DOI: 10.1111/j.1570-7458.2007.00618.x

Blackwell Publishing Ltd Stable isotopes as markers to investigate host use by Rhyzopertha dominica Rizana M. Mahroof* & Thomas W. Phillips Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA Accepted: 24 July 2007

Key words: Bostrichidae, Coleoptera, carbon isotope, C3 or C4 plant, corn, diet switching, elemental markers, lesser grain borer, nitrogen isotope, oak, stored products pest, wheat

Abstract Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) is a serious worldwide pest of stored cereal grains that also has the ability to breed in non-agricultural host plant material. Stable isotope signatures (concentrations of isotopes) were used as internal tissue markers to determine dietary differences among adult R. dominica and to make inferences about source habitats of field-trapped insects. Adult R. dominica collected near granaries or from non-agricultural forested sites near Stillwater, OK, USA, and insects reared on selected hosts under laboratory conditions were studied to determine the carbon and nitrogen isotope signatures. Laboratory-reared R. dominica showed δ13C (stable isotope ratio of carbon) values similar to the host on which they developed with an enrichment of about 1‰ in the

insect body. Insects reared on seeds of wheat and oak, which have C3 photosynthetic pathways, showed much depleted δ13C values (–23.7 and –26.2‰, respectively) in comparison to insects reared

on seeds of corn, a C4 photosynthetic plant (–11.3‰). A majority of the field-collected R. dominica δ13 showed C values similar to expectations for a C3 host. However, a few field-collected insects had δ13 C signatures similar to the C4 plant-reared insects in the laboratory experiment. Stored grain of C4 crops were lacking at many of the sample field sites. These results suggest that R. dominica occurs on

either C3- or C4-based hosts in the field, and point to utilization of non-grain C4 plants as hosts. Our studies indicated that 13C isotope is a reliable marker to infer types of hosts used in the feeding history of R. dominica.

grain storages. The relationship between the abundance Introduction and occurrence of R. dominica in agricultural and non- The lesser grain borer, Rhyzopertha dominica (F.) agricultural habitats is poorly known. Non-agricultural (Coleoptera: Bostrichidae), is a long-lived, destructive habitats may provide temporary alternate food sources, or pest of stored wheat and other cereal grains worldwide serve as overwintering sites for R. dominica in the absence (Cotton, 1956; Cuperus et al., 1986; Pederson, 1992; Fields of a major food supply from stored grains. & Phillips, 1994; Mayhew & Phillips, 1994; Vela-Coiffier Rhyzopertha dominica are strong fliers (Winterbottom, et al., 1997). Rhyzopertha dominica is thought to have 1922; Hagstrum, 2001) and, therefore, populations in originated from the Indian subcontinent, but is now different habitats may mix over large spatial scales. cosmopolitan in distribution (Potter, 1935). The insect Immigration from the outside non-agricultural sources was first recorded in the USA in 1861 (Schwardt, 1933) can lead to rapid colonization of this pest in grain storages and is distributed in all states where grain is grown (Cogburn, 1988). Current pest-management programs (Subramanyam & Harein, 1989). Work by Cogburn (1988), for R. dominica are designed to manage insects only in Fields & Phillips (1994), and Edde et al. (2005) showed the grain inside storage structures such as bulk grain that R. dominica has been trapped in distinctly different bins (Reed et al., 1995; Sloderbeck et al., 2002). A detailed habitats, such as those from the periphery of grain understanding of the occurrence of this species in diverse elevators or in forested sites many kilometers away from environments may have important implications in terms of pest management. Management efforts could *Correspondence: E-mail: [email protected] then be directed more appropriately to breeding sites or

© 2007 The Authors Entomologia Experimentalis et Applicata 125: 205–213, 2007 Journal compilation © 2007 The Netherlands Entomological Society 205

