Does access to Opuntia humifusa cactus seeds affect egg production in the cactus bug, Narnia femorata (Hemiptera: Coreidae)?

Anthony Riggio and Christine Miller

University of Florida Department of Entomology and Nematology

Abstract

Animals face challenges in acquiring the resources necessary for their survival and reproduction.

Our study considered Narnia femorata, a species of true-bug which feeds on the fruits of the

Opuntia humifusa cactus. We sought to determine if N. femorata is feeding on the seeds contained within O. humifusa cactus fruits, information which could help explain environmental and social factors affecting the life histories of true bugs. We compared the egg production of N. femorata breeding pairs provided O. humifusa fruits with the seeds intact with breeding pairs provided O. humifusa cactus fruits where the seeds were physically removed. We found that significantly more eggs were laid in the presence of fruit, as predicted from the results of prior studies showing the importance of proteins and lipids to egg production in insects. Our results suggest that N. femorata feeding on cactus fruit seeds will produce more eggs than those which do not. Therefore we posit that O. cactus fruit seeds are an important food resource for Narnia femorata because the seeds persist within the fruits throughout much of the annual growing season and contain higher concentrations of proteins and lipids relative to other O. cactus tissues.

Keywords: Narnia femorata, Opuntia, Cactus, Reproduction

Introduction

Animals are not always able to acquire the resources necessary to maximize their survival and reproduction (Batzli & Lesieutre 1991). There are food resources present within environments that can enhance the fitness of those animals that selectively feed on them (Batzli

& Lesieutre 1991). In herbivorous insects, these resources affect reproductive success by altering egg production and the choice of sites where eggs are laid (Awmack & Leather 2002).

Our study considered N. femorata, a species of cactus-feeding bug commonly found on the cactus stems (known as cladodes) and fruits of Opuntia cactus in the Southwest United States of America and Central America (Baranowski & Slater 1986). Narnia femorata was accidently introduced to Florida in the mid 20th century and has since formed many established populations statewide on native and introduced Opuntia cactus species (Baranowski & Slater 1986). Narnia femorata has emerged as a model species for studying how environmental variation influences sexual selection and female fecundity (Procter et al. 2014; Addesso et al. 2014; Miller et al.

2013). While it is well documented that N. femorata can feed on the fruit of the Opuntia humifusa cactus, it has not been determined whether these insects are feeding on the skin, pulp, or seeds of these fruits. This information is essential for understanding how seasonal changes in animal physiology and behavior may be linked to changes in Opuntia cactus phenology. For example, previous work has shown that N. femorata females change their mating behavior depending upon the presence or absence of cactus fruit (Addesso et al. 2014).

Interestingly, within these same habitats, the feral hog, Sus scrofa, and the coyote, Canis latrans, have adapted to the seasonal availability of food resources (including Opuntia cactus fruits) by varying their food preference and mating behaviors (Taylor & Hellgren 1997; Arias-

Del Razo et al. 2011).

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At the Ordway-Swisher Biological Station in North Central Florida, Opuntia humifusa fruit development begins in April (Miller, unpublished data). These fruits develop rapidly in late spring and early summer and begin to ripen in July (Miller, unpublished data). O. humifusa cactus fruit ripening has three distinct stages: an initial increase in dry weight (seeds and skin), followed by hardening of the seeds, and finally, fruit pulp swelling (Inglese et al. 1999). In the southwest United States, unripe fruits develop seeds early in the fruit development process

(Inglese et al. 1999). In North Central Florida where this study was conducted, the Opuntia cactus fruit seeds remain undeveloped and do not begin gaining significant dry mass until early summer (Miller, unpublished data). O. humifusa cactus fruits do not completely ripen until

October and November (Miller, unpublished data). Opuntia cactus fruit development is affected by competition with developing cladodes and environmental factors including temperature, sunlight and rainfall (Inglese et al. 1999). Opuntia cacti are rapidly denuded of their ripe fruit by a variety of vertebrate species (Miller, unpublished data). As a result, N. femorata and other animals that rely on Opuntia cactus fruit as a food source have had to adapt to its transient nature.

