Rootworm Resistance to Environmentally Friendly Farming Practices

Interview conducted by Alessandro PiroliniJul 28 2015

Dr. Joseph L. Spencer, Principal Research Scientist and Research Program Leader in Insect Behavior at the University of Illinois at Urbana-Champaign, speaks to AZoCleantech about how the behavior of corn rootworms has changed in recent years and how this is affecting their resistance to crop rotation techniques.

How long have crop rotation-resistant corn rootworms been around for?

Let me begin by noting that annual crop rotation was viewed as an ideal solution to corn rootworm problems by the early economic entomologists who first dealt with rootworm pests.  In the last quarter of the 19^th Century, Stephen A. Forbes, of the Illinois Natural Survey, studied the biology of the species we now know as the northern corn rootworm (NCR), Diabrotica barberi, and realized that their ecology was tied to the continuous availability of corn.

Historically, female rootworms laid their overwintering eggs almost exclusively in cornfields and when their offspring emerged from the egg the following summer, they had to find corn roots nearby or else they would die.

It was obvious to Forbes and his colleagues that by not growing corn two or more years in a row in the same field (continuous corn), the rootworm lifecycle would be broken and populations would not be able to build up in a field. Growing corn in an annual rotation with a crop that was not a host for rootworm larvae (Crop rotation) was an obvious solution to rootworm problems.

If everyone had followed Forbes’ crop rotation recommendation (Forbes 1883), we might not know rootworms as pests; however, it is not that simple. There are good reasons why farmers would want to grow continuous corn (e.g. to produce grain to feed livestock or to secure maximum profit from the land) rather than annually rotating corn production with that of a non-host plant, like soybean, on which rootworm larvae cannot develop.

Because there was always continuous corn available somewhere, corn rootworms persisted as pests. Where corn was grown in rotation with soybean (or another non-host crop), it was possible to grow corn with little risk of rootworm larval injury.

Rootworm Resistance to Environmentally Friendly Farming Practices

Diabrotica virgifera, the western corn rootworm. The adult stage of the western corn rootworm (shown searching for pollen on corn silk) is the target of ARS' first areawide integrated pest management program for corn. Image credit: Tom Hlavaty / Wikimedia Commons.

Diabrotica virgifera virgifera larvae. Image credit: Scott Bauer, USDA / Wikimedia Commons.

Western corn rootworm (Diabrotica virgifera virgifera LeConte) on corn (Zea mays). Image credit: Whitney Cranshaw, Colorado State University / Wikimedia Commons

The first crack in the armor of crop rotation was noted in the late 1920s and documented in the literature by an Illinois Natural Survey Scientist, John Bigger (Bigger 1932). He noted that short rotations were not effectively controlling the NCR, but that longer rotations were still effective. The mechanism of this resistance was not understood at the time.

More than 30 years later, University of Minnesota entomologist, Dr. Huey C. Chiang, noted that a small proportion of NCR eggs were capable of remaining in diapause for two winters or more (Chiang 1965). Eggs capable of this ‘prolonged diapause’ could remain as eggs in the soil during the year when the rotated field was planted with soybean and hatch after two winters when the field was again planted with corn.

Prolonged diapause may explain what Bigger had observed. In the following years, the proportion of the NCR egg population that underwent prolonged diapause grew, and it was shown that the proportion of NCR eggs capable of prolonged diapause was related to the local intensity of crop rotation (Levine et al. 1992). Where crop rotation was most widely adopted, prolonged diapause was the most problematic.

In the 1980s, reports of larval corn rootworm injury in rotated corn led to surveys of rootworm injury in rotated cornfields across Illinois (Steffey et al. 1992). There was concern that NCR prolonged diapause was becoming a more serious problem. The true extent of rootworm damage was found to be limited but greatest in central and eastern Illinois, regions where the corn and soybean crop rotation predominated and NCR were most common.

The apparent NCR explanation for root injury in rotated corn was challenged by observations of western corn rootworm (WCR), Diabrotica virgifera virgifera injury in rotated corn from a small but expanding area in east-central Illinois (Levine and Oloumi-Sadeghi 1996, Levine et al. 2002).

