INTRODUCTION AND LITERATURE REVIEW
Diabrotica Chevrolat (Coleoptera: Chrysomelidae: Galerucinae) is a largely
Neotropical genus (Wilcox 1972) that includes some of the most destructive insect pests
In North America, the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, and the northern corn rootworm (NCR), Diabrotica barberi Smith & Lawrence, pose significant threats to maize (Zea mays L.) These pests are responsible for substantial economic losses, with estimated annual impacts ranging from $1 to $2 billion in the United States due to yield reduction and control expenses.
(Mitchell 2011) Western corn rootworm was an invasive pest of maize in Europe since 1992 (Bača 1994) and now this pest was introduced to 29 European countries (CABI 2017) In
Europe, the damage costs caused by western corn rootworm have been estimated to be €472 million annually (Wesseler and Fall 2010)
Both western corn rootworm and northern corn rootworm have a univoltine life cycle with having one generation per year and overwinter as eggs (Ball 1957, Branson and Krysan
Eggs are laid in the soil during an obligate diapause from late July to September and remain there until hatching the following late May and early June Pupation takes place in the soil, with adult emergence occurring from late June to early July.
(Murphy et al 2010) to late August (Hughson and Spencer 2015)
The western corn rootworm thrives in temperatures ranging from 15°C to 31.5°C, with an optimal development range of 21°C to 30°C (Jackson and Elliott, 1988) Male western corn rootworms generally develop faster than females, taking approximately 26.3 days to reach adulthood at 24°C, compared to 28.9 days for females The development times for male larvae are 4.8 days for the first instar, 4.3 days for the second instar, and 9.4 days for the third instar, while females take 5.3, 4.9, and 10.4 days, respectively The head capsule widths for the first, second, and third instar larvae range from 200-260 μm, 300-400 μm, and 440-560 μm (Hammack et al., 2003) The pupal stages last 7.8 days for males and 8.4 days for females (Jackson and Elliott, 1988).
10 days ranges from 0.18 mg (Oyediran et al 2004) to 0.24 mg (Clark and Hibbard 2004) when western corn rootworm reared on maize under greenhouse conditions while larval dry weight to
20 days averages between 1.05 mg (Oyediran et al 2004) and 1.68 mg (Clark and Hibbard
2004) When larvae fed on maize, adult head capsule width is 1.23 mm while adult dry weight is 1.81 mg (Oyediran et al 2004)
Corn rootworm larvae primarily feed on maize roots, with a limited ability to survive on certain grasses This larval stage is the main contributor to the damage caused by corn rootworms, leading to significant yield reductions Their feeding can disrupt the flow of water and nutrients, increase susceptibility to pathogens, and compromise the structural stability of plants, which may result in lodging that complicates harvesting Additionally, adult corn rootworms feed on the leaves, pollen, and silks of maize, further impacting crop health and productivity.
High-density adult populations can lead to yield reductions by clipping ear silks before anthesis, ultimately decreasing pollination and impacting crop production.
1992) Western corn rootworms are protandrous wherein males typically emerge 5 to 6 days before females in the field (Branson 1987, Quiring and Timmins 1990, Hughson and Spencer
The development of resistance of corn rootworms
The management of western and northern corn rootworms has become increasingly challenging due to their adaptability and resistance to various control methods Both species have independently evolved biotypes that can withstand crop rotation practices, particularly when maize is alternated with non-host crops like soybean.
Glycine max (L.) Merr., commonly known as soybean, serves as a host for western corn rootworm females that lay their eggs in these fields The gut microbiota of this pest may enhance its resistance to soybean defense compounds Meanwhile, northern corn rootworm exhibits an 'extended diapause,' allowing its eggs to hatch more than a winter after being laid, which helps them adapt to local crop rotations This adaptation has become problematic in several Midwest states, including Minnesota, South Dakota, and Iowa, as northern corn rootworm larvae perish without host plants after hatching Both western and northern corn rootworms have developed resistance to various chemical insecticides, including organochlorides, organophosphates, carbamates, and pyrethroids, posing significant challenges for pest management The latest control strategies involve transgenic maize hybrids that express insecticidal crystalline toxins to combat these resistant pests.