Access to Opuntia cactus fruit positively affects the development and reproduction of N. femorata. The absence of these fruits slows the growth of N. femorata nymphs (Nageon de

Lestang & Miller 2009), resulting in adults of smaller size (Gillespie et al. 2011) and with a reduced ability to produce eggs (Nageon de Lestang & Miller unpublished data). Similarly, adult

N. femorata females fed a diet of solely Opuntia cactus cladodes have been shown to lay fewer eggs than females fed a diet of Opuntia cactus cladodes and fruits (Nageon de Lestang & Miller

2009; Miller et al. 2013). The increased fecundity, accelerated development, and enlarged size of

N. femorata provided O. humifusa cactus fruit suggests that these fruits are a high quality

2 resource that female N. femorata can use to enhance multiple life history traits, including reproduction (Miller et al. 2013).

Opuntia cactus fruits are comprised of three primary tissue types, the pulp, skin, and seeds (Russel & Felker 1987). Each of these tissue types contains a unique composition of nutrients (Russel & Felker 1987). Dried Opuntia humifusa cactus seeds are composed of approximately 12% protein and 7% lipid, two macronutrients critical for growth and reproduction in insects (Russel & Felker 1987). The O. humifusa fruit pulp and fruit skin have a substantially lower percent composition of these two macronutrients, ranging from 5-8% protein and from 1-2% lipid in dried samples (Russel & Felker 1987). With a dry weight composition of

7.8% protein and 1.8% lipids, Opuntia cactus cladodes are nutritionally similar to the Opuntia fruit pulp and skin (Hernandez-Urbiola, Perez-Torrero, & Rodriguez-Garcia 2011). In addition to containing higher concentrations of these macronutrients, O. humifusa cactus fruit seeds also contain the micronutrients phosphorous, iron, and zinc in substantially higher abundance than other O. humifusa tissues (Sawaya et al. 1983; Hernandez-Urbiola, Perez-Torrero, & Rodriguez-

Garcia 2011).

Proteins are recognized as important food resources for growth and reproduction in the diets of most plant-feeding insects (Wheeler 1996; Rodrigues et al. 2008; Bernays & Chapman

1994). In insects, nitrogen deficiency causes a decrease in developmental and reproductive rates through endocrine-mediated mechanisms and nervous tissue suppression (Wheeler 1996). Lipid deficiency has also been shown to affect the developmental and reproductive processes in many insects through the inhibitory actions of hormones (Wheeler 1996). Oogenesis is one of the processes that will not be activated when there is a lack of the necessary nutrients, resulting in females who lay fewer eggs (Wheeler 1996). Such starvation induced responses result in

3 dramatic changes in development and fecundity that differ from the gradual decline in oogenesis and growth that occurs in ageing females (Wheeler 1996).

We hypothesized that N. femorata would produce more eggs when allowed to feed on O. humifusa cactus fruit seeds then if these insects are not given access to this resource. This is thought to occur because relative to other O. humifusa cactus fruit tissues, the seeds have the highest percent composition of proteins and lipids. The O. humifusa cactus fruit seeds would provide N. femorata access to nutrients that do not exist in such abundance in other O. humifusa cactus tissues. Therefore we predicted that N. femorata breeding pairs provided O. humifusa cactus fruits with the seeds intact should produce more offspring than breeding pairs provided fruits with the seeds removed.

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Materials and Methods

Colony Development

Thirty N. femorata individuals were collected on 5/6/2013 from a population existing within the Ordway-Swisher Biological Station. This population is dispersed among Opuntia humifusa cacti within a xeric scrub habitat at an elevation of approximately 40 meters above sea level along the north shore of Ross Lake (29°42’ 22” N, 81° 59’36” W).

From the 30 N. femorata collected on 5/6/2013, 10 males and 10 females were randomly selected using a random number algorithm (www.random.org), sexed and assigned a number 1-

20. A random number algorithm (www.random.org) was then used to randomly pair the 10 males and 10 females into 10 Breeding Groups. These insects were kept in a 27°C heated incubator with a 14 hour light, 10 hour dark photoperiod. These breeding pairs were watered, fed, and observed every 7 days after this. Clutches were removed weekly from each of these 10

Breeding Groups and placed in an adjacent heated incubator chamber with the same conditions.

These “clutch groups” were given an alphabetical designation based on what breeding group they came from and the order they were laid. These 35 “clutch groups” were given water as well as O. humifusa cactus cladodes and ripe O. humifusa fruit. The O. humifusa fruits and cladodes were collected from several dispersed cactus patches along Ross Lake in the Ordway-Swisher

Biological Station (29°42’ 22” N, 81° 59’36” W). O. humifusa cactus cladodes were collected on

5/26/2013 and kept under a full-spectrum fluorescent light in a 27°C heated incubator with a 14 hour light, 10 hour dark photoperiod. Ripe O. humifusa cactus fruits were collected on 5/26/2013 for colony rearing and on 7/26/2013 for treatment. All O. humifusa cactus fruits were stored in a sealed refrigerated container at 3°C. Those O. humifusa cactus fruits used in the experiment were randomly assigned to a treatment group and manipulated as described below.