Subsequent analyses of rootworm eggs and adults collected from affected fields by Dr. Eli Levine of the Illinois Natural History Survey confirmed that the WCR, was the culprit behind the damage (Levine and Oloumi-Sadeghi 1996, Levine et al. 2002). Thus, like their NCR cousins, the WCR had developed resistance to crop rotation; however, this time the mechanism was not intrinsic to the egg, but was due to a change in the egg-laying behavior of the females.

Rather than only laying eggs in cornfields, rotation-resistant WCR females had relaxed egg laying fidelity and deposited their eggs in corn and soybean fields (and other crops growing adjacent to cornfields).

This change in behavior, which began as an isolated problem in a single field in 1987, spread to multiple counties and by 1995 was responsible for up to 50% yield loss in 9 counties in east-central Illinois and 15 counties in west central Indiana (Levine et al. 2002). In the following decade, rotation resistance continued to spread eventually affecting portions of Wisconsin, Michigan, Ohio, Iowa, Missouri and the Canadian Province of Ontario.

So there has been a behavioral shift in the rootworm allowing them to become more resistant to crop rotation?

Yes, rotation resistance is a problem of behavior. Broad adoption of annual crop rotation (ca. 98% of corn acres were rotated in the area of Illinois where the problem first arose (Onstad et al. 2001)) imposed a very strong selection against females who laid eggs only in cornfields.

We know from sampling rotated soybean fields in areas without rotation resistance, that we can always find a few WCR in soybean fields (and other locations outside of corn). Clearly, there was variation in female fidelity to cornfields that was already present in the population when they first entered Illinois in 1964 (reaching east central Illinois in the late 1960s).

Natural selection, working with that variation and the selection pressure imposed by near universal adoption of crop rotation, resulted in evolution of a rotation-resistant population in ca. 20 years (Levine et al. 2002).

A consequence of this change in behavior was a massive daily flux of WCR beetles moving between corn and soybean fields and the presence of high WCR abundance in soybean fields—the average number of WCR adults collected per 100 sweeps through the soybean canopy with an insect net reached >250 beetles in some counties.

In addition to local and field-to-field movement at the height of plant canopy, and higher elevation dispersal flights by a proportion of newly-mated females, vast numbers of airborne WCR were likely drawn into convective storms and carried long distances before they were deposited along with rain. Movement across all of these scales helped to spread the rotation resistance problem (Onstad et al. 1999; Isard et al. 2000, 2004).

Another very evident behavior of rotation-resistant WCR is the occurrence of soybean herbivory. Rotation-resistant WCR beetles in soybean fields will feed on soybean foliage. At times the level of herbivory can skeletonize portions of leaves on most plants across the fields.

This is very curious behavior, since WCR do not gain any significant nutritional benefit from this feeding and would die if forced to feed on soybean tissue for more than a few days. In fact, the anti-herbivore defensive chemicals (proteinase inhibitors) produced by soybean plants inhibit the digestion of soybean tissues by the WCR. Soybean foliage behavior is also observed among the scarce WCR in soybean fields from areas without rotation resistance—it was not a new behavior.

The prevalence of seemingly maladaptive behavior was difficult to explain. However, when we learned that soybean-feeding individuals were more likely to move around and lay eggs than corn-feeding beetles, this induced mobility provided a mechanism for getting egg-laying beetles back into cornfields where they could find nutritious foods that support egg development (Mabry and Spencer 2004; Mabry et al. 2004).

Only later, when we began to examine the biology of rotation-resistance at a physiological and molecular level, would we learn about some special adaptations of the rotation-resistant population.

Western Corn Rootworm (WCR) Beetles in Rotated Corn and Soybean Fields. Video Credit: Entomological Society of America / YouTube

What yield losses can occur as a result of corn rootworms?

Destruction of root tissue by feeding corn rootworm larvae is the most significant impact of a heavy rootworm infestation. For every circlet of roots (i.e. a node of roots) destroyed by larval feeding, a 15% reduction in yield is possible (Tinsley et al. 2013).