206 Mahroof & Phillips overwintering sites outside of the grain storages (Ramirez- A broad aim of our research is to investigate the dispersal Martinez et al., 1994). of R. dominica among habitats by defining the food Movement of animals among habitats and food sources resource-use pattern and the dietary history of dispersing has recently been investigated using methods involving insects. To accomplish this aim, we developed three tissue assays of certain elements (Hobson, 1999; Hobson objectives for the work reported here. The first objective et al., 1999; Callaham et al., 2000; Hobson et al., 2004). was to determine the carbon and nitrogen isotope ratios in Analyses of trace elements or stable isotopes of common adult R. dominica reared on selected laboratory diets. The elements to identify prior food sources, and perhaps with second objective was to investigate whether the carbon and information about prior habitats of phytophagous insects, nitrogen isotopic ratios of the adult R. dominica undergo follows from the old saying ‘you are what you eat’ and changes when the diet of adult insects were different from modified by McLean et al., (1979) ‘we are where we eat’, in the larval diet in the laboratory. The third objective was which isotope signatures currently in the body tissues may to use laboratory data to infer the host plant sources of provide distinctive information about isotope signatures R. dominica captured around grain storage facilities and in of host plant sources or habitats of the host plants and forested sites in OK, USA. the insects (Peterson & Fry, 1987; Ostrom et al., 1997; Callaham et al., 2000; Tigar & Waldron, 2002; Hobson Materials and methods et al., 2004; Hood-Nowotny & Knols, 2007). Higher plants can be categorized into three groups Rhyzopertha dominica reared on three hosts based on photosynthetic pathways: C3 plants, C4 plants, Eggs from adult R. dominica were collected from the and crassulacean acid metabolism plants (Ehleringer & laboratory colony that was established in 2000 at the Monson, 1993). The ratio of two naturally occurring stable Stored Products Research Laboratory in the Department isotopes of 12C and 13C in the environment is known as δ13C of Entomology and Plant Pathology at Oklahoma State (isotope ratio of carbon) (Farquhar et al., 1989). Plants University, Stillwater, OK, USA. About 500 10-day-old with a C4 photosynthetic pathway have higher proportion adult R. dominica were transferred to transparent plastic 13 × × of C in the photosynthate than C3 plants (Chamberlain boxes (31 17 8 cm), each containing a mixture of 50 g et al., 2004). Insects developed or fed on diets with certain of whole wheat kernels and 10 g of bleached wheat flour. δ13C values may reflect these same values of δ13C in their Adults were incubated at 29 °C, 60% r.h., and a photoperiod body tissues, cuticle, or other body parts (Bowden et al., of L12:D12 for a 24-h ovipositional period. The contents 1984). The δ13C isotope ratios measured in insects collected of the boxes were sifted using a set of sieves, 1400-μm from different habitats provide an indirect approach for mesh (USA no. 14), 850-μm mesh (USA no. 20) (Seedburo delineating food sources. Measurements of δ15N (stable Equipment Co., Chicago, IL, USA), and 212-μm mesh isotope ratio of nitrogen) values may also hold considerable (USA no. 70; Fisher Scientific, Madison, WI, USA) to promise for associating individuals with particular food collect the eggs. sources and habitats (Mizutani et al., 1991). Differences in Hard red winter wheat, Triticum aestivum L. (Poaceae), the δ15N among insects may be caused by differences in seeds of chinquapin oak (acorn), Quercus muhlenbergii diets (Owens, 1987), variation in organic materials in soil Engelm (Fagaceae), and shelled yellow corn, Zea mays L. (Hobson, 1999), or related to the physiological status of (Poaceae) were selected as hosts for rearing larvae of the insects in question (Gannes et al., 1997). R. dominica. Wheat was selected because it is a C3 plant and The patterns of δ13C or δ15N in adult phytophagous and it is the most common cereal grain grown and stored in carnivorous insects are sensitive to dietary changes on large quantities in the southern Great Plains of USA. Corn a relatively shorter time scale (Ostrom et al., 1997). Adults is a C4 plant that is a suitable host for R. dominica; it is of R. dominica are long lived, highly mobile, and can utilize also grown and stored in large quantities in other parts of various host tissues from a variety of plant species (Edde & the USA, but is typically not infested by R. dominica in

Phillips, 2006a). Rhyzopertha dominica larvae may breed in storages. Chinquapin oak is a C3 plant predominantly one species of plant, dispersing adults may subsequently found in wooded or forested sites in Oklahoma, and recent feed on a very different species of plant, and patterns of work found it to be a suitable larval food source for stable isotopes may vary throughout an individual’s R. dominica (Edde & Phillips, 2006a). Wright et al. (1989) lifetime. Therefore, an important step in applying δ13C or previously reported that R. dominica successfully survived δ15N isotope ratio data for the study of host use history in in acorns collected from nests of eastern woodrat, R. dominica adults is to determine if isotopic shifts occur Neotoma floridana (Smalli). when food sources change from those used by larvae to Acorns (seeds) from chinquapin oak, Q. muhlenbergii, those fed upon by dispersing adults. were collected from the natural wooded sites around