Recent studies have shown that the effectiveness of Bacillus thuringiensis (Bt) Berliner against western corn rootworm has diminished due to the pest's developing resistance (Gassmann et al 2011, Zukoff et al 2016, Ludwick et al 2017) As Bt corn is designed to combat both western and northern corn rootworms, the observed rise in northern corn rootworm populations in fields with Bt traits raises concerns that this species may also adapt to Bt corn and other pest management approaches.
To address the emergence of resistance to Bt products, the United States Environmental Protection Agency (EPA) mandates insect resistance management plans for corn rootworms that focus on monitoring resistance development to various Bt proteins Currently, four Bt proteins, including Cry3Bb1, are being utilized in these efforts.
The EPA has approved Cry34/35Ab1, mCry3A, and eCry3.1Ab for commercial use, making them essential for agricultural pest management To monitor the development of resistance in corn rootworm populations, toxicity bioassays using artificial diets are crucial (Siegfried et al 2005) Additionally, on-plant assays complement these diet-toxicity assays, enhancing resistance monitoring efforts (Meihls et al 2008, Nowatzki et al 2008, Gassmann et al 2011, Zukoff et al 2016).
Artificial diets for rearing insects offer significant advantages for research, education, and as nutritional supplements for other organisms These diets typically require less labor and space (Beck and Stauffer 1950) and reduce the time needed for host plant rearing (Vanderzant et al 1956) Additionally, they provide logistical benefits such as ease of transport, simplified preparation and presentation, minimized storage requirements, and lower production costs Ultimately, the objective of using an artificial diet should be to cultivate "normal" insect development.
5 insects that are behaviorally and physiologically similar to wide-type insects (Lapointe et al 2010b, Cohen 2015)
Diet development for the southern corn rootworm, Diabrotica undecimpunctata howardi, began with modifications to existing formulations aimed at other pests This generalist species feeds on over 100 plant species, has a short life cycle, and lacks a diapausing stage Sutter et al (1971) adapted the diet originally designed for pink bollworm, Pectinophora gossypeilla, after assessing the growth of southern corn rootworm on various established diets used for other phytophagous insects, including the European corn borer, Ostrinia nubilalis, and the tobacco hornworm, Manduca sexta.
The southern corn rootworm's initial diet formulation was adapted from the pink bollworm diet by altering the agar source and reducing its quantity, resulting in an agar-based diet with wheat germ and casein that supported development from egg to adult without affecting morphology, egg production, or viability, although development was slower compared to those reared on maize roots Subsequent improvements by Rose and McCabe involved modifying ingredient concentrations and replacing linseed oil with corn oil, leading to a diet that enhanced larval development over the original Sutter et al diet, yet still did not achieve the developmental rates seen with maize roots.
Marrone et al (1985) developed an improved diet for southern corn rootworm development that parallels to maize by changing concentration of ingredients in the Sutter et al
After six generations of selection, the Sutter et al diet was modified to enhance larval performance by increasing raw linseed oil and sucrose while reducing antimicrobial agents and potassium hydroxide This improvement was based on evaluating the impact of individual diet ingredients, which were varied from 10% to 100% Additional ingredients such as corn meal, zucchini, and various protein sources were tested as alternatives, but changes to the diet, excluding wheat germ, did not enhance larval growth Notably, formalin was identified as detrimental to growth, with its reduction leading to increased bacterial contamination Furthermore, no differences were observed in susceptibility to carbamate insecticides between the diet-adapted southern corn rootworm strain and the maize strain.
