Michael B. Sady, Western Nevada Community College, Carson City, NV 89703 USA
Send comments to:  mbsady@wncc.nevada.edu

James N. Seiber, Center for Environmental Sciences and Engineering, University of Nevada, Reno, NV 89557 USA
Current Address: USDA-ARS 800 Buchanan St., Albany, CA. 94710 USA

^*A portion of this report was presented at the Nevada Environmental Conference,
University of Nevada, Reno, 30 September - 01 October 1996.

Abstract
The blue milkweed beetle Chrysochus colbaltinus LeConte (Coleoptera: Chrysomelidae) has been studied since 1992 living on selected host milkweed (Asclepiadaceae) populations of Asclepias eriocarpa Benth. in California, and Asclepias speciosa Torr. in western Nevada. The adult whole beetles appear to contain little to no toxic cardenolides typically produced by milkweeds when collected in the wild on hosts of A. eriocarpa in coastal foothills of California, and no detectable cardenolide when collected in the wild on hosts of A. speciosa in western Nevada. The egg cases of C. colbaltinus collected from the same milkweed patches contained moderate to high amounts of cardenolide when found on A. eriocarpa, but no detectable cardenolide when found on A. speciosa. Since the leaf cardenolide content for the former milkweed is 20 fold higher than the latter, the use of cardenolides for chemical defense in this chrysomelid may be host-dependent. The population structure of C. colbaltinus reveals a propensity to aggregate in very dense numbers, emerging in an exponential growth rate in the late spring on several A. speciosa patches studied in western Nevada. The adult beetle also appears to utilize minimal amounts of plant resource for ovipositioning and herbivory. A study of headspace components of the adult beetle reveals that common fatty acids may serve as important semiochemicals for aggregation.
 

Key Words
Chrysomelidae. Host. Asclepias. Fatty Acids. Cardenolide. Chrysochus colbaltinus.
 

Introduction
Defensive chemicals that are biosynthesized by leaf beetles and used at some life stage have been studied (Hilker 1992). Cardenolides serve a protective role in species of Chrysomelidae both in adult beetles and eggs (Hilker 1994). While some chrysomelids biosynthesize cardenolides of varying types and quantity (Dobler and Rowell-Rahier 1994; Hilker et al. 1992), other chrysomelid species appear to exploit host plant cardenolides for a putative chemical defense (Isman et al. 1977a; Sady 1994).

In North America Chrysochus spp. is reported by Arnett (1985) to feed on dogbane (Apocynaceae) preferentially to milkweed (Asclepiadaceae). Both of these lactiferous plant families have variable cardenolide content (Seiber et al. 1983). The eastern North American beetle species C. auratus Fabricius, is ordinarily found on a host of Apocynum spp. (Williams 1988; 1991), while the western North American species, C. colbaltinus (spelling of the species binomial conforms to Arnett 1985), is found on a host of Asclepias spp. (Isman et al. 1977a; Root 1986; Sady 1994). In this study we concentrate on the western blue milkweed beetle and comparing cardenolide content for populations of the beetle feeding on two species of milkweed that differ greatly in their cardenolide content and biogeographic range.

Eastern and mid-western North American milkweed leaf beetle species found on Asclepias spp. tend to have a high degree of fidelity to small isolated patches of their primary host plants (Dickinson 1992). In this study we present data on four populations of C. colbaltinus living on hosts of A. speciosa in western Nevada that we have monitored since 1992 for their rate of emergence, aggregation, ovipositioning and herbivory patterns.

Also we report on the analysis of headspace chemical components of female adult C. colbaltinus that may influence the aggregation of beetles into the small dense patches they occupy on some host milkweeds populations.

Currently we have an interest in studying the microbial influence, if any, with respect to the plant - herbivore interaction. This work is of interest because of our unpublished observations, and the presence of bacteria (Flynn and Vidaver 1995) and mycoplasmalike organisms (Griffiths et al. 1994) in North American milkweeds. We would like know whether the herbivores of milkweeds infect, or are infected by, the host milkweed. In Tetraopes spp. feeding on showy milkweed in western Nevada gram positive rod bacteria were found in the gut contents. The latex of the host milkweed had bacteria of similar morphology (Evett and Austin 1998). In 1999 C. colbaltinus will be examined for bacteria.