Stable isotopes and host use by Rhyzopertha dominica 207

Stillwater. Acorns were cleaned, shelled, and the cotyledons elemental analyzer coupled to a Thermo Finnigan delta were chopped into small pieces (10 mm3). The pieces of S isotope ratio mass spectrometer (NIWA Science, acorn were air dried prior to use in the experiment. The Christchurch, New Zealand). Stable isotope ratios were moisture content of wheat, acorn, and corn used in the reported in per mil (‰) units. One ‰ unit represents a experiment was measured on dry weight basis and was one-part-per-thousand difference from a standard and found to vary from 11 to 13%. Wheat, split pieces of acorns, was calculated using the formula: and corn were pulverized separately using a mechanical δ × grinder and sifted using an 850-μm meshed sieve. Whole Delta ( )SA = [RSA/RST – 1] 1000, wheat kernels, pieces of acorns, or corn (40 g) were mixed with 10 g of the respective pulverized materials and where RSA is the measured isotope ratio of the unknown transferred to 950-ml glass jars. About 200, 24-h-old eggs sample and RST is the defined isotope ratio of the primary collected as described above were introduced to each jar standard. containing different host materials. Rhyzopertha dominica Primary standards accepted by the National Institute of 13 were reared at 29 °C, 60% r.h., and L12:D12 until adult Standards and Technology (NIST) for δ C is Pee Dee 13 12 emergence. Two weeks after adult emergence, adults were Belemnite (PDB) with C/ C abundance of 0.0112372, 15 15 14 separated from the diet, transferred to an air-tight container, and for δ N is Atmospheric Air (AIR) with N/ N and stored in a freezer for further use. Samples of diets abundance of 0.0036765. Internal standards of known used for rearing R. dominica were also stored in the freezer isotopic signatures, calibrated against accepted NIST for further analyses. This rearing procedure was repeated standards, were included before every run for calibration 10 times with fresh diets and newly laid R. dominica eggs. purposes. The internal standard for carbon was graphite (δ13C = –28.1‰ relative to PDB), and for nitrogen it was δ15 Adult Rhyzopertha dominica subjected to diet-switching N2 gas ( N = –2.8‰ relative to AIR). 13 15 Rhyzopertha dominica larvae were reared on seeds of either The enrichment of δ C or the δ N from diet to insect wheat, oak (acorn), or corn as described above. Soon after body for the laboratory-reared R. dominica was calculated emergence, a total of 60 adults was separated from each based on the following formulae: diet. Out of these 60 adults, 20 were assigned to each of the δ13 Δ δ13 δ13 same diets used for larval rearing, and allowed to feed. For C Enrichment insect-diet = Cinsect – Cdiet, and δ15 Δ δ15 δ15 example, adults emerged from wheat were separated into N Enrichment insect-diet = Ninsect – Ndiet. groups and transferred to 40 g of wheat, oak, or corn for adult feeding. Similarly, R. dominica emerged from oak were The analyses of both isotope signatures for laboratory diets transferred to acorn, wheat, or corn, and adults emerged were conducted for 11 samples and for laboratory-reared from corn were transferred to corn, wheat, or oak. These R. dominica were carried out on 11 individuals from each adults were introduced as a group to 120-ml glass jars, and diet. Carbon and nitrogen isotope data from insect or diet were maintained for three more weeks at 29 °C, 60% r.h., were analyzed separately by one-way analysis of variance and L12:D12. At the end of the 3rd week, all adults were (ANOVA) using the general linear model (GLM) procedure separated from diets and stored in a freezer until further of SAS (SAS Institute, 2003). Least square means were used use. Ostrom et al. (1997) showed that the shift in isotope for separating treatment means at α = 0.05. Significant ratios due to diet-switch stabilizes within 16 days of diet differences between insect body isotope values and corre- change. The whole procedure was repeated 10 more times. sponding diet isotope values for each diet were determined by independent t-tests (PROC TTEST) at α = 0.05 level. Stable isotope analyses and reporting Stable isotope analysis was carried out for the three Stable isotope analysis for Rhyzopertha dominica subjected to laboratory diets and for the laboratory-reared R. dominica. diet-switching Frozen insects were rinsed in double distilled water and δ13C and δ15N were determined for R. dominica subjected dried in an oven (Cole Parmer Instruments Co., Chicago, to the diet-switching experiment. The analyses of both IL, USA) at 60 °C for 48 h. Pulverized diet was also dried isotopes in adults for this experiment were conducted on at 60 °C for 48 h. The whole body of a single dried adult 11 individuals from each diet. Isotope data from this insect or 1 mg of dried powdered diet was used to determine experiment were subjected to one-way ANOVA using the δ13C and δ15N. Both isotopes were analyzed in the GLM procedure of SAS (SAS Institute, 2003). Least Stable Isotope Ratio Facility for Environmental Research square means were used for separating treatment means (SIRFER) at the Department of Biology, University of at α = 0.05. The comparisons were carried out for a given Utah, Salt Lake City, UT, USA, using a Carlo Erba 1108 first diet (larval diet) among various secondary diets (adult 208 Mahroof & Phillips diets), while the first diet was kept fixed using a SLICE University (36°07′N; 097°08′W); the Peavey Grain Co., option in a LSMEANS statement. Catoosa, OK (36°19′N; 095°78′W); Central Oklahoma Services, Douglas, OK (36°28′N; 097°67′W); and Central Field trapping of Rhyzopertha dominica adults Oklahoma Services, Marshall, OK (36°15′N; 097°61′W). Four-unit Lindgren funnel traps (PheroTech, Delta, BC, Trapping was carried out during two periods in consecu- Canada) were deployed in selected forested sites and grain tive years, from July to October 2005 and May to August storage facilities near Stillwater. Collection cups beneath 2006. Traps were hung on polyvinylchloride pipe stands, funnel traps were baited with no-pest strips (United 1.7 m above the ground at a rate of three traps per site with Industries Corp., St. Louis, MO, USA), which release a minimum of 35 m between traps. Collection cups on the volatile dichlorvos, an insecticide to kill captured insects. traps were emptied and trapped insects were collected on All traps were baited with the synthetic R. dominica a weekly basis, and at the same time, traps were serviced by aggregation pheromones dominicalure 1 (DL1) and replacing new fresh pheromone lures and new pieces of dominicalure 2 (DL2). Rubber septa lures (no. 11.5; Fisher, no-pest strip. Trapped R. dominica were returned to the Pittsburgh, PA, USA) were prepared before use by soaking laboratory and stored in a freezer until further use. Stable in hexane for 12 h, then in dichloromethane for 12 h, and carbon and nitrogen isotope analyses were carried out for air dried for 24 h. Septa lures were then fabricated in the field-captured beetles as described above. The analyses for laboratory by applying a hexane solution of the two field-captured R. dominica were carried out on 11 separate synthetic pheromones with a micropipette at a ratio of 1:1:2 beetles from each site, except for beetles from Douglas and (DL1:DL2:hexane). A total of 5 mg of each pheromone was Marshall, for which only three beetles were analyzed due to applied to each septum. an inadequate number of insects trapped. Field data were Traps were deployed in two selected forested sites, interpreted by plotting δ13C values against the same beetle’s Pasture II (PII; 36°03′N; 097°10′W; ≈607 ha) and on land δ15N values and compared with the laboratory data. adjacent to Lake Carl Blackwell (LCB; 36°07′N; 097°13′W; ≈268 ha) west of Stillwater. Predominant plant species at Results both sites were Q. muhlenbergii; post oak, Quercus stellata Wangenh; hackberry, Celtis occidentalis L.; loblolly pine, Stable isotope analysis for laboratory-reared Rhyzopertha dominica