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The “clutch groups” were checked weekly. Any N. femorata that eclosed into the adult stage were sexed and then isolated in individual containers for 7 days until they reached sexual maturity. These adults were given O. humifusa cactus cladodes and kept in a 27°C heated incubator with a 14 hour light, 10 hour dark photoperiod.

After this period, adults from each isolation date were given a number and these numbers were used in a random number algorithm to pair males and females (www.random.org). Each pairing became known as a “Testing Group”. After being randomly paired and assigned a number as one of the 75 Testing Groups, each group was sequentially assigned to one of the following two treatment descriptions or the control and provided fruit accordingly. Regardless of the treatment type, all “Testing Groups” were given soil, cactus cladodes, and sufficient water.

The treatment labeled “waxed, seedless fruit” consisted of a ripe O. humifusa cactus fruit that had the pedicel and all seeds removed. The treatment labeled “waxed fruit” consisted of a ripe O. humifusa cactus fruit that had the pedicel removed but no seeds removed. Unaltered O. humifusa cactus fruits were provided to 25 N. femorata “Testing Groups” to serve as the control.

Treatment Design

To assess the effects of a diet of ripe Opuntia humifusa cactus fruit without seeds, the seeds need to be removed in an innocuous manner. These methods concern the removal of the proximal end of the pedicel and in the case of treatment “waxed, seedless fruit”, the subsequent removal of the seeds from the ripe O. humifusa cactus fruit. Throughout the entirety of this procedure, gloves were worn to reduce human injury and to control the spread of disease between fruits. To insure asepsis, the necessary instruments, an Angular Semi-Blunt Mall Probe and Seeker, Hamilton Bell forceps and scalpel (Blade #21) and a metal tablespoon, were dipped

6 in 200ml of 6.00% Clorox bleach solution between dissections. The first incision was necessary for both treatment “waxed, seedless fruit” and treatment “waxed fruit” and required using the forceps for stabilization of the fruit body while the scalpel made a transverse incision through the proximal end of the pedicel. This removed approximately the 5mm of the fruit where it would attach to the cladode, an incision which opened the locule.

For treatment “waxed, seedless fruit”, the Angular Semi-Blunt Mall Probe and Seeker were then positioned at the now opened proximal end of the locule, parallel to the transverse axis of the O. humifusa fruit body and at a tissue depth of about 1mm, between the endocarp and the seeds. After the positioning of the Mall Probe and Seeker, it was inserted longitudinally into the proximal end of the locule for approximately 30mm. The angled margin of the Mall Probe and

Seeker was turned toward the transverse axis. Following positioning and insertion, the Mall

Probe and Seeker was rotated 360°, enveloping the seeds, and stripping the connective tissue between them and the endocarp. The O. humifusa seeds were then removed from the fruit body and placed on a receptacle by the forceps. A light source was used to examine each ripe O. humifusa fruit and confirm complete seed removal for treatment “waxed, seedless fruit”.

For both treatment “waxed fruit” and “waxed, seedless fruit”, the ripe O. humifusa fruit had the internal tissues of the locule exposed by the dissection. These tissues needed to be sealed to prevent intrusion of fungal, bacterial, or viral pathogens. Paraffin wax was the sealant used to prevent microbial intrusion into the ripe Opuntia humifusa cactus fruit after seed removal. This wax has a demonstrated history of preventing microbial infection and slowing degradation in a wide variety of fruits. (Ayers et al. 1964) Application of paraffin wax to ripe O. humifusa cactus fruit has been documented to reduce dehydration symptoms and protect the external appearance and overall color of the fruit (Berger et al. 2002). A tube of paraffin wax of a burgundy color

7 similar to ripe O. humifusa cactus fruit was chosen for this procedure. The wax was fractured into 20mm x 20mm x 2mm sheets and placed on a spoon pre-sterilized in 200ml of 6.00%

Clorox bleach solution. The spoon was heated, liquefying the paraffin sheets and bringing the liquid to a near boil (approximately 300°C). Following this, the transverse incision site of the O. humifusa fruit was embedded into a minimal amount of paraffin wax liquid for approximately 1 second. After this time, the fruit was moved to a sterile surface and allowed to cool, cementing a thin layer (0.5 mm) of paraffin wax to the proximal fruit surface. From here, additional heated but malleable paraffin wax (approximately 100°C) was applied to the transverse circumference using sterile, disposable paintbrushes. This paraffin wax was allowed to cool to room temperature (21°C), completely sealing the incision site.