Materials and methods

Plant and beetle collection and census.
Adult beetles and egg cases of C. colbaltinus (voucher specimen FMNH-Z18,400) were collected in the field on host milkweed of A. eriocarpa (voucher specimen RENO-69190) in Monterey county California USA, and on A. speciosa (voucher specimen RENO-67804) in Douglas county Nevada USA during the spring and summer months. Gender sorting of the adult beetles was accomplished by finding copulating pairs in embrace for at least 1 minute and assuming the male to be mounted on the dorsal side of the female. Verification of this technique was confirmed by aedeagus dissection. Whole adult beetles and egg cases were stored at -20 C for the cardenolide studies, and at 20 C in a headspace chamber for 1 week until senescence for the GC-MS analysis. Daily census of beetles on host milkweed populations in western Nevada took place around noon on 14 randomly chosen patches of A. speciosa in Douglas county, Nevada since 1992. Beetles, herbivory patterns, and egg cases ovideposited, along with total host milkweed plants, stems, and leaves, were counted and assessed during each visitation at each patch site.
 

Chemical analyses of whole beetles and egg cases.
The whole beetles and egg cases stored at -20 C were thawed and dried in an oven at 40 C for 48 hours and then placed in 2 ml spectral grade methanol for 24 hrs to extract cardenolide. The total cardenolide content was determined on three or more beetles or samples of eggs using the methods of TNDP spectroassay reported in Isman et al. (1977a) and Seiber et al. (1983). (As of 1996 the Seiber lab has been exploring the use of supercritical fluid extraction of milkweed plant parts and herbivorous insects to improve yields of cardenolide with results that appear promising but have yet to be reproduced). Egg cases analyzed included the fecal covering and the eggs within, usually between 8 to 10 eggs per clutch. Approximately 12 live female adult C. colbaltinus beetles collected in summer of 1995 on a host of A. speciosa in Douglas county, Nevada were placed in an empty 250 ml amber headspace chamber at 20 C without food for 1 week. Volatile components were analyzed by injecting a 2 ml sample of the headspace atmosphere from the chamber into a gas chromatograph - Varian 3400 equipped with a DB-5 column (J & W Scientific) set at a programmed temperature range of 50 to 310 C, and coupled to a mass spectrometer - Finnigan MAT SSQ710 scanning masses m/z 35 to 300. The GC-MS software library identified two major peaks on the gas chromatogram at a retention time of approximately 16:00 and 17:50 minutes to be hexadecanoic (palmitic) and octadecanoic (stearic) acids, which was confirmed by reference to authentic standards of both acids. GC-MS analysis of the headspace atmosphere of a live male beetle confined to a 20 ml headspace vial showed the same two GC peaks for the two fatty acids given above for female beetles.
 

Results
The cardenolide content of whole beetles and egg cases of C. colbaltinus are compared and contrasted on two different host milkweeds in Table 1. The beetle and eggs cases have no detectable host cardenolide when collected on A. speciosa either in California (Isman et al. 1977a), or in western Nevada (this report). However the cardenolide content is low but detectable in some C. colbaltinus beetles and quite significant for the egg cases collected on A. eriocarpa in California. This might suggest that the 20 fold difference in the leaf cardenolide content in A. eriocarpa and A. speciosa provides a host-dependency to any putative chemical protection afforded the chrysomelid on these hosts, or other asclepiad hosts.
 
The gas chromatogram of headspace components of female adult C. colbaltinus is consistent with that of an internal standard for palmitic and stearic acids. The mass spectrum showed characteristic fragments of m/z M⁺ 43, 57, 73, and 129, and m/z M 256 and 284 for palmitic and stearic acids respectively (McLafferty and Turecek 1993).
 
Three patches of A. speciosa in western Nevada have been subjected to a census since 1992. The data suggests that the beetle appears to utilize only a small portion of available milkweed patches and the smaller and less phenologically developed plants for herbivory and ovipositioning (Table 2). There were three particularly dense populations of beetles on three different host patches of A. speciosa which conformed to an exponential growth pattern with respect to the emergence/appearance of beetles in the late spring approximating a doubling time of about 2.5 to 3 days (Table 3). The populations of these dense patches of beetles nearly doubled the second year , but reverted to very low population numbers in the third year. The adult beetles emerge in the late spring in western Nevada cued to a thermally dependent signal as warming in western Nevada varies greatly from year to year (Table 4).
 