Pinus taeda L.; Eastern redbud, Cercis canadensis L.; and There were significant differences (F2,30 = 3291.9, P<0.0001) Eastern red cedar, Juniperus virginiana L. (Edde & Phillips, for δ13C values among wheat, oak, and corn seeds, and also δ13 2006b). Traps were also deployed around the periphery of (F2,30 = 882.1, P<0.0001) for C in the R. dominica adults the following grain handling facilities; the Stored Products reared on these three diets (Table 1). The R. dominica from Research and Education Center (SPREC), Oklahoma State corn had a higher δ13C signature value, which differed

Table 1 Mean (SEM) values of δ13C and δ15N for different diets and Rhyzopertha dominica reared on these respective diets, and the enrichment of δ13C and δ15N from diet isotopes to insect isotopes

Isotope (relative Type Diet isotope R. dominica Enrichment 1,2 2 3 Δ 4 to standards) of food values (‰) isotope values (‰) Difference insect-host (‰) δ13C (‰ PDB) Wheat –23.87 (0.05)b –23.69 (0.15)b ns 0.18 (0.15) Oak –26.15 (0.04)a –25.28 (0.31)a * 1.08 (0.31) Corn –11.24 (0.04)c –11.29 (0.28)c ns 0.05 (0.20)

δ15N (‰ air) Wheat 3.98 (0.13) 9.06 (0.36) * 5.08 (0.29) Oak 4.03 (0.19) 8.45 (0.61) * 4.20 (0.62) Corn 4.41 (0.16) 8.86 (0.35) * 4.71 (0.29) 1Means were based on n = 11 insects. 2Values for δ13C in a column followed by different letters are significantly different (ANOVA, P≤0.05). Differences for δ15N were not significant (P>0.05). 3Difference between diet and insect isotope for a given type of food. The * indicates significant differences between insect isotope and diet isotope for a given host at P≤0.05 (paired t-test); ns, not significant. 4The enrichment for δ13C and δ15N were calculated using the following formulae: Δ = δ13 δ13 Enrichment insect-diet Cinsect – Cdiet (n = 11), and Δ = δ15 δ15 Enrichment insect-diet Ninsect – Ndiet (n = 11). Stable isotopes and host use by Rhyzopertha dominica 209

insect body varied from 4.20 to 5.08‰ (Table 1). The δ13C values for the whole bodies of R. dominica and food sources showed that insect body δ13C values were similar to those of their diets as revealed in the scatter plot (Figure 1A); however, δ15N values of adult R. dominica were highly variable irrespective of their food sources (Figure 1B). Results also showed that the δ15N values among beetles raised on the same diet differed considerably.

Stable isotope analysis for diet-switched Rhyzopertha dominica The results of stable carbon and stable nitrogen isotope analyses of R. dominica reared on a single diet or two diets are summarized in Table 2. The δ13C between R. dominica when both larvae and adult stages were reared on a single diet (larval diet), and when larval stages reared on one diet and adults fed on the second diet (adult diets) were δ13 significantly different (F8,35 = 15.72, P<0.0001). The C values for larvae reared on wheat and adults fed on wheat were significantly different (P<0.05) from those for larvae reared on wheat and adults fed on corn. However, δ13C values of R. dominica reared on wheat and transferred to wheat or oak were not significantly different (P>0.05) from each other (Table 2). Similar results were also found for R. dominica larvae reared on oak and adults transferred to secondary diets. Conversely, the δ13C values of R. dominica with both larvae and adults reared on corn were significantly higher (P<0.05) than values of R. dominica Figure 1 Scatter plots of natural carbon and nitrogen isotope reared on corn and transferred to either wheat or oak. Thus, values for the laboratory-reared Rhyzopertha dominica. (A) δ13C and (B) δ15N values for the individual body signatures adult R. dominica reared as larvae on one photosynthetic of R. dominica and their respective diets. Isotope values type of diet (C3 or C4) and then fed as adults on the are expressed in per mil units (‰), see text for details.