Complete destruction of the three primary nodes could reduce yield by 45% (Tinsley et al. 2013). Adults can also impact potential yields if they feed on corn silks during pollination; severe 'silk clipping' can interfere with fertilization leading to fewer kernels per year.

What are the differences between rotation-resistant and susceptible rootworm beetles?

There are quite a few differences between the populations; however, most of the distinguishing characteristics are difficult to easily or quickly assess. This has been a frustration about studying resistant rootworms, without molecular genetics and physiological measurements we cannot quickly distinguish between isolated rotation-resistant and rotation-susceptible individuals.

One of the most obvious differences is the large rotation-resistant WCR populations that are present where there is low landscape diversity and most of the land is either corn or soybean fields. Where there is more crop diversity, especially more continuous corn acreage, the advantage of having low egg-laying fidelity to cornfields is lost since many cornfields will be corn year after year.

Even where WCR are abundant in cornfields, few WCR adults are seen in adjacent soybean fields. According to models, the gene flow between rotation-resistant and rotation-susceptible WCR will be too great to sustain rotation-resistance once rotated corn is grown over less than 80% of the landscape (Onstad et al. 2001).

Rotation-resistant WCR have less fidelity to cornfields and are more mobile in the environment than rotation-susceptible WCR. Unlike their rotation-susceptible relatives, rotation-resistant WCR spend much more time in soybean fields (and in other non-host fields) and move frequently between corn and soybean fields. This is a fundamental difference, but one that is only evident when sampling populations of individuals (Levine et al. 2002).

Because of some biochemical adaptations (i.e. greater expression of Cathepsin L proteinase to counter soybean-produced proteinase inhibitors), rotation-resistant WCR are able to withstand exposure to the soybean plant's anti-herbivore defences longer than rotation-susceptible WCR (Curzi et al. 2012). This advantage is hypothesized to grant them more time for activity in soybean fields before they are affected by soybean antiherbivore defences, creating more opportunities for egg-laying.

The same biochemical adaptation to soybean herbivory also confers a survival advantage on rotation-resistant WCR, they can live longer when forced to exist in a soybean environment. The relative abundance of the microbial community in the WCR gut is different between rotation-resistant and rotation-susceptible WCR beetles (Chu et al. 2013).

There are significant differences in gene expression patterns between rotation-resistant and rotation-susceptible WCR beetles when they are feeding on soybean tissue. When the different populations are feeding on corn tissues, there are not major differences in gene expression (Chu et al. 2014).

How are cathepsin levels in rotation-resistant rootworms related to the difficulty to control their numbers?

Cathepsins are a class of proteinases, they are involved in breakdown of proteins during digestion. Rootworm beetles produce a variety of different cathespins. When a soybean plant is attacked by an herbivore, they produce proteinase inhibitors that interfere with the insects' ability to digest the proteins in the plant tissues.

We discovered that rotation-resistant and susceptible beetles respond differently to the chemical defences of the soybean plant. When fed on soybean leaves, resistant WCR had higher background levels of the proteinase Cathepsin-L and increased their production of Cathepsin-L 3x-4x more so than susceptible WCR.

The rotation-susceptible WCR increased production of a different cathepsin, Cathepsin-B, in response to soybean feeding. However, Cathepsin-B is less effective than Cathepsin-L in the presence of the soybean defense, resulting in reduced survival of susceptible WCR while feeding on soybean. Production of a less vulnerable proteinase, Cathepsin-L, by rotation-resistant WCR buys them more time in soybean fields for activities like egglaying (Curzi et al. 2012).​

Why do you believe rotation-resistance is particularly prevalent in areas dominated by corn and soybeans?

Rotation resistance evolved in WCR because of the intense selection that crop rotation imposes on the population. Historically, there was widespread adoption of crop rotation across the region that became the epicentre of rotation resistance (Onstad et al. 2001). The WCR is a species whose biology is adapted to the presence of corn in the same location year-after-year.

Crop rotation disrupts the year-to-year availability of corn at one location. In a rotated cornfield, WCR females that have perfect egg-laying fidelity to cornfields will lay their eggs where corn will not be planted the following year—all of their offspring will starve and die when they emerge in a soybean field.