The first published diet specifically for western corn rootworm, adapted from a diet for southern corn rootworm, was developed by Pleau et al in 2002 This diet incorporates a variety of ingredient sources, including carbohydrates like wheat germ and corn meal, proteins such as casein and ground lima beans, various sugars including D-sucrose and honey, and oils like corn and peanut oil Additionally, it features sterols, cellulose, plant adjuvants such as lyophilized corn roots and zucchini, along with formalin and potassium.
DIET IMPROVEMENT FOR WESTERN CORN ROOTWORM LARVAE
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the U.S and Europe, posing high control costs Current diet formulations for WCR larvae in toxicity bioassays lack public availability of a diet that mimics the physiological traits of larvae on corn roots We present a novel diet formulation that enhances weight gain, larval development, and survival rates, outperforming the existing public diet Utilizing response surface methods and n-dimensional mixture designs, our formulation achieved a two-fold increase in weight gain and improved survival and molting rates from 93% and 90% to approximately 99% at 11 days This standardized growth medium for WCR larvae will aid in research consistency and regulatory compliance.
The western corn rootworm (WCR), scientifically known as Diabrotica virgifera virgifera LeConte, is the most significant insect pest affecting maize in the United States and parts of Europe The larvae predominantly feed on maize roots, leading to substantial damage and posing a serious threat to maize crops.
Western corn rootworm (WCR) feeds on maize plant silks, potentially leading to significant yield reductions if infestations occur before anthesis Annually, crop losses and efforts to mitigate damage from WCR are estimated to cost around $2 billion Managing WCR remains a persistent challenge for both the industry and corn growers, as this pest has developed resistance to various management strategies, including chemical insecticides and crop rotation practices.
2002, Gray et al 2009), and transgenic maize which expresses insecticidal proteins derived from
Bacillus thuringiensis (Bt) has been widely used for managing Western corn rootworm (WCR), but recent studies indicate that field-evolved resistance to all currently available single-gene Bt proteins has emerged This resistance poses significant challenges for effective pest management strategies.
Research aimed at preventing resistance development necessitates the ability to rear Western Corn Rootworm (WCR) Despite the preference for a standardized rearing method for research and regulatory purposes, one has yet to be established for WCR, leading to the use of both plant and diet rearing systems in bioassays This lack of standardization results in significant variations in life history parameters and toxicity outcomes across different systems, complicating comparisons and limiting the interpretability of results Developing an artificial diet that facilitates the normative development of WCR larvae, akin to those reared on maize roots, would represent a significant advancement towards a standardized bioassay method.
The initial artificial diet for the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was developed to facilitate its rearing, making it a viable alternative to the western corn rootworm (WCR) in research This diet, primarily composed of agar, wheat germ, and casein, successfully supported the growth of SCR from egg to adult stage; however, it led to slower development and lower fecundity compared to those insects raised on natural diets.
Research by Rose and McCabe (1973) and Marrone et al (1985) modified the diet formulation for SCR by increasing raw linseed oil and sucrose while reducing antimicrobial agents and potassium hydroxide These adjustments led to developmental rates for SCR comparable to those of larvae fed on corn roots, but this outcome was only achieved after six generations on the new diet, indicating potential for further formulation enhancements.
Pleau et al (2002) developed the first published diet for rearing Western Corn Rootworm (WCR) larvae, modifying earlier diets created for Southern Corn Rootworm (SCR) (Sutter et al 1971, Marrone et al 1985) Key adjustments, including optimizing pH, eliminating formalin, and incorporating corn root powder, resulted in nearly double the weight of WCR larvae compared to the SCR formulation This diet has been utilized in diet-toxicity bioassays to assess the baseline susceptibility of various WCR populations to the Cry3Bb1 toxin (Siegfried et al 2005) To maximize the diet's effectiveness for researchers, regulatory bodies, and industry, enhancements are necessary to ensure bioassays can accurately differentiate between susceptible and resistant insects, support molting, and be publicly accessible, ultimately mimicking the development of WCR when fed on corn roots (EPA 2014, 2016b, Ludwick and Hibbard 2016).
