Jon C. Bedick

Distribution, Ecology and Population Dynamics of the American Burying Beetle [Nicrophorus Americanus Olivier (Coleoptera, Silphidae)] in South-central Nebraska, USA

The endangered American burying beetle, Nicrophorus americanus Olivier, was previously widespread throughout eastern North America. In the past century numbers of this beetle have drastically declined and currently remnant populations are known from only six states despite intensive surveying efforts conducted for the last nine years. Efforts aimed at discovering and managing remnant populations have been generally limited by a lack of knowledge concerning N. americanus biology. We used baited pitfall traps to define the range of the Gothenburg, Nebraska population of N. americanus. Using mark-recapture techniques, we estimate that the annual Gothenburg population consists of more than one thousand individuals, meeting the recovery plan criterion to become the third breeding population in the Midwest region. Beyond estimates of population size and range, we present novel data on seasonal and daily activity, sex ratio, age-grading and foraging distances. In 1995 and 1996, the Nebraska population was univoltine and female biased, with over-wintering mature beetles emerging in early June and teneral beetles emerging in August. Nocturnal activity was highest in the third and fourth hours following sunset but was not strongly correlated with temperature. During foraging, beetles travel up to six kilometers, but the majority of our recaptures occurred at distances of less than 0.5 km, suggesting that distances between traps be increased to ensure independence of sampling units. This information will allow future work on captive breeding, re-introduction and genetic studies.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini.

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.

Insect cellular reactions to the lipopolysaccharide component of the bacterium Serratia marcescens are mediated by eicosanoids

Nodulation, which begins with the formation of cellular microaggregates, is the predominant cellular defense reaction to bacterial infections in insects. We suggested that these reactions to bacterial infections are mediated by eicosanoids. The lipopolysaccharide (LPS) component of some bacterial cells stimulates defense reactions in mammals and insects. Here, we report on experiments designed to test the hypothesis that eicosanoids mediate microaggregation reactions to LPS. Injections of LPS (purified from the bacterium, Serratia marcescens) into larvae of the tenebrionid beetle, Zophobas atratus, stimulated microaggregation reactions in a dose-dependent manner. Treatments with eicosanoid-biosynthesis inhibitors immediately prior to LPS challenge sharply reduced the microaggregation responses. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase and lipoxygenase reduced microaggregation, supporting our view that microaggregate formation involves lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 30min after injection, and microaggregation was significantly reduced, relative to control insects, over the following 90min. The dexamethasone effects were reversed by treating LPS-injected insects with the eicosanoid precursor, arachidonic acid. These findings indicate that cellular defense reactions to a specific component of bacterial cells are mediated by eicosanoids, and open up new possibilities for dissecting detailed hemocytic actions in insect immune reactions to bacterial infections.

Pre-oral digestion: A phospholipase A2 associated with oral secretions in adult burying beetles, Nicrophorus marginatus.

We report on a phospholipase A2 (PLA2) found in the oral secretions, but not midgut contents, of the burying beetle, Nicrophorus marginatus. PLA2 is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids (PLs), an essential step in digestion and absorption of essential polyunsaturated fatty acids. Like the digestive PLA2s known from mammalian systems, and the one described insect digestive PLA2 the N. marginatus oral secretion PLA2 depends upon Ca2+ for full activity. However, unlike most digestive PLA2s, the N. marginatus enzyme is only partially inactivated in the absence of Ca2+. The PLA2 in N. marginatus oral secretions was influenced by altering the enzyme reaction conditions, including reaction time, protein concentration, pH, and temperature. Standard reaction conditions for assessing enzyme activity include 1.0 μg protein/μl incubated at pH 9.0 for 30 min at 28°C.