Females with less than perfect fidelity to cornfields, that lay even a small portion of their eggs outside of the cornfield (i.e. in soybean fields), have deposited their eggs in fields where there is a high probability that corn will be planted the following year. If the reduced fidelity is inherited, this situation will generate successive generations with increasingly reduced fidelity to cornfields.

The fact that WCR expressed a variety of cathepsins with more or less activity in the presence of the soybean proteinase inhibitors provided an additional avenue for selection to fine tune the relationship.

The study you were involved with helps to deepen the understanding of the complex variables that result in rotation-resistant rootworms. How can the findings of this study be applied to the reduction of rotation resistance in bugs?

In the last several years, our understanding of mechanisms that contribute to rotation resistance has grown tremendously, the Chu et al. (2015) publication is a testament to how much we have learned in just a few years.

I think one of the most startling revelations was the discovery that microbes play an important role in rotation resistance. Before Curzi et al. (2012) and Chu et al. (2013), our view of the WCR interacting with corn and soybean was quite simple—perhaps if we knew we were facing a conspiracy of WCR + microbiota from the beginning we could have reached our conclusions much sooner? The other very important discovery was the variety of differentially regulated genes present in rotation-resistant vs. rotation-susceptible WCR.

Though the focus of Chu et al. (2015) was on gene modules (groups of many genes sharing similar or related functions) that were transcriptionally correlated with the expression of rotation-resistance and not specific genes, the broad functional categories associated with the modules offer tantalizing clues about functions or processes that are related to rotation resistance.

For instance, we found differentially regulated groups of genes involved in detoxification and genes involved in transport of metabolic products, lipids, sterols and drugs in and out of cells.  Some of these genes have been linked to insect resistance to other toxins.

Other modules had differentially regulated genes involved in immune function, this is intriguing given what we know about the role of microbiota in the WCR adaptation to crop rotation. I and my colleagues hope is that this information might be used to develop specific diagnostic tools that would allow the rapid identification of resistant populations.

A new management solution tailored to the specific vulnerabilities and unique gene regulation patterns of the rotation-resistant WCR is not very likely in the short term. The potential root injury consequences of associated rotation resistance have been managed quite effectively for over a decade with Bt corn hybrids. Future generations of rootworm resistant GMO corn hybrids are where insights into the biochemistry of both resistant and susceptible WCR might be applied.

The occurrence of field-evolved resistance to two Bt traits in some rotation-resistant WCR populations in Illinois will present difficult challenges and choice to corn producers facing a very formidable rootworm foe. Ultimately, we must learn to apply our available management tactics (Bt corn hybrids, insecticides and crop rotation) in a more integrated fashion.

Just as Stephen Forbes based his successful crop rotation recommendation on a deep appreciation of rootworm biology, I believe that the sustainability of WCR management tools of the future will benefit from as deep an understanding of the latest in rootworm biology, biochemistry and gene expression.

