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). 1 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. 4 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.