Treatment Presentation

Fruit was prepared for the treatment group immediately before it was presented.

Presentation of the fruit to the treatment group involved first sequentially assigning each randomly assigned testing group to the treatment “waxed, seedless fruit”, the treatment “waxed fruit” or the control. After assignment, the testing groups were ready for the fecundity study and received their treated or controlled fruit type, which was placed into their “deli” container. The

“deli” containers are cylindrical and measure 15 centimeters tall with a diameter of 11.5 centimeters. The “deli” container lids are circular and have a central square opening (6cm x 6cm) where a 1mm aperture nylon mesh can be positioned to allow for ventilation. During this study, testing group pairs were provided fresh cactus cladodes and fruit and adequate water. Testing

Groups were also kept in a heated incubator, at a temperature of 27°C and a 14 hour light, 10 hour dark photoperiod. They were examined weekly, and during each examination, the number

8 of clutches and total number of eggs was recorded. Following these recordings, the eggs were removed from the container and disposed of. Testing Groups were examined approximately weekly in the afternoon on nine dates between 7/26/2013 and 9/22/2013. The dependence of N. femorata survivorship on the presence of fruit seeds was analyzed using a “χ2 Test for

Independence” on Microsoft Excel 2010 Version 14.0.7116. The cumulative number of eggs produced over the five week period was compared for each treatment and the control with a

“Single Factor Analysis of Variance” test also using Microsoft Office Excel 2010 Version

14.0.7116.

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Results

Fifty eight of the 75 total pairs (77%) survived the five week study, 25 pairs (96%) from the control group, 19 pairs (76%) from the “waxed fruit” treatment, and 14 pairs (56%) from the

“waxed, seedless fruit” treatment. Survivorship of Narnia femorata adults was shown to be dependent on whether the breeding pairs were given Opuntia cactus fruit with seeds or Opuntia cactus fruit where the seeds had been removed (χ2=7.12, α<0.01). Considering all breeding pairs,

N. femorata breeding pairs produced on average 5.42 eggs (range: 0-60) per week over the 5- week period. 37 breeding pairs (49% of the total) laid no eggs, with 13 of those pairs (35% of all females that laid no eggs) from the “waxed, seedless fruit” treatment group.

The presence of O. humifusa cactus fruit seeds had a positive effect on egg production.

The results of the “Single Factor Analysis of Variance” test indicated that when pairs were provided “waxed, seedless fruits”, females laid significantly fewer eggs relative to when they were provided a “waxed fruit” treatment (P<0.001, Table 1). As predicted, on average females laid more eggs when provided a diet of O. humifusa cactus fruit with the seeds intact (waxed fruit: 9.3 ± 2.25 eggs; Figure 1) than when provided O. humifusa cactus fruit with the seeds removed (waxed, seedless fruit: 0.186 ± 0.186 eggs; Figure 1). Female N. femorata from the control group pairs laid on average 6.8 ± 2.11 eggs per week over the 5-week study.

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Discussion

We found that egg production in N. femorata is dependent upon access to the O. humifusa cactus fruit seeds. As we predicted, all treatment groups and the control had minimal egg production during the first week. This is because N. femorata does not reach sexual maturity and produce eggs until approximately 2 weeks after eclosion into the adult stage (Addesso et al.

2013). After the breeding pairs matured and acclimated to their respective conditions, there was a significant difference in egg production between N. femorata given access to Opuntia cactus fruits with the seeds intact and those given Opuntia cactus fruits where the seeds had been removed (Table 1). Egg production ceased after the first week for those breeding groups with access to O. humifusa cactus fruit with the seeds removed (Figure 1). Egg production increased during the second week for both the control and the “waxed” fruit treatment (Figure 1). The gradual decline in egg production observed in the control group and the “waxed fruit” treatment group between weeks two and four is most likely attributable to the aging of the breeding pairs

(Figure 1) (Wheeler 1996). There was a significant increase in the mortality rate for those Narnia femorata breeding groups given access to Opuntia cactus fruit with the seeds removed. We believe that the same nutrient-limited mechanisms that restrict growth and egg production affect many other life history traits of Narnia femorata and may explain the significant difference in mortalities observed.

Protein is a limiting nutrient in the diets of many plant feeding true bugs (Wheeler 1996).

