ABSTRACT ZHA, CHEN. Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae

ABSTRACT

ZHA, CHEN. Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae. (Under the direction of Allen Cohen.)

Mold control is one of the most vital issues in insect rearing systems. Mold outbreaks can alter the nutritional value of the diet, do harm to the insects, and even threaten the health of people who work in insectaries. Antifungal agents are widely used in insect diets to suppress the growth of mold; however, the potential detrimental effects of antifungal agents on insects are always a concern. When there is a high level of antifungal agents in artificial diets, the growth, development, survivorship and fecundity of insects are often negatively affected. To study the mechanisms underlying those deleterious effects, nutritional indices were determined for two representative lepidopterans, a butterfly and a moth, Vanessa cardui and

Heliothis virescens larvae reared on different concentrations of three widely used antifungal agents: methyl paraben, potassium sorbate and sodium propionate. These antifungal agents were administered in concentrations of 1,000, 5,000, and 10,000 parts per million (ppm). The results show that a high level of antifungal agents in the diets of these insects will suppress nutrient absorption and increase metabolic costs. Relative consumption rates and digestibility increased with increasing antifungal agent concentration, possibly to compensate for the declines in absorption and metabolism. Silk production and frass management by Vanessa cardui larvae were reduced in the presence of the highest level of antifungal agents.

Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae

by Chen Zha

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirement for the Degree of Master of Science

Entomology

Raleigh, North Carolina

2013

APPROVED BY:

______Allen C. Cohen Yasmin J. Cardoza Chair of Advisory Committee

______David Orr Clyde Sorenson

BIOGRAPHY

Chen Zha was born April 11th, 1989 in Anhui province, China. His love of insects revealed itself during his childhood. He studied life sciences in University of Science and Technology of China. After having received his BS degree, he shifted his major into entomology under the direction of Dr. Allen C. Cohen at North Carolina State University. His first research effort in entomology focused on the influences of antifungal agents on insect nutritional ecology, and he plans on working with chemical ecology of bed bugs and getting a PhD in entomology at Rutgers University.

ACKNOWLEDGEMENTS

I would like to thank everyone who helped me along the way, my advisor Dr. Allen Cohen, fellow graduate students, professors and staff, and my parents who provide me economical support studying abroad. Special thanks go to Drs. Yasmin J. Cardoza, David Orr and Clyde

Sorenson for being members of my graduate committee and for providing much help and advice to guide my work. I also thank Dr. Fred Gould for providing H. virescens eggs from his lab colony, and Sandra Paa for egg collection and technical support in rearing H. virescens. Finally, I thank Dr. Consuelo Arellano for helping with the statistical analysis of my data.

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TABLE OF CONTENTS

LIST OF TABLES ...... v

LIST OF FIGURES...... vi

Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae ...... 1 INTRODUCTION ...... 2 MATERIALS AND METHODS ...... 16 RESULTS ...... 27 DISCUSSION ...... 30 REFERENCES ...... 46

LIST OF TABLES

Table 1. The ingredients of YC diet ...... 63

Table 2. Effects of antifungal agents on V. cardui feeding and growth ...... 64

Table 3. Effects of antifungal agents on H. virescens feeding and growth ...... 65

Table 4. Percent V. cardui larvae fed on diet columns contain different concentrations of antifungal agents ...... 66

Table 5. Percent H. virescens larvae fed on diet columns contain different concentrations of antifungal agents ...... 67

Table 6. Percent larvae fed on different diet columns with different pH ...... 68

LIST OF FIGURES

Figure 1. The effects of different concentrations of 3 antifungal agents on frass production ...... 69

Figure 2. The effects of different concentrations of 3 antifungal agents on insect weight gain ...... 70

Figure 3. The effects of different concentrations of 3 antifungal agents on consumption rate (CR) ...... 71

Figure 4. The effects of different concentrations of 3 antifungal agents on relative consumption rate (RCR) ...... 72

Figure 5. The effects of different concentrations of 3 antifungal agents on approximate digestibility (AD) ...... 73

Figure 6. The effects of different concentrations of 3 antifungal agents on the efficiency of conversion of ingested food to biomass (ECI) ...... 74

Figure 7. The effects of different concentrations of 3 antifungal agents on the efficiency of conversion of digested food to biomass (ECD) ...... 75

Figure 8. Comparison among the 3 antifungal agents on frass production ...... 76

Figure 9. Comparison among the 3 antifungal agents on insect weight gain ...... 77

Figure 10. Comparison among the 3 antifungal agents on consumption rate (CR) ...... 78

Figure 11. Comparison among the 3 antifungal agents on relative consumption rate (RCR) ...... 79

Figure 12. Comparison among the 3 antifungal agents on approximate digestibility (AD) . 80

Figure 13. Comparison among the 3 antifungal agents on the efficiency of conversion of ingested food to biomass (ECI) ...... 81

Figure 14. Comparison among the 3 antifungal agents on the efficiency of conversion of digested food to biomass (ECD) ...... 82

Figure 15. Freeze dried diet sample of 10,000ppm methyl paraben treatment, showing the presence of white methyl paraben crystals which insects avoided to ingest ...... 83

Figure 16. “Frass management” of a V. cardui larva, showing that the frass was attached with silk and kept away from diet ...... 84

Figure 17. A comparison of V. cardui frass management behavior between 1,000 and 10,000ppm potassium sorbate (KS1 & KS3) treatments ...... 85

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Evaluating the Dynamics of Anti-Fungal Compounds in Lepidoptera Larvae

C. Zha

Department of Entomology, North Carolina State University Campus Box 7613, Raleigh, NC 27695

INTRODUCTION

Background on antifungal agents in insect diets

Laboratory rearing of insects has significant importance for studying their nutrition, biochemistry, behavior and other biological processes (Vanderzant 1974). Mass production of insects is an important process for biological control and sterile insect technologies

(Knipling 1955; Klassen & Curtis 2005), feeding other animals (Versoi & French 1992), production of pharmaceuticals and recombinant proteins as bioreactors (Hughes and Wood

1998), and food for people (DeFoliart 1999). Insect artificial diets have been widely used for laboratory rearing and mass production of insects (Vanderzant 1974). Microbial contamination in artificial diet is one of the most serious problems in insect rearing systems.

Aspergillus niger, A. flavus, Cladosporium spp., Fusarium spp., Rhizopus nigricans,

Penicillium spp. and some yeasts and bacteria are among the most commonly found microorganisms in insect diets (Cohen 2003; Bell et al. 1980; Ouye 1962). Microbial infection can adversely affect insects by altering the nutritional value of diet (Bell et al.

1980), preventing insect feeding by forming a thick mat (Ouye 1962), generating toxins which are toxic to insects, such as ochratoxin A produced by Aspergillus and Penicillium

(Dowd 1989, Mühlencoert et al. 2004), and even present health hazards to people working in insectaries (Bell et al. 1980). Decreasing microbial colonization and increasing the time that insects can feed on uncontaminated diet have significant benefit for enhancing insect health, reducing the cost of diet ingredients and the labor involved with diet preparation (Inglis &

Cohen 2004).

Antifungal agents are commonly used to protect artificial diets from fungal contamination. For example, Ludemann et al. (1979) listed some commonly used and tested antifungal agents, including methyl paraben, sorbic acid, formaldehyde, sodium benzoate, butylparaben, potassium sorbate and other compounds, considering both the effectiveness on suppressing mold and their tolerance in target insects. A review of literature on diets by

Cohen (2003) drew a similar conclusion, that the predominant antifungal agents were sorbate compounds and methyl paraben. Both the acid form and salt form of some agents are used in diets; for example, benzoic acid is also used in the forms sodium benzoate and potassium benzoate, and sorbic acid is also used in the forms potassium sorbate and sodium sorbate

(Cohen, 2003). Hsiao and Hsiao (1974) added Streptomycin sulfate (primarily an anti- bacterial agent) and Benlate (Benomyl fungicide from DuPont) as antifungal agents into alfalfa weevil diet. Some antifungal agents are not only artificial diet additives, but can actually be generated in insect natural food. For example, one of the most widely used antifungal agents in diet, methyl paraben, has been demonstrated to be found in plants (Bais et al. 2003; Kannathasan et al. 2011).

With little or no antifungal agents in insect diets, the colonization of mold takes place rapidly, often within one day (Ludemann et al 1979; Holleley et al. 2008). The utilization of microbial inhibitors in artificial diet can significantly suppress microbial colonization, and increase insect survivorship, growth, and development (Roeder et al., 2009). Some chemicals, such as potassium sorbate and propionic acid are significantly effective against both fungi and bacteria (Inglis & Cohen, 2004), and formalin is also effective against nuclear-polyhedrosis virus in Cabbage looper (Vail et al. 1968). The efficiency of antifungal

agents is influenced by several factors. One of the most vital factors is concentration; the concentrations of antifungal agents need to be high enough to suppress mold growth effectively, and these effective concentrations vary among antifungal agents and pH levels.

At pH 5.0, the effective concentrations of several antifungal agents vary dramatically: methyl paraben (1,000ppm), propyl paraben (300ppm), benzoic acid (2,000ppm), sorbic acid

(800ppm), and propionic acid (800ppm) (Funke 1983 cited by Cohen 2003). Antifungal agents are usually more effective in microbial growth suppression when the pH is low, and the effective concentrations increase with higher pH values (Cohen 2003). Cruess and

Richert (1928) showed that sodium benzoate was more effective at pH values of 2.5 to 4.5, and much more benzoate was required at more neutral pH to suppress microbial growth. In a study by Tong and Draughon (1985), potassium sorbate was most effective, followed by sodium propionate, methyl paraben, and sodium bisulfate at pH 4.5; while methyl paraben and potassium sorbate became the most effective ones at pH 5.5. Hedin et al. (1974) pointed out that antifungal agents lost much effectiveness above pH 6.0-6.6, while the boll weevils,

Anthonomus grandis, did not tolerate pH much lower than 5.3. Thus, pH value of the diet can be a critical issue in insect rearing.

