Feeding Bioassays Research Articles

Preference and Performance of Black Cutworm (Lepidoptera: Noctuidae) on Poa Species

Abstract Black cutworm (Agrotis ipsilon Hufnagel) is a destructive pest of golf course turf. Previous work suggests that Poa pratensis ‘Midnight’ is resistant to black cutworm, but it is not known if resistance also exists among other Poa species and cultivars. This study compared preference and performance of black cutworm on Poa species and cultivars to a susceptible host plant, creeping bentgrass (Agrostis stolonifera L. ‘Penncross’) in laboratory choice feeding assays and rearing experiments. Seven-and 14-day-old larvae were used in performance feeding tests. Larvae reared on all Poa spp. exhibited significantly lower developmental and survival rates than larvae fed creeping bentgrass. Young (7 days old) larvae did not survive to pupation. Although older (14 days old) larvae pupated and eclosed on Poa, larval weight gain was significantly lower than those reared on creeping bentgrass. In choice feeding bioassays, regardless of larval stage, black cutworm preferred creeping bentgrass over all Poa species and cultivars. The results of this research suggest that although Poa species are generally resistant to black cutworm, the resistance varies among cultivars and insect growth stage.

The larvicidal activity of Bacillus thuringiensis Cry proteins against Thaumatotibia leucotreta (Lepidoptera: Tortricidae)

The susceptibility of one of the most important pests in southern Africa, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), to Bacillus thuringiensis Cry proteins was investigated. Cry proteins were produced in Escherichia coli BL21 cells that were transformed with plasmids containing one of the six cry genes encoding the Cry proteins used in the study. The larvicidal activity of each Cry protein against T. leucotreta neonate larvae was evaluated using the droplet feeding bioassay method with the volume imbibed by neonate larvae determined by fluorescence spectroscopy. Bioassays were performed using crude extracts and semi-purified Cry inclusion bodies of BL21 transformants, with the concentration of the Cry proteins determined by a novel protein quantitation strategy. For each BL21 transformant, a better fit of the data to the probit model was obtained for the semi-purified Cry inclusion bodies than for the crude extracts. Cry9Aa and Cry1Ca had significantly lower larvicidal activity against T. leucotreta neonate larvae than the other evaluated Cry proteins (Cry1Ac, Cry2Aa, Cry1Aa, and Cry1Ab). Cry2Aa and Cry1Ac had significantly higher larvicidal activity against T. leucotreta neonate larvae than the other tested Cry proteins. The study is important because it establishes a benchmark for the susceptibility of T. leucotreta to different Cry proteins, which is a key step in the evaluation of Cry proteins for potential use in T. leucotreta control programmes.

Toxicity and horizontal transfer of chlorantraniliprole against the Asian subterranean termite Coptotermes gestroi (Wasmann): effects of donor:recipient ratio, exposure duration and soil type

The effectiveness of chlorantraniliprole and other insecticides (bifenthrin, fipronil, indoxacarb, imidacloprid and chlorfenapyr) were tested against Coptotermes gestroi (Wasmann). Four experiments were conducted: a topical bioassay, a horizontal transfer study, an insecticide bioavailability test and a feeding bioassay. The topical bioassay showed that chlorantraniliprole was significantly less active to C. gestroi at 24 h post-treatment compared with the other insecticides tested. Nevertheless, it is likely that a lesser amount of chlorantraniliprole was required to cause 50% mortality of C. gestroi at 7 and 14 days post-treatment. The exposure duration and donor:recipient ratio affect the mortality of recipient termites. Mortality after exposure to chlorantraniliprole in sandy clay was significantly lower than in sand; however, by 14 days, > 90% of donor and recipient termites died in both substrates, irrespective of concentration. Fipronil and imidacloprid showed faster action, and high to moderate toxicity to C. gestroi. Termite workers also ceased to feed after exposure for 1 h to 50 mg kg(-1) chlorantraniliprole-treated sandy clay. Chlorantraniliprole demonstrated delayed toxicity at the lowest label rate (50 mg kg(-1) ) in sandy clay. Its slow action will enable greater transfer of toxicant between nestmates, while feeding cessation will promote greater social interaction between healthy and exposed termites.

Isolation of entomopathogenic Bacillus from a biodynamic olive farm and their pathogenicity to lepidopteran and coleopteran insect pests

The occurrence of Bacillus entomopathogenic bacteria on a Tunisian biodynamic farm was determined by examining 75 samples from olive tree ( Olea europaea L.) habitats. A total of 40 Bacillus isolates were characterized according to their phenotypic, physiological and biochemical parameters. Isolates of the species Bacillus subtilis, Bacillus mycoides, Brevibacillus brevis, Paenibacillus polymyxa, Bacillus licheniformis, Bacillus sp. (1), Bacillus sp. (2) and a standard strain Btk HD-1 were used separately in feeding bioassays on fresh artificial diet against larvae of lepidopterans Prays oleae (Bernard) and Palpita unionalis (Hübner) and coleopterans Hylesinus oleiperda (F.) and Phloeotribus scarabaeoides (Bernard), which are olive tree pests. Larvae were successfully reared on an artificial diet with 25 g powdered olive tree leaves. Compared to the control data, only Btk and the isolates of B. licheniformis, P. polymyxa and B. brevis were entomopathogenic. Larval mortality assessed 7 days post-treatment showed high mortality rates with Btk to lepidopteran larvae (86.6% for P. oleae and 80.9% for P. unionalis) and low mortality against coleopteran pests. B. brevis isolates showed high mortality rates against P. oleae (up to 67.9%). B. licheniformis isolates caused up to 59.2% larval mortality for P. oleae and 43.6% for P. unionalis. Highest coleopteran mortality was achieved by P. polymyxa isolates (up to 55%). According to the 16S rDNA results, isolates of each of the three entomopathogenic strains were similar. Proteins in the strain supernatants were toxic to P. oleae larvae with LC50 values of 10.0 ( B. brevis), 12.5 ( B. licheniformis) and 37.6 μg/ml ( P. polymyxa). Also, P. polymyxa showed an LC50 of 12.4 mg/l against P. scarabaeoides. Our results suggest that entomopathogenic Bacillus present locally in the biodynamic farm could be used in biological control programmes of olive tree pests.