Efforts to enhance the artificial diet for SCR and WCR focused on measuring biochemical and physiological parameters to evaluate the impact of dietary changes on insect performance Typically, researchers altered one diet component at a time, assessing insect performance after extended feeding periods This conventional approach necessitates large factorial experiments with numerous treatment combinations and simultaneous variable variations, complicating the interpretation of results (Ruohonen and Kettunen 2004).
The one-ingredient-at-a-time (OIAT) approach is insufficient for identifying the optimal blend of ingredients in a mixture, as it confounds the effects of proportion and amount (Niedz and Evens 2016) Variations in any ingredient's proportion simultaneously alter the proportions of all other ingredients To address this issue, Henri Scheffé developed mathematical models in 1958, known as Scheffé models or Scheffé polynomials, specifically for modeling responses to mixtures The advent of response surface modeling (RSM) and advanced software has streamlined these computations (Anderson and Whitcomb 2004, Lapointe et al 2008) RSM, utilizing multivariate geometric design for mixtures, has proven invaluable for enhancing and optimizing insect diets (Lapointe et al 2008, Lapointe et al 2010b, Pascacio-Villafán et al 2014, Cohen 2015).
Recent advancements in insect diet formulations have utilized multi-dimensional mixture designs to pinpoint key ingredients that significantly influence insect development These mixture experiments serve as a specialized form of response surface experimentation, where the ingredients act as input variables and various life history traits are measured as responses By deriving mathematical equations, researchers can predict how different diet components affect insect responses, leading to enhanced formulations Validation experiments are then performed to confirm that these new diets achieve the anticipated performance improvements.
This research aimed to optimize the ingredient composition of the Pleau et al diet by employing multivariate geometric mixture designs and response surface modeling, as outlined by Lapointe et al (2008), to identify and assess the ideal proportions of the components.
25 key components in the diet for improved larval performance (larval survival, development, and weight) while limiting diet contamination
WCR eggs (non-diapausing strain) were obtained from the USDA-ARS Plant
At the Genetics Research Unit in Columbia, Missouri, soil was prepared in a Petri dish using 70 mesh sieved soil and incubated at a constant temperature of 25 °C in darkness Following the hatching of several larvae, remaining eggs were removed from the soil using a method outlined by Pleau et al (2002), which involved washing the soil through a 60 mesh sieve with water The eggs underwent a surface treatment with undiluted Lysol® for 3 minutes, followed by triple rinsing with distilled water Subsequently, the eggs were treated with 10% formalin for another 3 minutes and rinsed three times with distilled water before being pipetted onto coffee filter paper.
Industries, Sheboygan, WI) held inside a 16 oz Solo ® deli cup with a plastic lid (product
#LG8RB-0090 and #DM16R-0090, Solo Cup Company, Lake Forest, IL) containing 5 to 6 vent holes made by a number zero insect pin, and incubated at 25 ° C until hatch
The diet formulation was adapted from Pleau et al (2002), ensuring all glassware and containers were sterilized using approximately 0.5% sodium hypochlorite or exposed to UV light for 5 minutes before use To prepare the solution, distilled water and agar (product #A7002, Sigma-Aldrich) were combined in a 400 ml glass beaker, which was then microwaved for 2 minutes until boiling.
26 poured into a blender (Hamilton Beach, Inc., Model 51101BZ) The agar solution was cooled to
The study utilized various high-quality ingredients including wheat germ (Bio-Serv, product #1661), casein (Bio-Serv, product #1100), cellulose (Bio-Serv, product #3425), and sucrose (MP Biomedicals, product #04821721) Additionally, corn root powder from Monsanto, a specialized salt mix (Bio-Serv, product #F8680), a comprehensive vitamin mix (Sigma-Aldrich, product #V1007), and methyl paraben (Sigma-Aldrich, product #H5501) were incorporated to enhance the nutritional profile and stability of the formulation.