Other factors, such as the composition of other diet components are also involved in the effect of antifungal agents. Brock and Buckel (2004) found that sodium propionate inhibits growth of Aspergillus nidulans on glucose but not on acetate. On the other hand, Isao and Lee (1998) found that the fungicidal activity of sorbic acid against Saccharomyces cerevisiae was enhanced 64-fold by the synergistic effect of` polygodial. However, antifungal agents are usually expected to be stable without reacting or binding with other

dietary components, or being degraded by the process of heat treatment (Hedin et al. 1974).

The effectiveness of antifungal agents can also change through time. Ludemann et al (1979) pointed out that most anti-fungal agents will not be 100% effective for 2 weeks. Last but not least, the strain of the mold must be considered in evaluating the response to antifungal agents. Gifawesen et al. (1975) studied a strain of Aspergillus niger which was more resistant to methyl paraben than other stains. They tested the effectiveness of several other chemicals against that strain. More recent studies have explored the biochemical and genetic mechanism underlying the resistance of mold to antifungal agents (Plumridge et al. 2008,

Plumridge et al. 2010).

Antifungal agents with varied modes of action have been used in insect diets.

Considerable research on the mechanisms of antifungal agents has been done, and many mechanisms may be postulated for a single agent. For example, methyl paraben has a broad spectrum, and is particularly useful against molds and yeasts, by inhibiting membrane transportation and mitochondrial functions (Soni et al. 2002). Sorbic acid and sorbates show most profound inhibition effects on spore germination, and catalase inhibition in molds was concluded to be the mechanism of sorbate action (Troller 1965). Yousef and Marth (1983) suggested that sorbates inhibit the transfer of nutritional substances (e.g. glucose) into the fungal cell. Propionates cause accumulation of propionyl-CoA in fungi, which inhibits enzymes mainly involved in glucose metabolism (Brock & Buckel 2004). Phosphoric acid inhibits microbial cells by enhancing the concentration of H+ (Russell and Gould 1991, cited by Andow & Stodola 2001).

It is important to know, not only the effectiveness, but also the modes of action of specific antifungal agents. First, knowing the mechanisms underlying their antifungal effects help researchers avoid antimicrobials that might also be toxic to the target insects. Second, using groups of antifungal agents that have different mechanisms from one-another can increase the likelihood that all fungal contaminants will be controlled, even those with resistance to a given single mechanism. Third, by using antifungal agents with different modes of action, the designer of the antifungal “cocktail” can select antifungal agents with complementary functions which could lead to reduction in the amounts of each individual agent, thereby reducing the potential of harm to the target insect. The strategy of using mixtures of antifungal agents to derive synergistic benefits is discussed further here.

Antifungal agents with different functional mechanisms are often used together in diets to achieve better control (Andow 2001). Kishaba et al. (1968) studied 7 antifungal agents and demonstrated that no agent was effective enough against mold when used singly; this finding demonstrated the efficacy of striving to find the proper combination of different antifungal agents to suppress mold far more effectively through a combination of different modes of action. By inhibiting various metabolic pathways of fungal cells, antifungal agents can suppress mold germination and growth as well as toxin biosynthesis such as ochratoxin A

(Tong & Draughon 1985), better protecting insects from fungal toxins. Such synergistic strategies offer promise for effective and economical mold management in rearing systems.

Also, because the factors that inhibit mold growth may also have detrimental effects on insect metabolism, understanding the modes of action can help us select antifungal agents that will not adversely affect target insects. For example, many chemicals with highly

effective mold inhibiting properties, such as 2’-benzoyloxycinnamaldehyde (Kang et al.

2007), plagiochin E (Wu et al. 2008), and various proteins (Bormann et al. 1999; Trindade et al. 2006), would be harmful to insects because the targets of these mold inhibitors are chitin synthetic pathways. Clearly, these chitin synthesis pathways are also important for insects themselves. Arakawa and Sugiyama (2002) found that a chitin synthesis inhibiting antibiotic, nikkomycin Z, can promote nuclear polyhedrosis virus infection in silkworm larvae.

The side effects of the antifungal agents which are widely used in insect artificial diets can be significant. This is due to their complex and often non-specific modes of action and potential adverse effects on insect metabolism. Although there is little literature discussing the interactions specifically between antifungal agents and insect cells at the molecular and biochemical levels, the presence of metazoan toxicity on widely used antifungal agents can be expected (Brock and Buckel 2004; Buazzi and Marth 1991). Thus, the usage of antifungal agents in diets has potential risks which need to be considered carefully.

There is abundant information indicating that several widely used antifungal agents have detrimental effects on insects. These studies indicate that increasing the level of antimicrobial agents in diets there can inhibit insect growth and development. Furthermore, high levels of antimicrobial agents can be toxic to insects and result in high mortality (Singh

& House 1970a). Methyl paraben and formalin significantly decreased fecundity of Lygus hesperus females, while benzoic acid, propionic acid, and sorbic acid reduce biological fitness of Lygus hesperus(Alverson & Cohen 2002). Many antimicrobial food additives can reduce the size of insects feeding on the diet, and size of the insect is negatively related to the

level of antimicrobial agent concentrations (Singh and House 1970b). Because effects of antimicrobial agents can differ from one species to another (Cohen 2003), the detrimental effects of antifungal agents on different insects should be studied carefully and treated case by case. For example, 0.15% (1,500 ppm) methyl paraben plus 0.2% (2,000 ppm) sorbic acid were used in the diet for Heliothis zea (Brazzel et al. 1961), but would cause lethal effect on write-fringed beetle larvae (Bass & Barnes 1969). Boush et al. (1968) found that when there were certain levels of sorbic acid in diet, the survivorship of Attagenus megatoma and

Trogoderma parabile were not affected, but the filial generation would fail to survive beyond

1st instar.

The utility of nutritional indices in insect nutritional ecology studies

Nutritional ecology has been the subject of hundreds of studies of insects, with the works of Waldbauer 1968 and Gordon 1968 laying the foundation for numerous other studies

(Cohen 2003). Slansky (1990) demonstrated the utility of using nutritional indices to study host plant resistance, leading to dozens of subsequent studies that focused on feeding ecology measurements through use of efficiency indices. Slansky and colleagues produced a considerable body of work in using the nutritional indices for a variety of topics. For example, a series of studies on diluted food feeding behavior (Rueda et al. 1991; Wheeler &

Slansky, 1991; Slansky&Wheeler, 1991; Slansky & Wheeler, 1992). Bauce et al. (1994) used nutritional indices to study the relationship between foliage age and host plant resistance.

Brewer and King (1979) compared the nutritional value of different Heliothis diets with nutritional indices. Cohen and Patana (1984) used nutritional indices for a comparison

between artificial diet and natural food, and demonstrated that even a Heliothis zea lab colony with hundreds of generations reared on artificial diet can return to consumption of a natural food; they also showed that the nutritional indices of Heliothis zea fed with natural food (green beans) were higher than those with artificial diet. Henn and Solter (2000) used nutritional indices to study gypsy moth Lymantria dispar infected with microbial pathogens and to look for possible lethal mechanisms. Several researchers, such as Herbert and Harper

(1987), Vyjayanthi and Subramanyam (2002), have applied nutritional indices in silkworm toxicology studies. Various nutritional indices have been used to treat quantitatively the nature and mechanisms of insects in response to their foods. Such studies include consumption index (CI), relative consumption rate (RCR), relative growth rate (RGR), approximate digestibility (AD), conversion of ingested food (ECI), and conversion of digested food into biomass (ECD) (Waldbauer 1968; Scriber & Slansky 1981), with some nomenclature modifications made by Woodring et al. (1979). Some common nutritional indices are explained below.

Consumption rate

Consumption rate reflects how insects feed on their food. If insects do not grow well on a particular diet, one possible reason is that the amount of food ingested is too small. A low food intake can be attributed to deficiency of feeding stimuli or presence of feeding deterrents (Gordon 1968). Consumption rate is also related to the nutritional quality of the diet. Insects can increase the consumption of food and maintain a stable pupal weight when the nutritional quality is poor (Wheeler & Slansky 1991; House 1965); the increasing relative

feeding rate may compensate for the lowered assimilation efficiency (Blake and Wagner

1984). Altered consumption rate can play a significant role in insect growth and fitness; for example, the increasing of consumption rate on nutritionally poor diet can lead to deleterious chemical accumulations to a toxic dose (Slansky & Wheeler 1992). Several parameters were introduced to measure diet consumption (Scriber 1977), including the consumption rate:

mg (dry wt) food ingested CR , and relative consumption rate: day

mg food eaten RCR mg larval biomass day

Approximate digestibility

Approximate digestibility is a parameter which measures the efficiency of digestion of a given diet. Previous studies demonstrated that AD was reduced by feeding on a nutritionally poor diet. For example, younger instars often have a higher AD because they tend to ingest less indigestible crude fibers (Bailey 1976, Barah et al. 1989); however,

Slansky and Wheeler (1991) conducted an experiment on velvetbean caterpillars, Anticarsia gemmatalis which showed that the AD of nutrients was not affected significantly by the non- nutritional cellulose additives. Low food digestibility can be also attributed to a lack of or inhibition of essential hydrolytic enzymes (Gordon, 1968). Some environmental factors as well as gender and age of the insect are also correlated with AD, which will be discussed later.

The approximate digestibility (AD) or absorption efficiency is calculated as:

E F AD (Approximate Digestibility) E

where E=the amount of food ingested and F= the amount of frass produced, with both referring to dry weights.

Conversion of ingested food

Efficiency of conversion of ingested food to biomass (ECI) is a quantitative measurement of the utilization of food the insect ingested, and will rise and fall with AD and

ECD. The ECI can reflect several important parameters in insects’ nutritional ecology, including anti-metabolites and digestive organization (such as peritrophic complexes, extra- oral digestion, or digestive system structural peculiarities—all discussed by Cohen 2003).