Discussion
This study and previously cited work suggest that the blue milkweed beetle does not appear to biosynthesize cardenolides as other chrysomelids do, but may utilize host milkweed cardenolides when they are present in significant concentration. A. eriocarpa may be an evolutionarily advanced milkweed showing an apparent phylogenetic progression toward biosynthesis of more toxic and complex cardenolides, which, moreover, become increasingly concentrated in the latex, vis-a-vis A. speciosa (Farrell et al. 1992). Furthermore, in Tetraopes spp. (Coleoptera: Cerambycidae) the gap may have been bridged in plant chemical defense by circumventing it with unique feeding behaviors (Farrell et al. 1991). More information on C. auratus feeding on dogbane in a variety of biogeographically different settings would be helpful in order to further elucidate the diffuse character of host specificity in Chrysochus spp. As with most asclepiadaceous herbivores interspecific cardenolide content of host plants cannot alone account for the variability found in insects, even between populations feeding on the same plant part (Isman et al. 1977b). Interest is robust concerning the evolutionary significance of host shifts influenced by a change in plant chemistry (Secord and Kareiva 1996; Becerra 1997). Recent reports in Chrysochus, employing different methods and experimental appoach than those described here, demonstrate that it may well be the secretions vis-a-vis the whole beetle that contain the most significant quantities of toxic cardenolides and other defensive chemicals (Dobler et al. 1998).
 
The propensity of C. colbaltinus adults to aggregate in very dense patches in a given region where over 100 of them at a time will utilize only a very small portion of the plants in a patch could be a selective characteristic to increase mating success (Dickinson 1992). But it is enigmatic why some populations of beetles emerge, after overwintering in the larval stage on the host roots, as adults in late spring, doubling in numbers the next generation, and drop off to very few adults in the ensuing years. The host does not seem to change in size or nature, yet the population approaches extinction. Other milkweed beetle studies with Tetraopes spp. suggest a similarly short colonization and extinction population structure limited to a few years (McCauley 1989).
 
Finally despite their almost ubiquitous presence in nature, long-chain fatty acids appear to be of importance as behavioral cues for arthropods (Hibbard et al. 1994). Whether this is true for C. colbaltinus requires yet more controlled laboratory studies of beetle behavior to verify if palmitic and stearic acids, or yet to be discovered chemicals as a part of our current research interests, provide semiochemical behavioral aggregation cues that keep the dense patches of these beetles maintained throughout their short month or so of life. And what of unconscious olfactory signals that are presently being studied and their influence on plant - herbivore or animal - animal interactions (Hines 1997)?
 
We are also evaluating whether induced responses to herbivory may play a role in the milkweed - herbivore relationship as has been reported in other plant - insect relationships (Agrawal 1998).
 
Research in a variety of lab groups focusing on genetic make-up of the leaf beetles (Farrell 1998) and their hosts should also provide valuable insights on the host specificity in chrysomelidae.
 

Acknowledgments
This work was supported in part by the Nevada-NSF-EPSCoR Trec program which is funded by the UCCSN and matching grants from the National Science Foundation OSR-9353227 and OSR-9553369. We appreciate the assistance of Jim Lenoir and Loretta Raley of the Seiber lab, Dan Summers of the Field Museum of Natural History of Chicago, Brian Harris of the Natural History Museum of Los Angeles County, Christy Malone of the University of Nevada, Reno Herbarium, Adrian Wenner of U.C. Santa Barabara and Alice and Astrid Sady in the field collection and population census. We are particularly grateful for the watercolor painting of milkweeds and herbivores and the line drawing of the population and seasonal variations with milkweed phenology done by graphic artist Chris Olesen. We would also like to extend our thanks to the Summer 1997 Biology 280 Field Ecology class (taught by M. Sady at WNCC) for their data and comments. Prof. Gary Evett of WNCC and Fred Austin, a student of WNCC are acknowledged for their interest and work in identifying bacteria associated with beetles feeding on milkweeds in western Nevada. Finally we are very appreciative of the technical assistance of Leonard MacKey of WNCC, and Sherry Berg and Corky Goldade of Bently Nevada Corporation (and students of M. Sady at WNCC) in setting up the internet web site.

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