A New Sampling Protocol for the Endangered American Burying Beetle, Nicrophorus americanus Olivier (Coleoptera: Silphidae)

Abstract The endangered American burying beetle, Nicrophorus americanus Olivier, presently occurs in small and widely scattered areas on the periphery of its former range. As part of the recovery plan for this insect implemented by the U.S. Fish and Wildlife Service (USFWS), we conducted surveys in Nebraska to ascertain its presence. Additionally, we examined various trapping methods and procedures in the course of our survey work. We compared the effectiveness of different trap designs, trap placement, timing of trapping, types and amounts of bait, and monitoring traps. We also developed methods to reduce beetle mortality in traps. Our work offers more effective sampling procedures for N. americanus and suggests the current USFWS sampling protocol should be modified. New procedures include larger traps, moist soil substrate, a soil berm to the edge of the trap, longer trapping season, and increased distances between traps. A moist soil substrate, in combination with a shade-giving rain cover, precludes the necessity of servicing all traps by 9 AM each day (as mandated in the current protocol), a task that is logistically difficult or impossible if there are many traps. The sampling procedures we have identified as most appropriate for N. americanus will also improve sampling for other silphid species.

Eicosanoids mediate nodulation reactions to bacterial infections in larvae of the butterfly, Colias eurytheme.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. Treating larvae of the butterfly Colias eurytheme with the eicosanoid biosynthesis inhibitor dexamethasone, strongly impaired nodulation reactions to bacterial infections. The influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. An eicosanoid biosynthesis system in C. eurytheme larvae is documented. Specifically, the presence of eicosanoid-precursor polyunsaturated fatty acids in tissue phospholipids was determined, an intracellular phospholipase A2 that can release arachidonic acid from tissue phospholipids was recorded, and eicosanoid biosynthesis, registered as conversion of exogenous radioactive 20:4n-6 into eicosanoids, was observed. These findings support the hypothesis that eicosanoids mediate cellular immune responses to bacterial infections in these butterfly larvae, and more broadly, in most, if not all, insects.

Arachidonic and eicosapentaenoic acids in tissues of the firefly, Photinus pyralis (Insecta: Coleoptera)

We report on the presence of high proportions of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) in the tissue lipids of adult fireflies, Photinus pyralis. Arachidonic acid typically occurs in very small proportions in phospholipids (PLs) of terrestrial insects, ranging from no more than traces to less than 1% of PL fatty acids, while 20:5n-3 is often missing entirely from insect lipids. Contrarily, 20:4n-6 made up approximately 21% of the PL fatty acids prepared from whole males and females, and from heads and thoraces prepared from males. Proportions of 20:4n-6 associated with PLs varied among tissues, including approximately 8% for male gut epithelia, 13% for testes, and approximately 25% for light organs and body fat from males. Substantial proportions of 20:5n-3 were also associated with PLs prepared from male firefly tissues, including 5% for body fat and 8% for light organs. Because 20:4n-6 and 20:5n-3 are precursors for biosynthesis of prostaglandins and other eicosanoids, we considered the possibility that firefly tissues might produce eicosanoids at exceptionally high rates. Preliminary experiments indicated otherwise. Hence, fireflies are peculiar among terrestrial insects with respect to maintaining high proportions of PL 20:4n-6 and 20:5n-3.

Innate immune reactions stimulated by a lipopolysaccharide-like component of the alga Prototheca (strain 289).

We report on the influence of an LPS-like molecule (aLPS) from the pathogenic alga, Prototheca (strain 289) on insect and murine innate immune reactions. Insect innate reactions to infection include nodule formation, a process of entrapping bacterial cells in aggregates of hemocytes. We recorded eicosanoid-dependent, dose-related nodulation reactions to aLPS in hornworms (Manduca sexta). The insect reaction was attenuated by pre-incubating the aLPS with polymyxin-B. Conversely, the murine macrophages reacted to challenge with Escherichia coli LPS by secreting cytokines, but did not react to aLPS. We infer that, while highly conserved with respect to intracellular mechanisms of interaction, insect and mammalian immune surveillance systems differ in recognition of LPS molecular types.