The use of ECI can, for example, help to detect the presence of anti-metabolites which block nutrients and suppress absorption (Gordon, 1968). Peristalsis and the peritrophic complex also play important roles in digestive organization and nutrient absorption (Cohen 2003 also

Pechan et al. 2002), which will be explained further in the discussion. The ECI can be calculated without the weight of frass, thus the ECI provides a good parameter when the measurement of frass is not feasible, such as with subterranean insects (Moeser and Vidal,

2005) or liquid feeders (Cohen 2003).

The efficiency of conversion of ingested food to biomass (ECI) or growth efficiency is calculated as

P ECI (Efficiency of Conversion of Ingested Food to Biomass) E

where E=the amount of food ingested and P=weight gain, both referring to dry weights.

Conversion of digested food

Efficiency of conversion of digested food to biomass (ECD) is sometimes known as the metabolic efficiency; it is a measurement of how well the target insect has incorporated digested food into its own tissues. Metabolic rates, which include oxygen consumption, carbon dioxide production, and turnover of fuel molecultes in general, are directly related to food intake; highest metabolic rates occur with maximal feeding and most rapid growth

(Woodring et al. 1979). Thus, nutritional deficiency or the presence of antimetabolites, which may bind with the active sites of enzymes and interfere with biochemical reactions, can reduce metabolic efficiency; this phenomenon is discussed in detail by Gordon 1968.

Examples of such phenomena are inhibition by imidazole, discussed by Woolley 1959 and by

Pence 1963 and 3-acetyl pyridine discussed by Shyamala & Bhat 1958. Other biological or environmental factors such as age (Kern & Wegener 1984), temperature, body mass

(Gillooly et al. 2001), and interaction with other organisms (Alleyne & Beckage 1997, Huang et al. 2008) can also affect the metabolic index. It is also noteworthy that total metabolic efficiency can be lowered by increasing metabolic cost, for example, detoxification, secretion, enzyme production and other metabolically expensive metabolic processes. This topic will be discussed in detail in a later section of this paper.

The efficiency of conversion of digested food to biomass (ECD) or metabolic efficiency is calculated as

P ECD(Efficiency of Conversion of Digested Food to Biomass) E F

where E=the amount of food ingested, F= the amount of frass produced and

P=weight gain, all referring to dry weights.

Rationale for applying nutritional ecology on antifungal agents study

Nutritional ecology has emerged as a valuable approach to understanding relationships of insects to their trophic environment. The nutritional indices are a central component of nutritional ecology, revealing the relative value of various foods in the feeding regimen of insect species. Some examples of the versatility of the nutritional indices are presented here. Baker (1974) suggested the potential function of symbionts underlying a significant higher conversion efficiency of ingested food, and consequently increased growth rate while reducing food consumption. Panizzi & Slansky (1991) indicated the usefulness of nutritional ecology studies for pest management. Slansky (1986) pointed out the importance of nutritional ecology studies in parasitoids for biological control, and work has been done to investigate the nutritional ecology underlying host-parasitoid relationships using nutritional indices as important tools (Alleyne & Beckage 1997, Huang et al. 2008). The nutritional ecology of silkworms is a major aspect of study in the field of sericulture (Vyjayanthi and

Subramanyam 2002). Moeser and Vidal (2004) suggested that nutritional indices such as ECI make useful tools for indicating the suitability of different plant genotypes for pest development.

Surprisingly, considering the importance and great amount of work that has been done on both antifungal agents and insect nutritional ecology, the applications of nutritional indices on antifungal agent treatment effects on insects are relatively uncommon. Most work

on antifungal agents has focused on developing and modifying diet formulas (Shorey & Hale

1965), the effects on insect growth and fitness (Alverson & Cohen, 2002; Singh & House

1970a, 1970b; Bass & Barnes 1969), or the effectiveness against mold (Gifawesen et al.

1975). More research is needed to investigate the mechanism underlying the negative effects antifungal agents have on insect physiology and behavior. For example, if an antifungal agent is demonstrated to promote food intake, or have less negative effects on digestion, absorption and metabolism, then it could be considered a favorable choice for artificial diets.

Yûsuke et al. (2011) showed the utility of nutritional indices for comparing nutritional quality between diets; likewise, nutritional indices can also be used to compare effects of various antifungal agents on insect performance. Even if two antifungal additives yield insects of the same quality, the one with which insects have higher utilization of diet may be preferred in mass rearing. However, decisions need to be made on a case by case basis, and comparative research among different antifungal agents and insect species is necessary to facilitate this decision making.

Previous experience with Lepidoptera larvae in our rearing processes has resulted in anecdotal observations of lower growth and frass production in rearing trials where antifungal agents were at higher concentrations. In this study, we used nutritional indices to investigate how insect growth responds to different levels of antifungal agents. Two

Lepidoptera larvae (Vanessa cardui and Heliothis virescens) were used in the study. The rationale for selecting these species were 1) the fact that both V cardui and H. virescens are commonly reared on artificial diets (Cohen 2003), 2) the relatively large size of these insects made handling and measurements easy, allowing accurate measurements of food

consumption, frass production and growth rates, 3) the two species represented two divergent taxa within the Lepidoptera thus allowing comparisons to be made of responses of taxonomically disparate members of this order. The objective of this study was to determine if the harmful effects of high levels of antifungal agents on Vanessa cardui and Heliothis virescens) are due to reduction in (1) feeding, (2) digestive efficiency, (3) amounts of absorption of nutrients, (4) metabolic capabilities or (5) a combination of effects. The answers to these questions and the testing of the ensuing hypotheses will provide better insights into the mechanisms of antifungal agent impact on insects at a physiological or metabolic level. Knowing the relationship between antifungal agents and insect nutritional ecology can provide us better insight into the interactions between antimicrobial agents and the insects that they are being used to protect from mold growth. Such understanding will help us develop artificial diets more efficiently. For example, by studying a certain behavioral or physical process (feeding, digestion, assimilation or metabolism), comparison among different antifungal agents can be made without rearing the insects for multiple generations. It would also help researchers to be aware of the potential influences by antifungal agents in their study, for example, to avoid diets that contain digestion-suppressing antifungal agents while studying the digestion of a certain species.

MATERIALS AND METHODS

Insects

Vanessa cardui larvae used in this study were originally purchased from Carolina

Biological Supply Company (Burlington, North Carolina), and were reared on our YC diet

(Cohen modified Yamamoto Diet, unpublished observations) for three generations previous to the experiments. Although the painted lady butterflies in this study were obtained from

Carolina Biological Supply Company, the information about their origin was proprietary with that company.

Heliothis virescens eggs used in the study were YDK strain (Gould, 1995) obtained from a colony from Dr. Fred Gould’s insectary in Department of Entomology, North

Carolina State University. This colony was originally collected from Yadkin County, North

Carolina in 1988. The H. virescens are maintained on CSB (corn soy blend) diet, modified from Burton (1970), in the NCSU Entomology Department insectary. Specimens from this colony were demonstrated in pilot experiments to accept, develop, and grow on YC diet.

These preliminary experiments indicated that the use of YC diet as a base medium for delivering appropriate doses of antimicrobial agents was feasible. Also, in the pilot experiments, it was demonstrated that both species of insects in this study tolerated doses of antifungal agents that were adequate to suppress microbial growth. Because there was difficulty in preventing fungal growth in the rearing conditions used in the pilot studies unless the base mixture of 1,570 ppm of each antifungal, it was decided that all further diet

trials would be done with a starting (base) YC Diet that contained 1,570 ppm of each of the three antifungal agents.

Insect rearing

V. cardui were reared on YC diet in the lab for three generations, with a colony size of 30 to 40 individuals. The YC Diet was developed by Dr. Allen Cohen as an offshoot of the

Yamamoto Diet (1967) (unpublished information) as a broadly nutritive diet meant to support growth of multiple species of Lepidoptera (Table 1).

The V. cardui colony was started with approximately 20 early instars purchased from

Carolina Biological Supply Company (Burlington, North Carolina). Larvae were fed on YC diet until pupation. Pupae were hung with masking tape on the top of a butterfly cage (Item #

971521 Carolina Biological Supply Company, Burlington, NC) to facilitate the emergence of adults by allowing the newly emerged adults to hang from the top during sclerotization. This measure was taken to simulate natural pupation conditions in V. cardui, and was found to be a useful technique for achieving nearly 100% adult emergence from puparial cases.

Butterflies were fed on artificial nectar (water 65g, sugar 30g, honey 5g, potassium sorbate

0.1g). Leaves from potted mallow plants (Malva sp., Item # 144042 Carolina Biological

Supply Company, Burlington, NC) were used as oviposition sites for the adults. Eggs were washed from oviposition substrates and sanitized with 5% bleach solution (5 ml sodium hypochlorite in 95 ml tap water ), then hatched and reared communally in 8-cell trays

(Product # SMRT8 Bio-Serv, Frenchtown, NJ) filled with YC diet (approximately 50 eggs and 70g diet per cell). The trays were tipped 45° to allow frass to drop from diet, to keep the

diet clean and minimize mold outbreaks on the substrate. Third or early fourth instar larvae were transferred to new trays with density being lowered to 2 to 3 individuals per cell, and fresh food was provided.

Rearing and experiments for both of the two Lepidoptera species (V. cardui and H. virescens) were conducted in a rearing room at 28±1°C, 40-60% RH with a 14:10 L: D. Two space heaters were placed in two opposite corners in the rearing room. Two HEPA (High- efficiency particulate air) filtration systems (from Honeywell, Minneapolis, MN) were placed near to each heater, to remove air-bone mold spores, facilitate air circulation and disperse heat uniformly in the rearing room.

Experimental design

Feeding experiment

A randomized complete block design with 9 treatments was used for V. cardui.