Table 2 Mean (SEM) values of δ13C and δ15N for Rhyzopertha significantly (P<0.05) from those of wheat- or oak-fed dominica in the diet switching experiment. Larval stages were adults. Similarly, the δ13C values between wheat- or oak- reared on a single diet (larval diet) and adults were fed on either larval diet or one of the other two diets fed adults were also significantly (P<0.05) different from δ15 each other. No significant differences in N values were Larval Adult δ13C [‰ PDB δ15N [‰ air noted for diets (F2,30 = 2.0, P = 0.1) or beetles (F2,30 = 1.4, diet diet (SEM)]1,2 (SEM)]2 P = 0.2) reared on C3 (wheat and oak) vs. C4 plants (corn). Paired t-test showed no significant differences (P>0.05) Wheat Wheat –23.88 (0.30)a 9.47 (0.99) Oak –24.60 (0.50)a 9.18 (0.02) between insect isotope values and diet isotope values Corn –19.62 (1.13)b 7.63 (0.43) for carbon with wheat and corn, but those for oak-fed R. dominica and oak seeds were significantly different Oak Oak –26.28 (0.40)a 6.75 (0.49)b (P = 0.02). However, nitrogen isotope analyses showed Wheat –24.40 (0.68)a 10.70 (1.50)a significant differences (paired t-test, P<0.05) between insect Corn –19.93 (0.92)b 9.70 (0.21)a and diet isotope values for all types of hosts (Table 1). The carbon enrichment from food sources to insect body Corn Corn –11.57 (0.65)c 9.33 (0.55) was quite low and varied from a gain of 0.05–1.08‰ with Oak –20.73 (1.29)a 9.83 (0.26) an average gain of 0.44‰ among the three diets. The Wheat –15.53 (2.79)b 9.45 (0.59) 15 δ N values of R. dominica were highly enriched and 1Means were based on n = 11 insects. considerably different relative to the nitrogen in the food 2Values within a given larval diet in a column followed by sources. The nitrogen enrichment from food sources to different letters are significantly different (P≤0.05). 210 Mahroof & Phillips

Figure 2 Plot of δ13C and δ15N signatures of (A) laboratory-reared and (B) field-captured individuals of Rhyzopertha dominica. Vertical lines in (A) indicate δ13C mean values for the three laboratory diets. Isotope values are expressed in per mil units (‰). SPREC, Stored Products Research and Education Center; LCB, Lake Carl Blackwell; P II, Pasture II (see text for details of the locations). other type of diet had intermediate δ13C values between the forested site, LCB, and periphery of three grain adults developed from and then fed the same diet type elevators Catoosa, Douglas, and Marshall had δ13C body consistently. signatures similar to those fed on wheat or oak in the

Significant differences (F8,35 = 2.93, P = 0.017) were laboratory (ranged from –24.28 to –27.15‰). Adults observed for δ15N only when R. dominica larvae were collected from the forested site Pasture II showed δ13C reared on oak and adults were fed on either wheat or values that might have used both C3 and C4 host plants. corn (Table 2). Adults from the oak-based diet gained A majority of individuals from Pasture II had δ13C 15 significantly higher amounts of the heavier N isotope values related to C3 diets; however, results for two of the 11 by feeding on wheat or corn, resulted in higher δ15N in Pasture II beetles analyzed suggest that they may have fed diet-switched adults. Larvae of R. dominica reared readily on C4-based diets (Figure 2B). As with Pasture II beetles, on wheat or corn and when subjected to diet switching, no R. dominica from SPREC varied in δ13C value from –10.20 significant differences (P>0.05) were observed in the body to –27.15‰. SPREC beetles formed groups that are signature of δ15N from the larval host to the adult host. isotopically distinct with ranges of –10.20 to –12.51‰ like

C4 plants and –23.65 to –27.15‰ like C3 plants. Stable isotope analysis for field-captured Rhyzopertha dominica in comparison to laboratory data Discussion Scatter plots of δ15N against δ13C revealed that laboratory- reared insects clustered into two distinct groups based on Our first laboratory study clearly showed separation of their diets (Figure 2A). Scatter plots of δ15N against δ13C δ13C in R. dominica when they were raised from larvae on δ15 for field-captured R. dominica at six locations showed that a C3- or C4-based diet, yet N values were not informative individual beetles grouped into three isotopically similar with regard to diet type. Separation of the δ13C values clusters (Figure 2B). A majority of the field-collected of R. dominica raised on three diets under controlled R. dominica showed δ13C values similar to expectations for conditions into distinctive groups suggests that individuals a C3 plant-based diet for both larval and adult feeding. fed on photosynthetically different hosts can have substantial However, a few insects had δ13C signatures similar to those differences in their δ13C body signatures. Differences in δ13 expected for insects with a C4 plant-based diet, and two C that exist among C3 and C4 plants are biologically beetles from the SPREC site appeared intermediate between significant for isotopes of carbon assimilated by insects.

C3 and C4 types (–19.9 and –20.2). Adults collected from Thus, an analysis of the isotopic composition of an insect Stable isotopes and host use by Rhyzopertha dominica 211 body could be used to verify the source populations of knowledge, there were no corn fields or corn storages R. dominica, if the predominant plant species of an area within 10 km of Pasture II, and the relatively long distance can be determined. Although δ15N are successfully used in of this site from known corn storages suggests that corn some ecological studies (Callaham et al., 2000; Tigar & was not a host for these beetles. We must consider that