Access to proteins has been shown to enhance egg production in these insects (Rodrigues et al.

2008; Bernays & Chapman 1994). Plessis et al. (2012) found that the protein and lipid rich seeds of the sunflower, Helianthus annuus, enhanced egg production in another plant feeding true bug,

Nysius natalensis (Sosulski & Fleming 1977). Opuntia humifusa cactus fruit seeds are presumed

11 to provide N. femorata with access to proteins and lipids, two necessary nutrients with relatively less abundance in all other O. humifusa cactus tissues.

This study addresses many unanswered question in the biology of N. femorata.

According to Nageon De Lestang & Miller (2009), Narnia femorata nymphs without access to

Opuntia cactus fruits grow slower and eventually develop into smaller sized adults with reduced reproductive success (Gillespie et al. 2011; Nageon de Lestang & Miller unpublished data;

Addesso et al. 2013). Adult female N. femorata without access to the Opuntia fruits produce fewer eggs while males without access to the fruits have reduced testes size (Addesso et al. 2013;

Nageon de Lestang & Miller unpublished data). The results of this study offer a mechanism to explain how growth and reproduction in Narnia femorata is affected by the presence or absence of Opuntia fruits. We posit that the life history changes observed in these and other experiments examining the effects of Opuntia cactus fruits on Narnia femorata are due to insects feeding on the nutritious seeds contained with the Opuntia cactus fruit.

Narnia femorata feeding on ripe O. humifusa cactus fruits have larger gonads than those that feed on unripe O. humifusa cactus fruits (Nguyen & Miller 2011). However, it is unknown if

N. femorata is able to distinguish ripe from unripe O. humifusa fruits or if it will preferentially feed on one of the other. Because Opuntia cactus fruit choice (ripe vs. unripe) has an effect on reproduction in Narnia femorata, these seasonal feeding preferences would offer benefits to survival during the seasonal fluctuations in abundance of this food resource.

Understanding how the seasonal availability of O. humifusa cactus fruits causes changes in the reproductive behavior of N. femorata is important in determining the importance of this fruit as an available resource. The developmental periods of Opuntia cactus fruits vary by season and geographic location (Inglese et al. 1999; Miller unpublished data). While the timing of these

12 periods of growth and development is known for Opuntia cactus growing in the Southwestern

United States (Baranowski & Slater 1986), there is currently no published information on the seasonal growth patterns of O. humifusa cactus growing in the Southeastern United States. These seasonal growth cycles are a critical time for nutrient uptake by the Opuntia cactus fruit (Inglese et al. 1999).

Social cues also influence egg production in N. femorata (Miller et al. 2013).

An abundance of juvenile offspring has been shown to enhance egg production in female N. femorata more than the presence of a single Opuntia cactus fruit (Miller et al. 2013).

Because offspring will traverse over areas greater than a single Opuntia cactus cladode, the use of these social cues allows female N. femorata to assume the presence of high-quality food resources even when these resources are unavailable at their present location (Miller et al. 2013).

These social cues would improve the egg production of N. femorata in early spring, when O. humifusa cactus fruits begin to develop and may not be visible to all breeding females (Miller, unpublished data).

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Conclusion

The nutritional requirements and feeding strategies of N. femorata offer insight into the niches that can be exploited by phytophagous insects. This information may eventually aid in the control of pestiferous species with behaviors and life histories similar to N. femorata.

Furthermore, the role of Opuntia cactus fruit on the life history of N. femorata is not yet clearly understood. However, the rapid development and persistence of highly nutritious Opuntia cactus fruit seeds may explain how N. femorata is able to exploit this seasonal food resource.

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References

Addesso, K., Short, K., Moore, A., & Miller, C. (2013). Context-dependent female mate preferences in leaf-footed cactus bugs. Behavior, 151, 479-492.

Arias-Del Razo, I., Hernandez, L., Laundre J., & Myers, O. (2011). Do predator and prey foraging activity patterns match? A study of coyotes (Canis latrans), and lagomorphs (Lepus californicus and Sylvilagus audobonii). Journal of Arid Environments, 75, 112- 118.

Awmack, C., & Leather, S. (2002). Host plant quality and fecundity in herbivorous insects. Annual Review of Entomology, 47, 817-844.

Ayers, J., Kraft, A., & Peirce L. (1964). Delaying spoilage of tomatoes. Food Technology, 18, 100-103.

Baranowski, R., & Slater, J. (1986). Coreidae of Florida. Arthropods of Florida, 12, 27-28.