Treatments were three antifungal agents tested at three concentrations each. Six replications were performed, and blocks consisted of replication in time. Caterpillars of V. cardui were fed with YC Diet with a mixture of 3 antifungal agents in it: methyl paraben, potassium sorbate and sodium propionate. The standard level of each antifungal agent was 1,570ppm.

The concentration of only one of the three anti-fungal agents was varied in each treatment, thus there were 3 groups: methyl paraben group (i.e. the concentration of methyl paraben was changed from 1,000 to 10,000 ppm while the other two agents were 1,570 ppm), potassium sorbate group, and sodium propionate group. Three levels of each chemical, 1,000, 5,000, and 10,000 ppm, were tested.

Each replication contained 3 groups with 3 treatments in each group, and there were

10 samples for each treatment. For both species one extra trial was done before the experiment, and the number of days taken to see the first pupa was recorded. Experimental trials were run for one day shorter than the time required from hatching to pupation to maximize larval growth while avoiding any pupation, to ensure that all the larvae were feeding during the same experimental period. This measure was taken because of the observations of Waldbauer 1968 and Cohen and Patana 1984 that significant error was introduced if non-feeding periods were included in calculations of feeding, growth, and frass production.

Most anti-fungal agents will not be 100% effective for prevention of mold growth for

2 weeks (Ludemann et al, 1979). Because it took longer than 2 weeks for V. cardui to grow from neonate to wandering stage, V. cardui were hatched and reared communally in 8 cell trays until early 3rd instars, and then reared individually in 2 ounce dessert cups (Item #

SOLB200 Ace Mart Restaurant Supply, Texas). Each cup was filled with about 8 grams of diet, and 15 holes were perforated on the lid with a #2 insect pin to enhance air exchange during larvae growth and freeze drying process. Cups were stacked on each other (5 cups each stack) to save more space and get better organized, but each cup and each stack was randomly located. Air exchange was not influenced, since the holes were perforated along the margin of the lids, with diameters greater than the bottom of the cups. The entire cups were frozen at -20oC after 7 days, and cups were held frozen until they could be freeze dried.

The diets and treatments for H. virescens were the same as for V. cardui. H. virescens neonates were reared individually in 1 ounce dessert cups (Item # SOLP100 Ace Mart

Restaurant Supply, Texas) fitted with plastic lids. Each cup was filled with 4 to 5 grams of

YC Diet. Holes were not perforated until freeze drying to keep neonates from escaping.

During the wandering stage, H. virescens larvae will dig and mix the diet with frass to make a pupal chamber (Zha, personal observation). It is not feasible to separate diet from frass in these samples, so the cups were set upside down to prevent larvae from making pupal chambers in diet. The 30 cups of each group were fitted into a 30-well (5×6 wells) cup tray

(Product # 9040 Bio-Serv, Frenchtown, NJ) in the order of treatments (from 1,000 to 10,000 ppm), and then the trays were stacked on each other randomly and set upside down in the rearing room. The cups and their entire contents (uneaten diet, larvae, and frass) were frozen at -20oC after 11 days. The experiment was originally designed as a randomized complete block design as for V. cardui. Although trays were stacked randomly, cups on all trays were arranged in the same manner so that the 5,000 ppm treatment was in the middle of each tray, rendering these experiments as a stratified randomized block design. It should be noted that whatever non-random conditions might have been in effect, the greatest differences in all outcomes were between the 1,000 and 10,000 ppm treatments.

Cups contaminated with visible mold or bacteria growth were discarded to minimize the influence of microbes on the results. Dead individuals were discarded and mortality was not recorded. It seems noteworthy that H. virescens seemed to have higher mortality not due to anti-fungal agents, but the upside-down set up of rearing cups; some individuals failed to make their way to diet and died apparently due to starvation.

Frozen samples were freeze-dried at -60°C, 35 millitorr for 24 hours, and the dry weights of larvae, uneaten diet and fecal materials were recorded. Three variables were

measured: (1) dry weight of diet left in each cup; (2) dry weight of frass produced by each insect; (3) dry weight of each insect. Original dry weights of the diets presented to larvae were estimated by multiplying wet weights by the percentage of dry material in the diet.

Choice test on different concentrations of antifungal agents

To determine whether neonates can make choices among different concentrations of antifungal agents, a certain amount of single antifungal agent was added into diet. Five concentrations (0, 1,000, 1,570, 5,000, 10,000 ppm) of each antifungal agent (methyl paraben, potassium sorbate and sodium propionate) were tested at one time in multiple choice tests. Diet was poured into 8 cell trays and diet columns in the same volume were made with a No. 7 cork borer. The 5 diet cylinders (1.3 cm diameter ×2 cm high) of the 5 antifungal agent concentrations were placed in an 8 cell tray with equal distance and angle.

Twenty V. cardui or H. virescens neonates were introduced to the center of the cell right after hatching. Choice tests for each agent were replicated 20 times. The locations of diet cylinders in each cell were rotated clockwise each time to standardize environmental factors.

The trays were kept in the rearing room, under conditions described above, overnight and the number of neonates feeding on each diet was recorded the next day. Both V. cardui or H. virescens neonates seldom sat on the diet cylinders in preliminary experiments, so neonates which stayed near the diet at a distance less than their body length were recorded as “feeding on the diet”. Neonates sitting far away from all the diet or moving actively were not recorded.

Dead neonates were also not included in the data.

Multiple choice test on pH

To test if the acceptability of diet was related to pH, four diets with different pH were tested. Citric acid was used to alter the pH of the diet. To eliminate the possibility of the citrate anion affecting the diet’s palatability rather than pH, , the diet’s pH was altered by adding the same amount (weight) but different ratio of citric acid and potassium citrate mixture. This measure kept the concentration of the citrate ion relatively constant for all pH treatments. Four pH levels were tested, and the protocol was the same as the choice test on antifungal agents.

Comparisons of detrimental effects among the three antifungal agents

Nutritional indices of larvae reared in 5,000 ppm of each antifungal agent in the feeding experiment were used for comparison to determine the differences among the possible detrimental effects due to different antifungal compounds. Because the standard level of each antifungal agent was 1,570ppm, the 5,000 ppm antifungal agent treatments can be described as basic diet with additional 3,430 ppm of one antifungal agent, with other environmental factors randomized. Thus, the comparisons were set up using complete randomized block design, and treatments were 5,000 ppm of three different antifungal agents.

Six replications were performed, and blocks consisted of replication in time. The comparisons were done within each species separately.

Analysis

To obtain the dry weight of frass, eaten food, and net weight gain, the wet weight of initial diet in each cup was measured and converted to dry weight via dry diet to water ratios.

The wet weight of each early 3rd V. cardui was recorded and converted to dry weight based on the water content of the larvae. The water content of the larvae was determined by dividing the weight of individual freeze-dried 3rd instar larvae by the fresh weight of the same larva, which had been frozen prior to measurement. The mean of 10 3rd instar larvae was used in these calculations. The weights of H. virescens neonates were considered negligible in the context of the total weight gain (based on the observation that neonates weighed less than 100 µg, and the final larval weights exceeded several hundred mg). Ten freeze dried diet samples from each species were treated by oven-drying at 110°C, 35 min to calculate the average water content of dried food.

For each sample, these calculations were made:

The amount of food ingested = initial dry diet – remaining dry weight of diet

The amount of frass produced = dry weight of fecal materials

Weight gain = dry weight of freeze dried larva- dry weight of 3rd instar larva (V. cardui)

Or weight gain = dry weight of freeze dried larva (H. virescens)

Once weight gain, amount of fecal materials produced, and diet consumed data were obtained, we calculated nutritional indices to evaluate the feeding, digestion and assimilation process of the insects. The nutritional indices can reflect how differences in insects’ foods affect the insects. The relative consumption rate (RCR), approximate digestibility (AD), the

efficiency of conversion of ingested food to biomass (ECI) and the efficiency of conversion of digested food to biomass (ECD) are three commonly used nutritional indices (Scriber

1977, Waldbauer 1968).

The consumption rate (CR) is calculated as:

mg (dry wt.) food ingested CR day

The relative consumption rate (RCR) is calculated as:

mg food eaten RCR mg larval biomass day

The approximate digestibility (AD) is calculated as:

E F AD (Approximate Digestibility) E

The efficiency of conversion of ingested food to biomass (ECI) is calculated as:

P ECI (Efficiency of Conversion of Ingested Food to Biomass) E

The efficiency of conversion of digested food to biomass (ECD) is calculated as:

P ECD(Efficiency of Conversion of Digested Food to Biomass) E F

where E=the amount of food ingested, F= the amount of frass produced, P=weight gain, all referring to dry weights.

The calculation of nutritional indices can be problematic especially when several indices are calculated at the same time; Waldbauer (1968) pointed out that a method in measuring frass weight can be doubtful for measuring intake. Waldbauer’s comments about this potential error were in response to some researchers who considered the measurement of

frass a useful indication of the amount consumed. The natural losses in fresh diet, for example include the degradation of diet components and water evaporation. These losses would increase with time, and would amplify the discrepancy between the dry weight calculated from percent dry matter in diet and the actual percent dry matter of the food.

Waldbauer 1968 pointed out that the error from failure to consider natural losses in diets can lead to significant deviation from actual consumption. Generally speaking, magnification of introduced errors when calculating nutritional indices is far greater when the larvae eat only a small fraction of the food (Waldbauer 1968, Cohen and Patana 1984, and Schmidt 1986).

Thus, it is suggested that the amount of diet offered to the insects should be just enough for the insect to ingest while minimizing leftovers. However, this can be difficult in practice, because even insects feeding on the exact same diet can have dramatically different growth and food consumption rates. Providing small amounts of diet in rearing containers can speed up water evaporation, affecting diet quality, and nutritional indices measurements due to the altered moisture in food (Reese & Beck 1978).

