Differential gene expression in Perillus bioculatus nymphs fed a suboptimal artificial diet

Abstract

Fragments of two artificial diet up-regulated and two prey up-regulated transcripts were isolated from the predatory Pentatomid Perillus bioculatus using suppression subtractive hybridization. A BlastX search found similarities for two diet-upregulated clones, i.e., the tyrosine-3-monooxygenase gene and the gene for the chitin binding protein, Gasp. The probe generated from the tyrosine-3-monooxygenase clone hybridized to two transcripts 2.3 and 1.2 kb in size. The two transcripts were differentially regulated: the 2.3 kb transcript was upregulated in the first and late third instar diet-fed nymphs, whereas the 1.2 kb transcript was upregulated in the second and early third instar diet-fed nymphs. The Gasp gene was upregulated in late third instar nymphs. A positive correlation was found between levels of expression of the isolated genes and the number of generations the insects had been reared on the artificial diet.

Introduction

Insects face many environmental stresses, including the possibility of inconsistent quantity or quality of food sources required to support development and reproduction. Therefore, insects must have mechanisms to cope with fluctuations in their food. For predictable seasonal food shortage, insects employ the behavioral/physiological mechanisms of diapause (Danks, 1987; Tauber et al., 1986) to escape the shortage. For unpredictable changes in food quantity or quality, or in environmental factors affecting digestion, insects enlist a number of behavioral, biochemical and physiological mechanisms to respond to suboptimal nutrient intake (reviewed: Slansky and Scriber, 1985; Chapman, 1985; Chown and Nicolson, 2004). Such responses can be clearly observed at the molecular level. For example, altering the protein to carbohydrate ratio from the optimum induces a decrease in expression of the proliferating cell nuclear antigen gene in the gut of Locusta migratoria (Zudaire et al., 2004). Starvation and a high sugar diet induce specific sets of genes in Drosophila melanogaster larvae (Zinke et al., 2002).

Biocontrol programs employing augmentative and inoculative release strategies require large numbers of high-quality insects if they are to suppress their target pest. Large automated insectaries are producing one billion medfly pupae (Ceratitis capitata) per week and 150–500 million screwworm pupae (Cochliomyia hominivorax) per week for sterile insect release programs. In addition, procedures have been developed to produce 1.2 million fruit fly parasitoids (Fopius arisanus) per week (Bautista et al., 1999; McGraw, 2001; Robinson, 2002). One of the limiting factors for the broader adoption of biocontrol as a pest management approach is the cost of the beneficial insects, which can far exceed that of chemical control. An optimized artificial diet can greatly reduce the cost of producing beneficial insects. The development of an optimized diet for Chrysoperla carnea and Chrysoperla rufilaris reduced the cost per insect from $0.3587 to $0.00025 (reviewed: Tauber et al., 2000). The current strategy for diet development is to measure some physiological or biochemical parameter in order to quantify the effect of changes in the diet formulation on the insect's performance (Adams, 2000; Adams et al., 2002; Wittmeyer et al., 2001; Coudron et al., 2002; Coudron and Kim 2004). Diet components are changed one at a time and the insect's performance on the new formulation is examined. This endeavor is time-consuming, taking years to decades to perfect a diet, and many attempts end in failure. What is needed to accelerate diet development is a directed method that can provide a more informative feedback in order to target deficiencies in the diet formulation. Nutrigenomics, the examination of how nutrition affects gene expression patterns, offers not only a means to quantify an insect's response to a diet reformulation but also provides information on diet limitations.

Perillus bioculatus is a predaceous pentatomid native to North American (Knife, 1952) that attacks a number of different insect orders but has a preference for the coleopteran, Leptinotarsa decemlineata (Saint-Cyr and Cloutier, 1996). In order to mass rear this beneficial insect, development of an artificial diet was undertaken. The diet is functional in that it has supported development for over 30 consecutive generations (Coudron, unpublished data), but P. bioculatus reared on this diet are deficient in fecundity and developmental time as compared to prey-fed insects (Coudron and Kim, 2004). In order to develop molecular markers to aid in diet development, third instar P. bioculatus nymphs that were fed either the artificial diet or prey (Trichoplusia ni) were examined for differential gene expression. The isolated genes were then used for developmental studies comparing their expression among nymphal instars and between generations on the diets. The third instar was chosen for this investigation to insure sufficient feeding time to elicit differential gene expression and for size, decreasing the number needed to collect a total of 100 mg for RNA extraction. Another advantage to using third instars rather than adults is that having molecular markers isolated from such an early developmental stage decreases the screening time of new diet formulation.