Waldron, 2002) as an index to delineate food resources, there were many other potential C4 hosts for R. dominica in our studies showed δ15N was a poor indicator of R. dominica our forested sites. Seeds from grasses (Poaceae), many δ15 food sources. The N body signatures were highly species of which have C4 photosynthesis (Ehleringer & variable even when R. dominica were reared on a Monson, 1993), likely served as hosts for R. dominica in the homogeneous diet. These differences may be due to high forested habitats. Similar conclusions were drawn for variation in the diet itself. The δ15N enrichment in a host the larger grain borer, Prostephanus truncatus (Horn) plant could vary with variation in nitrogen storage [(Coleoptera: Bostrichidae) (Tigar & Waldron, 2002)], and patterns among tissues, variation in the organic materials, for five annual cicada species [(Homoptera: Cicadidae) or fertilizers that occur in soil (Hobson, 1999), with depth (Callaham et al., 2000)] when they were suspected to feed of the soil, and types of disturbance that occur in soil on C3 or C4 plants. (Nadelhoffer & Fry, 1988). Differences in N storage patterns Most Bostrichidae are wood eaters in both larval and of C3 and C4 plants or differences in the metabolism of C3 adult stages, attacking standing dead wood or recently and C4 plant-feeding insects (Callaham et al., 2000) may fallen trees (Potter, 1935). Although the original host also cause the variation in δ15N values. Apart from the plants of R. dominica cannot be known, R. dominica may individual δ13C values, based on diet to insect enrichment originally have fed solely on wood or parts of woody plants values, δ13C further proves to be a reliable elemental before making an adaptive shift to stored grain. When marker for tracing food resources, because carbon gain grain became available in bulk storages, R. dominica may from food to insect body was considerably low. However, have broadened its host range and adaptively evolved to the trend of highly increasing δ15N enrichment from diet to predominantly use stored grain. A closely related species, insect body, as would be expected in the conversion of P. truncatus, may have recently broadened its host range plant material to animal biomass, potentially prevents the from woody food sources, such as branches, roots, and ability to forecast food sources in various environment tubers, (Chittenden, 1911) to stored grain (Markham, 1990; based on δ15N. Substantial variations observed for nitrogen Ramirez-Martinez et al., 1994). Adults of R. dominica were isotope values in the current laboratory study along with trapped in remote forested sites, as recorded by Cogburn greater enrichment rate from diet to insect body reveal that (1988), Fields & Phillips (1994), and Edde et al. (2005), δ15N is not a suitable elemental marker for determining which suggests that large numbers of R. dominica inhabit food sources. forests. As adults are widely captured in non-agricultural We trapped a considerable number of beetles in the habitats, isolated from grain storage by many miles, and grain storage environments, which was expected due to R. dominica can complete its lifecycle on non-grain food proximity to stored wheat as breeding material for beetles. materials, we hypothesize that such habitats may harbor Thus, it follows that R. dominica with δ13C values similar to populations with individuals that invade stored grain after those of C3 plants in the grain storage environments may harvest. Habitats outside of stored grain may be used as likely have fed on wheat. It is clear from the field data that overwintering sites, but actual reproduction in these the majority of R. dominica captured around SPREC, habitats is limited, or beetles may actually reproduce in Pasture II, and most, if not all, of the adults captured from these habitats in sufficient numbers to be an important

Catoosa, Douglas, Marshall, and LCB fed on C3-based source population. The primitive wood-boring habit diets. However, a few adults caught around SPREC or may persist in natural populations; yet, without further Pasture II showed that larvae of these R. dominica likely fed on research on host utilization by R. dominica, we are unable