Besides the issues of natural losses and diet aliquots causing error in measuring nutritional indices, several other factors can contribute errors that may be cryptic. For example, food consumption by insects can be affected by frequent disturbance by researchers while changing the diet and removing frass (Waldbauer 1968). These factors may magnify the complexity of experiments, and decrease available sample size by failure in rearing insects on dehydrated diet. Stamp (1991) demonstrated that values for nutritional indices

(RGR, RCR, ECD) were reduced when there was <5% leftover; however, even as much as triple the food required by the experimental insects only affected RGR in a significant

manner. Thus, in our study more than enough food for larvae maximal growth was provided.

The wet weight of diet was recorded immediately after the molten diet was distributed to individual cups, and then converted to dry mass. The average water content in freeze dried diet was also measured to minimize the errors.

Determining these parameters, can help us determine if differences in success of insects using different foods are due to differences in feeding rates, digestive efficiency, absorption efficiency, and/or metabolic efficiency.

All data were analyzed in JMP 10(SAS Institute, Cary, North Carolina). For the variables in feeding experiment, i.e. frass weight, amount of diet eaten, weight gain, AD, ECI and ECD, analysis of variance (ANOVA) was performed to test for effect of antifungal agent concentrations on each of the variables of interest. All significant ANOVA effects were followed by Tukey HSD test for mean comparisons.

The number of neonates on each diet in multiple choice tests was recorded. The

Friedman Test was used to model the ratings of 20 judges on treatment, i.e. different concentrations of one antifungal agent.

For the comparisons of detrimental effects among the three antifungal agents, the variables were subjected to analysis of variance (ANOVA) for possible effect of treatment on

5,000 ppm of different antifungal agents. All significant ANOVA effects were followed by

Tukey HSD test for mean comparisons. An alpha level of 0.05 was used for all analyses.

RESULTS

Feeding experiment

The amount of frass produced, diet eaten, weight gain and nutritional indices as influenced by antifungal agents

Significant differences were detected among most of the treatments. Almost all parameters for V. cardui dropped significantly when the concentrations of antifungal agents increased except AD and RCR (Table 2, Figure 1-7). The amount of frass produced was reduced by diets containing 5,000 and 10,000 ppm methyl paraben (F=19.33; df=2, 161;

P<0.0001), and 10,000 ppm potassium sorbate/sodium propionate (F=26.85; df=2, 173;

P<0.0001. F=19.95; df=2, 171; P<0.0001). The weight gain of insects was reduced by diets containing 5,000 and 10,000 ppm methyl paraben (F=23.99; df=2, 161; P<0.0001), and

10,000 ppm potassium sorbate/sodium propionate (F=37.93; df=2, 173; P<0.0001. F=22.79; df=2, 171; P<0.0001). CR was reduced by 5,000 and 10,000 ppm methyl paraben (F=35.64; df=2, 161; P<0.0001), 5,000 and 10,000 ppm potassium sorbate (F=35.03; df=2, 173;

P<0.0001), and 10,000 ppm sodium propionate (F=16.91; df=2, 171; P<0.0001). RCR was increased by 5,000 and 10,000 ppm methyl paraben (F=5.43; df=2, 161; P=0.0052), 10,000 ppm potassium sorbate (F=9.17; df=2, 173; P=0.0002) and 10,000 ppm sodium propionate

(F=7.85; df=2, 171; P=0.0005). AD was increased by diet with 10,000 ppm methyl paraben

(F=3.20; df=2, 161; P=0.0432), 10,000 ppm potassium sorbate (F=3.59; df=2, 173;

P=0.0295), and 10,000 ppm sodium propionate (F=15.30; df=2, 171; P<0.0001). ECI dropped with 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=4.55,

df=2, 161; P=0.0120. F=11.46, df=2, 173; P<0.0001. F=12.08; df=2, 171; P<0.0001). ECD dropped with 10,000 ppm potassium sorbate/sodium propionate (F=9.04; df=2, 173;

P=0.0002. F=15.65; df=2, 171; P<0.0001).

Most parameters for H. virescens dropped significantly when the concentrations of antifungal agents increased except AD (Table 3, Figure 1-7). The amount of frass produced was reduced by the diets with 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=7.81; df=2, 151; P=0.0006. F=18.04, df=2, 116; P<0.0001. F=30.15; df=2,

137; P<0.0001). The weight gain of insects was also reduced in the diets with 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=10.48; df=2, 151; P<0.0001.

F=21.06; df=2, 116; P<0.0001; F=29.77; df=2, 137; P<0.0001). The CR was reduced by the diets with 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=7.65; df=2,

151; P=0.0007. F=17.42; df=2, 116; P<0.0001. F=28.91, df=2, 137; P<0.0001). The RCR was increased in insects fed diets with 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=7.56, df=2, 151; P=0.0007; F=8.64, df=2, 116; P=0.0003. F=9.84; df=2, 137;

P=0.0001). The AD was increased in insects fed diets with 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=4.70; df=2, 151; P=0.0105. F=14.42; df=2,

116; P<0.0001. F=26.19; df=2, 137; P<0.0001). The ECI dropped with diets containing

10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=10.30; df=2, 151;

P<0.0001. F=16.03; df=2, 116; P<0.0001. F=14.90; df=2, 137; P<0.0001). The ECD also dropped with diets containing 10,000 ppm methyl paraben/potassium sorbate/sodium propionate (F=9.95; df=2, 151; P<0.0001. F=18.70; df=2, 116; P<0.0001. F=21.83; df=2,

137; P<0.0001).

Choice test

Numbers of neonates feeding on different diets were significantly different among treatments. Feeding of V. cardui neonates was reduced by high levels of methyl paraben

(F=8.08; df=4, 19, 76; P<0.0001). Comparatively, potassium sorbate showed positive effect on diet acceptance by neonates (F=5.41; df=4, 19, 76; P=0.0007). The presence of sodium propionate in diet did not affect the choice of the neonates (Table 4).

The analysis of H. virescens feeding behavior showed that H. virescens neonates consumed less diet at high levels of both methyl paraben and potassium sorbate (F=4.31; df=4, 19, 76; P<0.0034. F=3.70; df=4, 19, 76; P=0.0083). Sodium propionate did not affect larval feeding choice (Table 5).

Four diets with different pH (5.5, 5.1, 4.8 and 4.5) were formulated via changing the citric acid to potassium citrate ratio. The analysis of both V. cardui and H. virescens feeding behavior on different pH showed no significant differences (Table 6).

Comparison of detrimental effects among the three antifungal agents

Vanessa cardui performed differently with the addition of different kinds of antifungal agents to the diet. V. cardui reared on a high level of methyl paraben had lower amount of frass, less weight gain, reduced CR, elevated RCR and reduced ECI (F=4.02; df=2,

176; P=0.0196. F=5.51; df=2, 176; P=0.0048. F=7.10; df=2, 176; P=0.0011. F=7.69; df=2,

176; P=0.0006. F=4.93; df=2, 176; P=0.0082). H. virescens reared on the three diets did not show any significant differences (Figure 8-14); i.e. the performance of H. virescens did not differ from each other when there was an extra high level of methyl paraben, potassium

sorbate, or sodium propionate, indicating a relative insensitivity to these agents by H. virescens.

DISCUSSION

The results indicate that, overall, high levels of antifungal agents may decrease the frass production and weight gain of the insect species in this study by reducing (1) consumption rate, (2) absorption rate and (3) metabolic capabilities. However, relative consumption rate (RCR) and approximate digestibility (AD) were unchanged or, more often, elevated with increased level of antifungal agents in diet. The nutritional indices are not independent from each other, and they are also influenced by insect age, gender, and various environmental factors (Waldbauer 1968). Most environmental factors in our studies were controlled to be the same for all trials, and the sex ratio should not be significantly different among all trials since larvae were assigned to each trial randomly. However, the individuals reared in the highest concentrations of all antifungal agents usually grew and developed slower and ended up with earlier stage larvae in the end of the experiment. Thus, the relationships between nutritional indices and insect development are discussed fully in the following discussion.

Reduced growth under high antifungal agent concentrations

Both V. cardui and H. virescens gained significantly less body weight and produced less frass when feeding in the highest concentration of all three antifungal agents tested in

this study. These effects may be caused by the adverse effects of high concentrations of antifungal agents, by reducing feeding, absorption and metabolism of the larvae, and extending larval development period. Similar results with other insect species were found in previous studies which showed that insect size and development rate can be reduced by high levels of antifungal agents (Singh & House 1970b, Alverson & Cohen 2002). In this study, both V. cardui and H. virescens larvae were frozen at the point when most larvae reared in the lower concentrations of antifungal agents had reached late 5th instar or even wandering stage. However, the larvae reared in the highest level of antifungal agents were usually still in 4th or even earlier larval stages. Thus, the reduced development rate of those larvae can directly result in less weight gain and fecal materials. In this study we did not allow all insects to reach the same development state but controlled the time of rearing instead. The main reason for this was that it would be impossible for many larvae reared in the highest antifungal agent concentration to reach late larval stages; the quality of diet would also deteriorate with time.

The consumption rate (CR) could be reduced for similar reasons. Previous studies indicated that daily food consumption is positively related to daily growth, and later instars ingested more food than did earlier instars (Bailey 1976, Poston et al. 1978, Cherry 1991).

Thus, consumption rate is directly related to the size of the larvae; since the individuals reared on diets with the highest concentration of antifungal agents are much smaller, their consumption rate is also lower than it is for larger individuals. Larvae may feed less in the presence of large amount of antifungal agents, acting as they might with distasteful allelochemicals. Reduction in feeding in response to common antifungal agents has not been

well-studied despite the long-standing observations (such as those of Singh & House 1970b) that antifungal agents are toxic in high concentration. It would strand to reason that toxicity would be related to reduction in feeding responses (Wang et al. 2007, Senthil-Nathan et al.