Batzli, G., & Lesieutre, C. (1991). The influence of high quality food on habitat use by arctic microtine rodents. OIKOS, 60, 299-306.

Berger, H., Mitrovic, A., Galletti, L., & Oyarzún, J (Eds,). (2002). Proceedings from the 4th International Convention on Cactus Pear and Cochineal: Effect of hot water and wax applications on storage life of cactus pear (Opuntia ficus-indica) fruits. Leuven, Belgium: International Society for Horticultural Science.

Bernays, E., & Champan, R. (1994). Chemicals in plants. In R. Champan (Ed.), Host-Plant Selection by Phytophagous Insects (pp. 14-68). New York, NY: Chapman & Hall.

Gillespie, S., Tudor, S., Moore, A., & Miller, C. (2011). Mating decisions vary according to environmental cues, but in different ways for males and females. Integrative and Comparative Biology, 51, 47.

Hernandez-Urbiola, M., Perez-Torrero, E., & Rodriguez-Garcia, M. (2011). Chemical Analysis of nutritional content of the prickly pads (Opuntia ficus indica) at varied ages in an organic harvest. International Journal of Environmental Research and Public Health, 8, 1287-1295.

Inglese, P., Barbera, G., & La Mantia, T. (1999). Seasonal reproductive and vegetative growth patterns and resource allocation during cactus pear fruit growth. HortScience, 34, 69-72.

Miller, C., Fletcher R., & Gillespie, S. (2013). Conspecific and heterospecific cues override habitat quality to influence egg production. PLoS One, 8, 2779-2796.

Nageon De Lestang, F., & Miller, C. (2009). Effect of diet on development and survivorship of Narnia femorata nymphs (Hemiptera: Coreidae). Florida Entomologist, 92, 511-512.

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Nguyen, L., & Miller, C. (2011). The use of multiple cues in male-male competition of the cactus bug (Narnia Femorata: Coreidae). University of Florida Undergraduate Thesis Database. Retrieved from http://www.honors.ufl.edu/apps/Thesis.aspx/Download/1090

Plessis, H., Byrne, M., & van der Berg, J. (2012). The effect of different host plants on the reproduction and longevity of Nysius natalensis. Entomologia, 145, 209-214.

Procter, D., Moore, A., & Miller, C. (2012). The form of sexual selection arising from male-male competition depends on the presence of females in the social environment. Journal of Evolutionary Biology, 25, 803-812.

Rodrigues, D., Soares, G., & Moreira, G. (2008). Feeding preference of Holymenia clavigera and Anisocelis foliacea marginella (Hemiptera: Corediae: Anisoscelini) in relation to intra- and interspecific features of host plants (Passifloraceae). Environmental Entomology, 37, 1323-1331.

Russel, C., & Felker, P. (1987). The prickly-pears (Opuntia spp., Cactaceae): A source of human and animal food in semiarid regions. Economic Botany, 41, 433-445.

Sawaya, W., Khalil, J., & Al-Mohammad, M. (1983). Nutritive value of prickly pear seeds, Opuntia ficus-indica. Plant Foods for Human Nutrition, 33, 91-97.

Sosulski, F., & Fleming, S. (1977). Chemical, functional, and nutritional properties of sunflower protein products. Journal of the American Oil Chemists’ Society, 54, 100-104.

Taylor, R., & Hellgren, E. (1997). Diet of feral hogs in western south Texas plains. The Southwestern Naturalist, 42, 33-39.

Wheeler, D. (1996). The role of nourishment in oogenesis. Annual Review of Entomology, 41, 407-431.

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Table 1. Results of analysis of variance for cumulative egg production by treatment (α=0.05 ) Source df F P Treatment 2 216.731 <0.001

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Figure 1. Mean number of eggs produced per week (± SE) by breeding groups in the control and each of the two treatment types.

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Control

Waxed Fruit 20 Waxed, Seedless Fruit

Week 2 Whole Fruit

15 Week 2 Waxed Fruit

Week 2 Waxed, Seedless Fruit

Week 3 Whole Fruit 10 Week 3 Waxed Fruit

Week 3 Waxed, Seedless Fruit

5

Week 4 Whole Fruit Mean Number of Eggs Per Pair Per Week Per Pair Per Eggs of Number Mean Week 4 Waxed Fruit

Week 4 Waxed, Seedless Fruit 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 5 Whole Fruit

Week 5 Waxed Fruit

Week 5 Waxed, Seedless Fruit

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