C4 foods in their local habitats. We speculate that the higher to postulate the current natural hosts in the forested sites. δ13C values observed for some SPREC adults is, because This species has been reared successfully on oak seeds and they originated from the nearby animal feed mill. The feed other woody substances in the laboratory by Edde & Phillips mill is situated less than 100 m south of SPREC and uses (2006a) and in the current study. Therefore, one of the corn for processing animal feed. Rhyzopertha dominica is possible diets for R. dominica in the forested site may be reported to damage corn in storages (Potter, 1935). How- the acorns from oak trees, a dominant group of plants ever, we have never observed R. dominica infesting corn available in these ecosystems. storages (TW Phillips, pers. obs.). In a non-agricultural Intermediate δ13C values could result when insects habitat like Pasture II, some adults also had δ13C body develop as larvae on a host of one photosynthetic type and signature values similar to those of C4-based diets. To our then feed as adults on a host from another photosynthetic 212 Mahroof & Phillips type, or on a host that otherwise differs in isotopic com- agricultural pest, and warrant further research on potential position. Intermediate δ13C values observed for R. dominica source populations of this species from non-agricultural captured around SPREC suggest the possibility that these habitats and on variation in its host use pattern. insects had mixed C3 and C4 hosts, or they used a host with δ13C between C and C plant. Our laboratory experiment 3 4 Acknowledgements on diet switching showed similar intermediate δ13C values (19.62 ± 1.13‰) for R. dominica whose larvae were reared The authors are grateful to Drs Richard Berberet and Kris on wheat and adults were fed on corn. Gates (1995) Giles for constructive comments on the earlier draft of this recorded that the R. dominica are capable of dispersing manuscript and to Bjorn Martin and John McLean for over long distances to attack grains in storages. Considering helpful discussions. Rhyzopertha dominica aggregation the close proximity between SPREC and the animal feed pheromones were kindly provided by Wendell Burkholder, mill, it is possible that some R. dominica may have fed on US Department of Agriculture (USDA), Madison, WI, a mixed diet of wheat and corn although we have never USA. This work was supported in part with funds from the encountered R. dominica-infesting corn in Oklahoma. USDA Risk-Avoidance and Mitigation Program (RAMP) Similar intermediate δ13C values were also observed by Tigar and the Oklahoma Agricultural Experiment Station. & Waldron (2002) for P. truncatus, a serious pest of corn. These authors suggested that the intermediate δ13C value (–17.3‰) proved the nature of P. truncatus to assimilate References a mixed diet, perhaps with corn, a C4 host and non-corn, Bowden J, Digby PGN & Sherlock PL (1984) Studies of elemental a C3 host. composition as a biological marker in insects. I. The influence The diet-switching study showed that δ13C and δ15N of soil type and host-plant on elemental composition of values of a species can vary when larvae are reared on Noctua pronuba (L.) (Lepidoptera: Noctuidae). Bulletin of identical diets but adults are fed on different diets. Our Entomological Research 74: 207–225. Callaham MA, Jr, Whiles MR, Meyer CK, Brock BL & Charlton RE analyses were carried out for the fresh-killed, whole body (2000) Feeding ecology and emergence production of annual of the insect including the gut, fat bodies, and other cicadas (Homoptera: Cicadidae) in tall grass prairie. Oecologia digestive tissues. These tissues may have retained carbon 123: 535–542. values from recently assimilated diet. Changes in diet can Chamberlain PM, Bull ID, Black IHJ, Ineson P & Evershed RP take anywhere from a few days to a few weeks to appear in (2004) Lipid content and carbon assimilation in Collembola: animal tissue (Hobson & Clark, 1992). Perhaps if the implications for the use of compound-specific carbon isotope isotopic values of elytra or wings could be determined analysis in animal dietary studies. Oecologia 139: 325–335. exclusively, these parts of the body may have chemical Chittenden FH (1911) Papers on insects affecting stored products, composition more representative of the larval host plant the larger grain borer. US Department of Agriculture Bulletin than other, soft body parts like the gut (Bowden et al., 96: 48–52. 1984). However, this approach is limited in our experiment Cogburn RR (1988) Detection, distribution and seasonal abundance of Sitotroga cerealella and Rhyzopertha dominica as due to the minute size of the R. dominica adult (3 mm long indicated by pheromone-baited adhesive traps. Proceedings, and 0.8 mm wide and dry body mass of ≈0.4 mg; US XVIII International Congress of Entomology, 3–9 July 1988, Department of Agriculture, 1986, RM Mahroof, pers. University of British Columbia, Vancouver, Canada. obs.) and the potential for an inadequate sample weight to Cotton RT (1956) Pests of Stored Grain and Grain Products. determine isotopes after dissection. Burgess Publishing Co, Minneapolis, MN, USA. In summary, our studies showed that natural carbon Cuperus GW, Prickett CK, Bloome PD & Pitts JT (1986) Insect isotopes can be used to infer host sources of field populations populations in aerated and unaerated stored wheat in Oklahoma. of R. dominica that may have developed on diets that are C3 Journal of the Kansas Entomological Society 59: 620–627. Edde PA & Phillips TW (2006a) Potential host affinities for the or C4 plants or on plants with intermediate carbon isotope values. The majority of insects in our field samples showed lesser grain borer, Rhyzopertha dominica: behavioral responses to δ13C values suggestive of development on a C plant. host odors and pheromones and reproductive ability on non-grain 3 hosts. Entomologia Experimentalis et Applicata 119: 255–263. However, several dispersing R. dominica were trapped that Edde PA & Phillips TW (2006b) Field responses of nontarget had δ13C values indicative of C plants. Beetles trapped in 4 species to semiochemicals of stored-product Bostrichidae. δ13 forested sites far from stored grain, and with C values Annals of the Entomological Society of America 99: 175–183. like C4 plants, very likely developed on non-grain C4 hosts. Edde PA, Phillips TW & Toews M (2005) Responses of Rhyzopertha Our data suggesting non-grain hosts for some field- dominica (Coleoptera: Bostrichidae) to its aggregation trapped R. dominica support the hypothesis for the pheromones as influenced by trap design, trap height, and retention of ancient host use patterns in this serious habitat. Environmental Entomology 34: 1549–1557. Stable isotopes and host use by Rhyzopertha dominica 213