2009). However, the choice tests in this study showed that high levels of antifungal agents did not always affect rates of feeding or total amounts consumed. For example, sodium propionate did not influence larval feeding choice in either V. cardui or H. virescens, while

V. cardui.actually had a significantly higher rate of consumption with the higher concentration of potassium sorbate. Therefore, some possible reasons other than reduction of palatability are worth considering. As mentioned previously, larvae reared on high concentrations of antifungal agents usually did not reach late 5th instar stage by the end of the experiment (as did the insects’ counterparts fed diets with lower levels of antifungal agents).

The CR increases greatly in later instars compared to younger individuals (Valles et al. 1996,

Trichilo & Mack 1989). This is mainly due to their limitation in regard to small size (less strength of mandibular muscles and narrower madibular gape), as insects have to molt and grow to have larger mouth parts to take in food and larger gut volume to hold a greater volume of food they have ingested. The relationship between size of larvae and the gape of their mouthparts is discussed by Cohen (2003).

It is possible that feeding behavior itself was not suppressed by the existence of antifungal agents. Instead, the antifungal agents may have direct detrimental effects on absorption or metabolism as they decreased digestion and/or absorption and subsequently,

ECI and ECD. Thus, the slow growth rate would affect CR indirectly by holding insects in earlier instars and smaller sizes.

Few studies have been done to investigate the palatability of antifungal agents on insects. Benedict et al (2009) found that methyl paraben in diet was avoided by Anopheles gambiae but not A. arabiensis. The Benedict et al. 2009 study is especially interesting because it shows that even closely related (congeneric, in this case) insects can have sharp differences in their response to additives in their diet. Our choice tests showed that different antifungal agents have effects on insect feeding behavior, and the effects may vary among species. Methyl paraben is a feeding deterrent for both V. cardui and H. virescens. Potassium sorbate reduced feeding response in H. virescens, but appeared to be reducing diet palatability in V. cardui. Sodium propionate showed neutral effect on both species. Another factor that must be considered is that the counterions in sorbate and propionate compounds

(potassium and sodium) in antifungal agents can affect insect feeding behavior negatively or positively. Another factor that could modify palatability of diet antifungal additives pH changes influenced by acid forms of compounds such as sorbic acid/sorbate salt, propionic acid/propionate salt, and benzoic acid/benzoate salt (Gothilf & Beck 1967, Dadd et al. 1982,

Cohen 2003). The influences of widely-used antifungal agents on feeding behavior are worth further study on case–by-case basis.

Increasing feeding and digestion under high antifungal agent concentrations

Interestingly, although the consumption rate (CR) dropped significantly as antifungal agents concentrations increased, the relative consumption rate (RCR) was actually increased.

While gross consumption necessarily increases as larvae get larger, RCR often declines with increasing insect age and the accompanying increase in body mass. Previous studies showed

that RCR is highest in early instars, then declines in later instars (Blake & Wagner 1984,

Valles et al. 1996). Since larvae in our high antifungal agent trials were usually smaller and stunted at earlier instars, it is not surprising that they had higher RCR values. Besides the age factor, a high RCR can compensate for a low ECI; the larvae may feed relatively more when they are not absorbing the food efficiently enough. Blake and Wagner (1984) found that feeding insects with foliage resulted in higher RCR, while artificial diet resulted in higher

ECI. Since high concentrations of antifungal agents reduced both ECI and ECD, the insects may offset that negative effect by feeding more intensively. In other words, the rate of feeding and amounts consumed by insects reared on high levels of antifungal agents is probably enhanced instead of suppressed.

Similar to RCR, AD of V. cardui and H. virescens is also increased with higher levels of antifungal agents. Approximate digestibility is another nutritional index that will decrease as the insect grows and develops to later instars. Waldbauer (1968) reviewed and calculated data from some previous studies (Hiratsuka 1920, Evans 1939, Davey 1954, Husain et al.

1946), and concluded that AD declined from first to last instars. Some later studies further proved the negative relationship between AD and age (Bailey 1976, Khalsa 1979, Barah

1989, Singh et al. 1991). Bailey (1976) and Barah et al. (1989) stated that the decreased AD was due to selective feeding in younger larvae; older larvae will ingest not only tender part of the leaves but also indigestible crude fibers, which decrease the overall digestibility. Insects in our study reared on artificial diet also showed declined AD with well developed individuals reared on low antifungal agents, so it was very likely affected directly by the presence of antifungal agents. However, Gordon (1959) also suggested that when an animal

doubled its weight and volume, the surface area of its digestive tract increased by only a factor of 1.8, so the digestibility would decrease as the animal grows. Therefore, the difference among insect ages in our study is again confounding.

The reason why high levels of antifungal agents can enhance AD is not clear. It is possible that high levels of antifungal agents may have detrimental effects on insect gut movement, for example, slowing down gut peristalsis, and consequently enhance digestibility by keeping food in gut for a longer time. This relationship between gut movement and digestion and absorption efficiencies is discussed by Pechan et al 2002 and by Cohen 2003).

However, it does not necessarily benefit the insect’s growth and development. While the available nutrient components in digesting food are supposed to decrease through time, it may be a more energetically beneficial strategy to eliminate partially digested food and focus on newly ingested food instead of digesting the eaten food thoroughly. To test this hypothesis, feeding the insects with a mixture of diet and dye, for example, bromophenol blue, could be useful in future experiments; the speed of food moving through gut can be visualized by observing the appearance of the colored frass (Krishna & Singh 1968).

Reduced ECI & ECD under high antifungal agents concentrations

In previous studies discussed above, an increasing CR and decreasing RCR and AD are related to larval ages and body mass. However, the relationship between ECI, ECD and age are complex. Waldbauer (1968) stated that there is an obvious tendency for the ECI to decrease with age based on reviewing a series of studies (Hiratsuka 1920, Evans 1939,

McGinnis & Kasting 1959, Johnson 1960, Friend et al. 1965), and suggested that the decline

of AD with age was responsible for the decline of ECI. Conversely, many other studies showed an increasing of ECI with age (Bailey 1976, Khalsaet al. 1979, Blake & Wagner

1984, Barah 1989). The increasing of ECI through age is possibly related to the factors that also lead to decline of RCR. Schroeder (1976) found that assimilation was enhanced by prolonged food deprivation. Scriber (1979) also showed that RCR was negatively correlated to ECI due to a “power and efficiency” trade-off, i.e. insects eat less when the assimilation is more efficient and vice versa. From those previous studies, we can suggest that ECI is closely correlated to both AD and RCR. Insects will absorb better if they digest well, so a low AD will decrease ECI; and a decreased RCR will make a higher ECI value. Since both

AD and ECI decrease with age, their effects on ECI would be more complex. Thus, later instars could have an increased, decreased or possibly fluctuating ECI values.

In this study, the ECI is significantly reduced in high antifungal agent trials. The increased AD is not responsible for the decline of ECI, since AD is thought to be positively correlated with ECI (Slansky & Feeny, 1977). Instead, the increased RCR in high antifungal agent trials is possibly triggered by declining ECI. The absorption of digested food is negatively influenced by a high level of antifungal agent, although the food may get thoroughly digested in the gut. Cohen 2003 discussed extensively the factors that influence rates and efficiencies of digestion and absorption. Among the factors that influence absorption are 1) exposure of the digested food to sites of absorption, especially receptors in the gut lining 2) possible interference at absorption sites by competing molecules 3) presence and concentration of diet components that facilitate absorption, and 4) the organization and characteristics of the digestive system, especially the peritrophic matrix (PM). At the onset

of the experiments discussed here, the hypothesis that antifungal agents—especially ones at high concentrations—might impact nutritional indices at one of the above levels of digestive system organization. It was hypothesized that a factor such as modification of the rate of peristalsis by an antifungal agent or influence by the agent on the characteristics of the PM

(such as thickness, number of layers, or possible damage to the PM’s integrity) could all impart a downstream effect on the digestion and absorption efficiency with respect to diet components. This issue was discussed by Pechan et al. 2002 regarding PM characteristics and influence on nutritional indices and by Cohen 2003 regarding the comprehensive characters mentioned above.

If the rate of peristalsis in the midgut was decreased by the high concentrations of antifungal agents, this could account for the increase in AD. Thus, food stayed in the gut for a longer time and was digested more thoroughly due to the increase in gut residence period.

At the same time, there would be a decrease in ECI because the reduction in gut movements, which, according to Cohen 2003 involves a churning action whose reduction would lead to a reduced amount of absorption. Another possible reason is that the activity of nutrient receptors in the gut is suppressed by antifungal agents; and since AD is enhanced, the activity of digestive enzymes is less likely suppressed by antifungal agents. This study points to the possible mechanism of direct effects of antifungal agents on gut receptors/absorption sites and would merit further study in future research.

Few studies have focused on the change of ECD with age. ECD is usually inversely related with AD and increases with advancing age and developmental progress (Khalsa et al.

1979, Barah et al. 1989, Singh et al 1991, Reese & Beck 1978). As more food is assimilated,

a smaller proportion of food is converted into biomass. ECD reflects metabolic efficiency, and can be reduced by lowered efficiency of conversion of digested food or enhanced metabolic cost. Metabolic efficiency can be reduced by antimetabolites, which may lower the efficiency of biochemical reactions by binding with the active sites of enzymes (Woolley

1959). Considerable work has been done to study antimetabolites in insects. For example,

Perry and Miller (1965) studied the effects of antimetabolites on growth and metamorphosis of housefly larvae. Shyamala and Bhat (1958) used antimetabolites to study the nutritional requirements of silkworms. Blaustein and Schneiderman (1960) did a brief survey of the potential antimetabolites on the development of giant silkmoth. However, the literature review for this study revealed no documentation of methyl paraben, potassium sorbate, or sodium propionate being documented as antimetabolites. Phytophagous insects are evolutionarily adapted to feeding on allelochemical-rich host plants, and several phytophagous species have been shown to develop effective detoxification enzyme systems

(Lindroth et al. 1991). High concentrations of antifungal agents may trigger detoxifying processes in the gut and bring extra metabolic cost, thus decreasing the net metabolic efficiency.