References

• Bigger, J. H. (1932). Short rotation fails to prevent attack of Diabrotica longicornis Say. Journal of Economic Entomology. 25, 196—199.
• Chiang, H. C. (1965). Survival of northern corn rootworm eggs through one and two winters. Journal of Economic Entomology  58, 470–472.
• Chu, C-C., J.L. Spencer, J.A. Zavala, and M.J. Seufferheld. 2013. Insect-microbiota interactions facilitate resistance to crop rotation in the western corn rootworm.  Proceedings of the National Academy of Sciences, USA 110(29), 11917-11922.  doi: 10.1073/pnas.1301886110
• Chu, C.-C., J.A. Zavala, J.L. Spencer, M.J. Curzi, C.J. Fields, J. Drnevich, and M.J. Seufferheld. In Press. Patterns of differential gene expression in adult rotation-resistant and wild-type western corn rootworm digestive tracts. Evolutionary Applications DOI: 10.1111/eva.12278
• Curzi,  M.J., J.A. Zavala, J.L. Spencer and M.J. Seufferheld. 2012. Abnormally high digestive enzyme activity and gene expression explain the contemporary evolution of a Diabrotica biotype able to feed on soybeans. Ecology and Evolution 2, 2005—2017.
• Forbes, S.A. 1883. The corn root-worm. (Diabrotica longicornis, Say) Order Coleoptera. Family Chrysomelidae. Illinois State Entomologist Annual Report 12, 10-31.
• Isard, S.A., J.L. Spencer, M. A. Nasser and E. Levine. 2000. Aerial movement of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae): Diel periodicity of flight activity in soybean fields. Environmental Entomology 29, 226-234.
• Isard, S.A., J.L. Spencer, T.R. Mabry and E. Levine. 2004. The influence of atmospheric conditions on high elevation flight of western corn rootworm (Coleoptera: Chrysomelidae). Environmental Entomology 33, 350—356.
• Levine, E., Oloumi-Sadeghi, H., and Fisher, J. R. (1992). Discovery of multiyear diapause in Illinois and South Dakota northern corn rootworm (Coleoptera: Chrysomelidae) eggs and incidence of the prolonged diapause trait in Illinois. Journal of Economic Entomology 85, 262–267.
• Levine, E., and H. Oloumi-Sadeghi. 1996. Western corn rootworm (Coleoptera: Chrysomelidae) larval injury to corn grown for seed production following soybeans grown for seed production. Journal of Economic Entomology 89, 1010-1016.
• Levine, E., J.L. Spencer, S.A. Isard, D.W. Onstad and M.E. Gray. 2002. Adaptation of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) to crop rotation: Evolution of a new strain in response to a cultural management practice. American Entomologist 48, 94-107.
• Mabry, T.R. and J.L. Spencer. 2003. Survival and oviposition of a western corn rootworm variant feeding on soybean. Entomologia Experimentalis et Applicata 109, 113—121.
• Mabry, T.R., J.L. Spencer, E. Levine and S.A. Isard. 2004. Western corn rootworm (Coleoptera: Chrysomelidae) behavior is affected by alternating diets of corn and soybean. Environmental Entomology 33, 860-871.
• Onstad, D.W., M.G. Joselyn, S.A. Isard, E. Levine, J.L. Spencer, L.W. Bledsoe, C.R. Edwards, C.D. DiFonzo and H. Willson. 1999. Modeling the spread of western corn rootworm (Coleoptera: Chrysomelidae) populations adapting to soybean-corn rotation. Environmental Entomology 28, 188-194.
• Onstad, D.W., J.L. Spencer, C.A. Guse, E. Levine and S.A. Isard.  2001. Modeling evolution of behavioral resistance by an insect to crop rotation. Entomologia Experimentalis et Applicata  100, 195-201.
• Steffey, K. L., Gray, M. E., and Kuhlman, D. E. (1992). Extent of corn rootworm (Coleoptera: Chrysomelidae) larval damage in corn after soybeans: search for the expression of the prolonged diapause trait in Illinois. Journal of Economic Entomology 85, 268–275.
• Tinsley, N.A., R.E. Estes and M.E. Gray. 2013. Validation of a nested error component model to estimate damage caused by corn rootworm larvae. Journal of Applied Entomology 137, 161—169.

About Dr. Joseph L. Spencer

Dr. Joseph L. Spencer is a Principal Research Scientist and Research Program Leader in Insect Behavior in the Illinois Natural History Survey at the University of Illinois at Urbana-Champaign.

Dr. Spencer received his Ph.D. (Entomology) from Michigan State University in 1994, after a Post Doc at the University of Arizona, he came to the Illinois Natural History Survey (INHS) at the University of Illinois in 1996 to study the behavior and biology of the rotation-resistant western corn rootworm.

Joe has spent 19 years investigating all aspects of western corn rootworm beetle biology and ecology with a special focus on adult movement and dispersal within, between, and above corn and soybean fields.

Since 2001, Dr. Spencer has studied rootworm interactions with Bt- corn hybrids.

He is currently studying the biology and behavior of Bt- and rotation-resistant western corn rootworms in Illinois. Joe is also an avid insect photographer and nature observer on the rare occasions when he is not in a cornfield.