Ehleringer JR & Monson RK (1993) Evolutionary and ecological Nadelhoffer KJ & Fry B (1988) Control of natural nitrogen-15 aspects of photosynthetic pathway variation. Annual Review of and carbon-13 abundances in forest soil organic matter. Soil Ecology and Systematics 24: 411–439. Science Society of America Journal 52: 1633–1640. Farquhar GD, Ehleringer JR & Hubick KT (1989) Carbon isotope Ostrom PH, Garcia MC & Gage SH (1997) Establishing pathways discrimination and photosynthesis. Annual Review of Plant of energy flow for insect predators using stable isotope ratios: Physiology and Plant Molecular Biology 40: 503–537. field and laboratory evidence. Oecologia 109: 108–113. Fields PG & Phillips TW (1994) The distribution and PCR-based Owens NJP (1987) Natural variation in 15N in the marine environ- fingerprints of Rhyzopertha dominica (F.) in Canada. Stored ment. Advances in Marine Biology 24: 389–451. Product Protection. Proceedings of the 6th International Pederson JR (1992) Insects: identification, damage, and detection. Working Conference on Stored Product Protection, 17–23 Storage of Cereal Grains and Their Products, 4th edn (ed. by April 1994, Canberra, Australia (ed. by EE Highley, J Wright, DB Sauer), pp. 435–488. American Association of Cereal HJ Banks & BR Champ), pp. 517–522. Chemists, St. Paul, MN, USA. Gannes LZ, O’Brien D & Martinez del Rio C (1997) Stable Peterson BJ & Fry B (1987) Stable isotopes in ecosystem studies. isotopes in animal ecology: assumptions, caveats, and call for Annual Review of Ecology and Systematics 18: 293–320. more laboratory experiments. Ecology 78: 1271–1276. Potter C (1935) The biology and distribution of Rhyzopertha Gates MW (1995) Population Dynamics of Lesser Grain Borer, dominica (F.). Transcripts of the Royal Entomological Society Rusty Grain Beetle, and Cephalanomia watersoni in Commercial of London 83: 449–482. Elevators. MS Thesis, Oklahoma State University, Stillwater, Ramirez-Martinez M, Alba-Avila A & Ramirez-Zurbia R (1994) OK, USA. Discovery of the larger grain borer in a tropical deciduous forest Hagstrum DW (2001) Immigration of insects into bins storing in Mexico. Journal of Applied Entomology 118: 354–360. newly harvested wheat on 12 Kansas farms. Journal of Stored Reed C, Herrman T, Higgins R & Harner J (1995) Questions & Products Research 37: 221–229. Answers about Aeration Controllers. Kansas State University Hobson KA (1999) Tracing origins and migration of wildlife Agricultural Experiment Station and Cooperative Extension using stable isotopes: a review. Oecologia 120: 314–326. Service, MF-2090. Manhattan, KS, USA. Hobson KA, Bowen GJ, Wassenaar LI, Ferrand Y & Lormee H SAS Institute (2003) The SAS System, version 9.1 for Windows. (2004) Using stable hydrogen and oxygen isotope measurements SAS Institute, Cary, NC, USA. to infer geographical origins of migrating European birds. Schwardt HH (1933) Life history of the lesser grain borer. Journal Oecologia 141: 477–488. of the Kansas Entomological Society 6: 61–66. Hobson KA & Clark RG (1992) Assessing avian diets using stable Sloderbeck PE, Subramanyam B & Higgins RA (2002) Management isotopes. I. Turnover of 13C in tissues. Condor 94: 181–188. of Stored Grain Insect, Part III,: Structural Sprays, Pest Hobson KA, Wassenaar LI & Taylor OR (1999) Stable isotopes Strips Grain Protectants and Surface Dressings. Kansas State (δD and δ13C) are geographical indicators of natal origins of University Agricultural Experiment Station and Cooperative monarch butterflies in eastern North America. Oecologia 120: Extension Service, MF-917. Manhattan, KS, USA. 397–404. Subramanyam B & Harein PK (1989) Insects infesting barley stored Hood-Nowotny R & Knols BGJ (2007) Stable isotope methods in on farms in Minnesota. Journal of Economic Entomology 82: biological and ecological studies of arthropods. Entomologia 1817–1824. Experimentalis et Applicata 24: 3–16. Tigar B & Waldron S (2002) Using new tools to track the larger Markham RH (1990) The role of quantitative ecology and systems grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bos- analysis in the biological control of larger grain borer. trichidae). Advances in Stored Product Protection. Proceedings Biological Control of the Larger Grain Borer. Proceedings of of the 8th International Working Conference on Stored Product the IITA/FAO Coordination Meeting, 2–3 June 1989 (ed. by Protection, 22–26 July 2002, York, UK (ed. by PF Credland, RH Markham & HR Herren), pp. 129–130. IITA, Cotonou, DM Armitage, CH Bell, PM Cogan & E Highly), pp. 396–401. Republic of Benin. US Department of Agriculture (1986) Stored Grain Insects, Mayhew TJ & Phillips TW (1994) Pheromone biology of the Agricultural Research Services. USDA, US Government Printing lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Office, Washington, DC, USA. Bostrichidae). Stored Product Protection. Proceedings of the Vela-Coiffier EL, Fargo WS, Bonjour EL, Cuperus GW & Warde 6th International Working Conference on Stored Product WD (1997) Immigration of insects into on-farm stored wheat Protection, 17–23 April 1994, Canberra, Australia (ed. by EE and relationships among trapping methods. Journal of Stored Highley, J Wright, HJ Banks & BR Champ), pp. 541–544. Products Research 33: 157–166. McLean JA, Shepherd RF & Bennett RB (1979) Chemoprinting by Winterbottom DC (1922) Weevil in Wheat and Storage of Grain X-ray energy spectrometry: we are where we eat. Proceedings of in Bags. A Record of Australian Experience during the War a Conference, Movement of Selected Species of Lepidoptera in Period (1915 to 1919). South Australian Government, Adelaide, the Southeastern United States, 9–11 April 1979, Raleigh, NC, Australia. USA (ed. by RL Rabb & GG Kennedy), pp. 369–379. Wright VF, Fleming EE & Post D (1989) Survival of Rhyzopertha Mizutani H, Kabaya Y & Wada E (1991) Nitrogen and carbon dominica (Coleoptera: Botrichidae) on fruits and seeds collected isotope compositions relate linearly in cormorant tissues and from woodrat nests in Kansas. Journal of the Kansas Entomo- its diet. Isotopenpraxis 27: 166–168. logical Society 63: 344–347.