Another kind of metabolic cost can be caused by a damaged peritrophic complex. The peritrophic complex is a very important membrane structure in the insect midgut, which holds the ingested food, protects gut cells from physical abrasion, and prevents the invasion of pathogens and parasites. Another function of the peritrophic membrane is to conserve digestive enzymes; for example, Bolognesi et al. (2008) found that damaging the peritrophic membrane in Spodoptera frugiperda resulted in a decline in ECD, and suggested that it was

due to failure in enzyme recycling. Chang et al. (2000) and Pechan et al. (2002) showed that

ECI and ECD of fall armyworm were reduced by resistant corn plant tissue containing a 33- kDa cysteine protease, which damages the peritrophic membrane. In our study, if a high level of antifungal agents has deleterious effect on the peritrophic membrane, then the metabolic cost in enzyme production and secretion will also reduce ECD.

Conclusion: the role of antifungal agents in insect nutritional ecology

In summary, a high level of the three antifungal agents (methyl paraben, potassium sorbate and sodium propionate) tested in the study showed a comprehensive impact on feeding, digestion, absorption and metabolism. Although the nutritional index values are correlated with insect size, the lag in development of larvae reared under highest antifungal agent level do not explain the observed changes in nutritional indices. We suggest that antifungal agents affect insect physiology in different levels of feeding dynamics and physiology. Antifungal agents may slow down gut peristalsis and increase the extent of digestion. It also seems plausible that ECI is reduced possibly by slowed peristalsis and interference with nutrient receptors. ECD is also reduced, possibly by increases in metabolic cost of the detoxification of antifungal agents and possibly toxin-induced increases in metabolic rate. As a response to decreased assimilation, more food is ingested, which increases RCR. Also although insects are feeding more and digesting more thoroughly, thus compensating for the loss in absorption and metabolism, their growth and development are still negatively influenced by high levels of antifungal agents overall. Further work on investigating whether antifungal agents influence gut movement, detecting nutrient receptors,

and identifying digestive and detoxifying enzyme activity are needed to reveal the specific mechanisms and draw more precise interpretations. It would also be valuable to know if the peritrophic membrane is influenced by antifungal agents.

The subject of this series of studies is the relationship between insects and antifungal agents that are commonly used in diets. Because we understand the tremendous importance of the relationships between insects and microbes, a brief discussion of insect/microbe relationships especially those pertaining to gut microbes is below. Dillon & Dillin 2004 reviewed the large body of information about the contributions from gut microbes including improving the ability of some insects to live on suboptimal diets, improving digestive efficiency, and providing of essential nutrients. Many widely used antifungal agents, including the three agents in our study, have relatively general modes of action, and can inhibit a wide spectrum of fungi and bacteria. We do not know the exact roles gut microbes in V. cardui and H. virescens play when insects feed on artificial diet. Our wheat germ based artificial diet with mineral and vitamin additives should be able to provide enough essential nutrients, but it does not necessarily mean that the role of gut bacteria is no longer significant.

Thus, the influences of antifungal agents on gut microflora still need further investigation.

Some research has been done to investigate the impacts of food to insect gut micro organisms. For example, Priya et al. (2012) found that the gut bacteria diversity of

Helicoverpa armigera varied greatly among insects collected from different crops. The nutritional value of diet can influence the performance of insect gut bacteria and even secondary symbionts (Chandler et al. 2008). The production of gut bacteria can be changed by food (Kane and Breznak 1991); even the role of gut bacteria can be altered by food. Vries

et al. (2004) found that Erwinia bacteria either benefited or parasitized their thrips hosts depending on the food provided. Tran and Marrone’s research (1988) showed that the total bacteria population in southern corn rootworms, Diabrotica undecimpunctata howardi gut was 100 times lower when fed on artificial diet compared to the ones fed on host plants, and they suggested that the dramatic change might due to the antibiotic agents in their diet. But even by using a combination of heat (≈ 80 °C) and antimicrobials, insect diet is not free from microbes (Sikorowski & Lawrence 1991). Since many insects can obtain their gut microbes from food (Sikorowski & Lawrence 1994), the gut microbes in lab colonies and wild populations could be dramatically different by feeding on food with different microbe communities. Many insect colonies have been kept in lab for generations and decades, for example, the H. virescens colony used in this study has been kept in lab conditions for 25 years, so it is possible that the colony has developed a unique gut microbe flora. The gut microbes which may be involved in our study contain so much complexity and uncertainty; based on our experimental methods, that possible changes in microbial flora were not possible to measure under our experimental conditions. However, the fact that the microorganisms in insect guts can be changed by diet should be emphasized, and it is worthy of study in future research.

Comparisons among the effects of different antifungal agents

Previous studies on detrimental effects of antifungal agents usually focused on measuring biological parameters such as biomass, mortality, and fecundity. In our study, we tried to evaluate the potential toxicity of different antifungal agents via nutritional indices.

Since the concentration of only one antifungal agent was varied each time and the other agents maintained at basic level of 1,570 ppm, both 5,000 and 10,000 ppm trials can be considered as having an extra amount of a given antifungal additive in basic diet and used in comparison. We chose the 5,000 ppm treatments to compare the effects of the 3 antifungal agents, since the results of 10,000 ppm methyl paraben treatments are questionable due to problems in solubility of very high concentrations of this antifungal agent. Although the

10,000 ppm methyl paraben treatment showed significant changes in nutritional indices, the results vary dramatically among individuals. Many larvae remained extremely small through the end of the trial, while some individuals managed to keep a normal growth rate. Compared to the high solubility of potassium sorbate and sodium propionate, methyl paraben has solubility much lower than 10,000ppm in water under room temperature (Merck index). With an 80°C water bath and constant agitation, the 10,000 ppm of methyl paraben can dissolve completely in hot agar/water solution. Delivering methyl paraben into water via ethanol is a more efficient way to dissolve methyl paraben thoroughly in hot water. However, in both methods methyl paraben crystals will form when the diet cools down. Even in the same batch of cups, the crystals can form differently among cups. Small crystals formed densely on the diet surface which deterred the larvae from feeding in some cups, while in other cups methyl paraben formed large clusters or aggregations, which created “low methyl paraben” regions for larvae to consume. In fact, selective feeding behavior was observed in diet with large methyl paraben crystals: there were “crystal clumps” dispersed randomly in diet, and there were signs of insects chewing around the crystals, making the crystals higher than the diet surface around them (Figure 15). A concentration as high as 10,000 ppm of methyl paraben

would never be used in usual diet formulae, but it called our attention to issues of solubility of certain other diet ingredients, including many acid forms of antifungal agents and Wesson salt mixture. Homogeneous artificial diet solution may not result in homogeneous solid diet, and heterogeneous diet may cause malnutrition and microbial colonization due to the lack of nutrients and antifungal agents or non-homogeneous distributions of diet components. Thus, this is an issue which needs to be considered carefully in diet formulation and modification in the insect rearing process.

Our study showed that in addition to the conventionally-measured factors (such as weight, fecundity, and survival); nutritional indices can be used in measuring the deleterious effects of agents. Generally speaking, an antifungal agent with more detrimental effects will cause higher RCR and AD, and lower CR, ECI and ECD compared to less harmful chemicals.

By comparing nutritional indices of insects reared on 5,000 ppm of each antifungal agent in diet, some differences were revealed among different agents. V. cardui reared on a high level of methyl paraben had lower amounts of frass, less weight gain, reduced CR, enhanced RCR and reduced ECI comparing to the other two antifungal compounds. Thus it would appear that methyl paraben is a harsher chemical than the other two antifungal agents for V. cardui.

Similar results were reported by Singh and House 1970 and Alverson and Cohen 2001, who found methyl paraben to be more toxic than several other antimicrobial agents. However, the value of methyl paraben is still considerable in terms of the stability of this antifungal agent and the relatively small amounts of this preservative required controlling mold.

In contrast to the findings on V. cardui, the three antifungal agents have similar effects to one-another in the tests with H. virescens. This may be caused by differences

among species or strains. Another possible explanation for the lack of significance the nutritional index parameters measured in the H. virescens trials is the relatively smaller sample size. Not a single cup of V. cardui was infested by mold during the whole process; however, the sample size of H. virescens was reduced due to mold infestation in all but the

10,000 ppm trials. Mold can be brought into the rearing room with H. virescens eggs, and the set up of H. virescens may facilitate mold colonization. Since H. virescens were assigned to each cup right after hatching, no holes were made on the lids to limit larval escape; thus the increased moisture inside the rearing cups facilitated mold growth. This point, about the strong relationship between high water activity and mold is discussed extensively by Cohen

2003.

As mentioned in the Introduction, the effect of anti-fungal agents varies dramatically with pH. Many anti-fungal chemicals are most effective at lower pH, and the effective concentrations must increase when pH is elevated. On the other hand, most insects do not do well with diets whose pH is below a species-specific threshold. The pH of YC diet used in the experiment is about 5.7~5.9. The anti-fungal agents used in YC diet are mostly the salt forms instead of acid forms, since salt forms are easier to dissolve in water. Consequently, it would be advantageous in terms of feeding behavior and microbial management to reduce the pH of the YC Diet for both V. cardui and H. virescens neonates, so the use of citric acid in YC diet to enhance mold suppression could be considered in future usage.

In summary, our analysis of nutritional indices and choice tests showed that effects of different antifungal agents vary between the two insect species included in this study.

Matching antifungal agents to species in a wide variety of insects, especially in terms of

effects of these agents on feeding behavior and growth can lead to substantial improvements in artificial diet-based rearing.

