did not produce any hatchlings, either because eggs were not laid or because eggs did not hatch. The Pot-Diet-I and Lettuce groups had the highest percentage of such pairs. Importantly, the number of mated pairs not yielding hatchlings was not significantly different for the Plant leaf, Pot-Diet-II and Let-Diet-II groups.

Effect of diet on parasitization by E. puttleri

Eggs produced by beetles reared on plant leaves, the Potato, Lettuce+Potato and modified Forrester (here-in-after referred to as the Forrester) diets were tested for their ability to support parasitization by, and the development of, E. puttleri. Percent parasitism was significantly higher for the control group than for the three experimental groups which, except for the Forrester group, exhibited approximately 50% parasitism (Table 3). In contrast, percent parasitoid emergence was significantly lower for the control group. Therefore, the percent of eggs producing wasp offspring was approximately equal for the control (51%) and Potato diet (49%) groups (Table 3, Column 5), and although this percentage for the Forrester-diet group was only 31%, values for the four treatment groups were not significantly different. Mean adult weights for all treatment groups were in the same range, and longevity, while not significantly different for the three artificial diet-reared groups, was significantly lower for the Forrester group as compared to the control group. Thus, eggs produced by beetles fed on plant leaves and on the Potato and Lettuce+Potato diets appear to be equally suitable for rearing E. puttleri. Eggs produced by Forrester diet-fed CPBs were somewhat inferior for rearing these wasps.