Frass management: a behavior influenced by high antifungal agent concentrations

Besides feeding rate, another behavioral change in V. cardui was also observed during these experiments. Healthy V. cardui individuals were observed to bind clusters of fecal pellets with silk and keep them away from the diet. We interpret this behavior as “frass management” (Figure 16). With a higher amount of antifungal agents in diet, less silk production and less frass management were observed (Figure 17). One possible explanation is that the limited growth of larvae reared in high antifungal agent concentrations reduced silk production, so the larvae did not have enough silk to manage their frass. On the other hand, the larvae reared on low antifungal agent diet usually settled down to feed at one spot and deposited their frass at one place. Thus, it is also possible that the larvae reared on high antifungal agent diet were not satisfied with the food and tried to escape instead of settling down to feed and deposit frass at one place. The functions of silk production in Lepidoptera are often related to facilitating movement, constructing nests, making cocoons, defense, and protection. Although Cohen noted the phenomenon of frass binding in rearing containers with cactus moth (Cactoblastis cactorum Berg (Lepidoptera: Pyralidae) (personal communication), this phenomenon has not been described in the literature, to our knowledge.

Recently, Kanazawa et al. (2011) reported the new role of silk in a social spider mite species

Stigmaeopsis longus, which displayed nest sanitation behavior with silk through the coordinated deposition of fecal materials. In our study, very similar behavior was observed in

individual lepidopteran larvae. Caterpillars may manage fecal materials by physically separating frass from food with silk, which can prevent or limit microbial contamination.

Beside its physical character, silks also have distinct chemical properties, including kairomonal functions, various silk peptides and proteins (Gauthier et al. 2004), immune function (Korayem et al. 2007) and antimicrobial activity (Pandiarajan et al. 2011). It is also plausible that the silk produced by V. cardui to manage frass has antimicrobial properties which can further prevent the contamination of frass other than physically separation function. On the other hand, no fecal management behavior was observed in H. virescens.

Our observations indicate that V. cardui is a good subject for further study of the sanitation function in lepidopteran silk production. This finding demonstrates that insect behavior can be altered by diet components in artificial rearing systems, and indicates the possibility of using behavioral parameters such as silk production to evaluate diet and insect rearing systems and support quality control in mass rearing processes.

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Table 1. The ingredients of YC diet. Dry mixture (trituration in a roller mill) and agar/antifungal agents solution are made separately. The diets in these experiments were combined by adding 33g of the dry mix to 200g of the boiled water with dissolved agar and the antifungal mixture, and then thoroughly blended with a variable speed hand mixer.

Ingredient Weight (g) Mixing Final Versions of Diets Wheat germ 154 Soy protein 70 Sucrose 62 Torula yeast 50 Rice (preparation) 25 Cannellini beans 25 Vanderzant Vitamins 10 Wesson salts 10 L-ascorbic acid 10 Cholesterol 5

Total dry mix (g) 421 33 (out of 421)

Agar 16 Methyl paraben 1.5 Potassium sorbate 1.5 Sodium propionate 1.5 Water 800

Water/agar/antifungal 820.5 200 (out of 820.5) agents mix (g)

Total Weight (g) 233

Table 2. Effects of antifungal agents on V. cardui feeding and growth.

Antifungal Conce Frass (mg) Weight gain CR RCR AD (%) ECI (%) ECD (%) agent ntratio (mg) (mg/day) (day-1) n, ppm Methyl 1000 231.5±10.57a 141.0±5.81a 80.1±2.66a 0.6±0.02b 60.0±0.91a 24.6±0.57a 42.0±1.34a paraben 5000 151.7±10.09b 92.9±5.93b 56.8±2.07b 0.8±0.05a 64.0±1.56a 21.8±0.98ab 37.3±2.41a

10000 142.0±13.80b 83.4±7.57b 51.8±2.96b 0.8±0.07a 64.9±1.91a 20.9±1.11b 35.5±2.64a

Potassium 1000 223.0±11.16a 137.7±6.26a 78.7±3.07a 0.6±0.02b 60.8±0.71b 24.4±0.45a 40.9±1.03a sorbate 5000 184.3±8.31b 116.7±4.26b 66.8±2.31b 0.6±0.01b 61.3±0.71ab 24.9±0.53a 41.4±1.22a

10000 132.8±6.00c 78.9±3.70c 50.6±1.54c 0.7±0.03a 63.5±0.89a 21.6±0.63b 34.9±1.32b

Sodium 1000 218.8±11.90a 132.2±6.88a 77.9±3.20a 0.7±0.05b 61.5±1.10b 23.2±0.78a 39.5±1.77a propionate 5000 186.3±10.58a 112.5±5.93a 69.1±2.93a 0.7±0.03b 62.8±0.85b 22.7±0.58a 37.0±1.26a

10000 126.0±8.91b 75.3±5.23b 54.4±2.47 b 1.0±0.10a 68.9±1.06a 18.6±0.84b 28.2±1.46b

Mean ± SEM followed by the same letter within columns are not significantly different (6 replications, P<0.05, Tukey HSD test [JMP 10]).

Table 3. Effects of antifungal agents on H. virescens feeding and growth.

Antifungal Concent Frass (mg) Weight CR RCR (day-1) AD (%) ECI (%) ECD (%) agent ration, gain (mg) (mg/day) ppm Methyl 1000 93.5±6.87a 56.1±4.17a 24.3±1.11a 0.5±0.03b 67.4±1.30b 19.7±0.85a 30.5±1.67a paraben 5000 91.5±6.41a 53.5±3.65a 24.6±1.08a 0.6±0.03b 68.2±1.24b 18.5±0.69a 28.3±1.38a

10000 62.5±5.12b 34.8±2.80b 19.5±0.89b 0.7±0.05a 72.6±1.32a 15.0±0.71b 21.6±1.29b

Potassium 1000 99.7±8.67a 58.7±4.61a 25.0±1.42a 0.5±0.04b 66.0±1.47b 20.2±0.88a 31.8±1.74a sorbate 5000 98.8±9.17a 52.3±4.51a 24.1±1.48a 0.5±0.03b 65.2±1.51b 18.6±0.82a 29.7±1.72a

10000 51.1±3.87b 28.6±2.29b 16.8±0.67b 0.7±0.05a 73.9±1.12a 14.4±0.69b 20.2±1.18b

Sodium 1000 100.5±8.13a 57.8±3.86a 25.8±1.41a 0.5±0.04b 66.4±1.50b 19.8±0.72a 30.9±1.41a propionate 5000 95.7±6.79a 56.7±3.58a 23.5±1.10a 0.5±0.03b 65.6±1.22b 20.8±0.75a 32.8±1.44a 10000 41.0±3.26b 27.4±2.20b 15.2±0.63b 0.8±0.07a 76.9±1.15a 15.3±0.84b 20.8±1.38b

Mean ± SEM followed by the same letter within columns are not significantly different (6 replications, P<0.05, Tukey HSD test [JMP 10]).

Table 4. Number of V. cardui larvae fed on diet cylinders containing different concentrations of antifungal agents.

Concentration of 0 1000 1570 5000 10000 antifungal agent (ppm) Larvae choice on 4.5±0.51a 3.4±0.48ab 4.5±0.52a 1.3±0.23b 0.9±0.20b methyl paraben

Larvae choice on 1.9±0.36b 2.7±0.48ab 3.6±0.38a 3.8±0.47a 3.2±0.36a potassium sorbate

Larvae choice on 3.4±0.47a 3.5±0.29a 3.4±0.29a 3.5±0.30a 3.1±0.36a sodium propionate

Mean ± SEM followed by the same letter within rows are not significantly different (P<0.05, Friedman test [JMP 10]).

Table 5. Number of H. virescens larvae fed on diet cylinders containing different concentrations of antifungal agents.

Concentration of antifungal agent 0 1000 1570 5000 10000 (ppm) Larvae choice on methyl paraben 3.8±0.43a 3.3±0.45a 3.0±0.32a 2.7±0.50ab 1.2±0.31b

Larvae choice on potassium 3.2±0.51ab 3.8±0.38a 3.9±0.39a 2.8±0.35ab 1.6±0.29b sorbate

Larvae choice on sodium 3.2±0.40a 2.8±0.30a 2.2±0.37a 2.5±0.37a 2.2±0.37a propionate

Mean ± SEM followed by the same letter within rows are not significantly different (P<0.05, Friedman test [JMP 10]).

Table 6. Number of larvae fed on different diet cylinders with different pH.

pH 5.5 5.1 4.8 4.5 V. cardui 4.4±0.6a 3.6±0.3a 4.3±0.5a 3.5±0.6a

H. virescens 4.2±0.4a 3.6±0.3a 3.9±0.2a 3.6±0.4a

Mean ± SEM followed by the same letter within rows are not significantly different (P<0.05, Friedman test [JMP 10]).

Figure 1. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on frass production. Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 2. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on insect weight gain. Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 3. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on consumption rate (CR). Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 4. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on relative consumption rate (RCR). Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 5. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on approximate digestibility (AD). Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 6. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on the efficiency of conversion of ingested food to biomass (ECI). Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 7. Effects of different concentrations of 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 1, 2, 3=1000, 5000, 10000ppm) on the efficiency of conversion of digested food to biomass (ECD). Different letters indicate significant differences across antifungal doses (Tukey’s test, P<0.05).

Figure 8. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on frass production. Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 9. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on insect weight gain. Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 10. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on consumption rate (CR). Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 11. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on relative consumption rate (RCR). Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 12. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on approximate digestibility (AD). Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 13. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on the efficiency of conversion of ingested food to biomass (ECI). Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 14. Comparison among the 3 antifungal agents (MP=methyl paraben, KS=potassium sorbate, NP=sodium propionate; 2=5000 ppm) on the efficiency of conversion of digested food to biomass (ECD). Different letters indicate significant differences (Tukey’s test, P<0.05).

Figure 15. Freeze dried diet sample of 10,000ppm methyl paraben treatment, showing the presence of white methyl paraben crystals which insects avoided ingesting.

Figure 16. “Frass management” of a V. cardui larva, showing that the frass was attached with silk and kept away from diet.

Figure 17. A comparison of V. cardui frass management behavior between 1,000 and 10,000ppm potassium sorbate (KS1 & KS3) treatments. The larvae fed on lower level of antifungal agents not only grew faster, but also produced more silk and managed their frass better than larvae fed on higher level of antifungal agents.