Phaseoloidin, a Homogentisic Acid Glucoside from Nicotiana Attenuata Trichomes, Contributes to the Plant’s Resistance against Lepidopteran Herbivores

Plant trichomes are known for their capability to produce and store secondary metabolites that protect plants from biotic and abiotic stresses. (1)H NMR studies on intact individual trichomes located on the leaf surface of Nicotiana attenuata revealed the presence of two major secondary metabolites: nicotine, the signature metabolite of the genus, and phaseoloidin, a homogentisic acid glucoside. This glucoside was reported originally from the seeds of Entada phaseoloides, and this is the first report of its occurrence in a Solanaceous plant. Artificial diet feeding bioassays with Manduca sexta and Spodoptera littoralis larvae, two important herbivores of N. attenuata, revealed that the ingestion of phaseoloidin negatively influenced caterpillar performance. This effect was more pronounced for the generalist, S. littoralis, than for the specialists, M. sexta.

Effect of artificial diet components on conspecific repellency of larval oral secretions from western spruce budworm (Lepidoptera: Tortricidae)

AbstractA choice feeding bioassay was used to investigate the effects of artificial diet components on the repellency of larval oral secretions from western spruce budworm, Choristoneura occidentalis Freeman, to conspecific larvae. Oral secretions from insects fed on agar supplemented with linseed oil were as repellent as secretions from insects fed on complete artificial diet. Secretions from insects fed on agar alone, agar and casein, or agar and wheat germ were not significantly more repellent than distilled water; neither was linseed oil alone. Linseed oil consists of glycerides of several fatty acids, which are likely metabolized quickly in the insect gut; repellency of oral secretions could be due to any of the related fatty acids or metabolites.

Ecotoxicological evaluation of three tertiary wastewater treatment techniques via meta-analysis and feeding bioassays using Gammarus fossarum

Advanced treatment techniques, like ozone, activated carbon and TiO 2 in combination with UV, are proposed to improve removal efficiency of micropollutants during wastewater treatment. In a meta-analysis of peer-reviewed literature, we found significantly reduced overall ecotoxicity of municipal wastewaters treated with either ozone ( n = 667) or activated carbon (=113), while TiO 2 and UV was not yet assessed. As comparative investigations regarding the detoxification potential of these advanced treatment techniques in municipal wastewater are scarce, we assessed them in four separate Gammarus-feeding trials with 20 replicates per treatment. These bioassays indicate that ozone concentrations of approximately 0.8 mg ozone/mg DOC may produce toxic transformation products. However, referred effects are removed if higher ozone concentrations are used (1.3 mg ozone/mg DOC). Moreover, the application of 1 g TiO 2/l and ambient UV consistently reduced ecotoxicity. Although activated carbon may remove besides micropollutants also nutrients, which seemed to mask its detoxification potential, this treatment technique reduced the ecotoxicity of the wastewater following its amendment with nutrients. Hence, all three advanced treatment techniques are suitable to reduce the ecotoxicity of municipal wastewater mediated by micropollutants and may hence help to meet the requirements of the European Water Framework Directive.

Growth variation among Bacillus thuringiensis strains can affect screening procedures for supernatant‐secreted toxins against insect pests

Supernatant-secreted proteins in Bacillus thuringiensis (Berliner) (Bt) with insecticidal activity provide an important source of information for discovery of new useful strains and/or entomotoxins. However, physiological variation among isolates might interfere in the detection efficiency of screening procedures on Bt collections. The aim of this study was to assess the magnitude of this variation in a sample of isolates from a tropical Bt collection, which was gauged through the assessment of their temporal patterns of growth and protein secretion in culture supernatants (SNs), as well as of the corresponding toxicity against fall armyworm (Spodoptera frugiperda, JE Smith). Feeding bioassays were performed, with larvae being treated with heated and non-heated total protein extracted from SNs collected at different culture times. Larva mortality and reduction in pupa formation were observed. Intra- and interisolate variations were observed in the temporal patterns of growth, quality and quantity of protein secreted, as well as in insecticidal activity of these SNs, based on larvae mortality and pupation rates. These results suggest that the insecticidal potential of certain isolates can be hidden if comparisons are done on the basis of the same number of cells in the culture and/or the same culturing time. Methods of screening Bt collections on the basis of feeding bioassays can be misleading with regards to identifying more promising isolates for biocontrol purposes if physiological differences are not considered. The consequences and implications of these findings for the development of experimental systems that depend on toxicity bioassays to identify alternative Bt strains and entomotoxins with practical applicability have been discussed.

Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities

Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis. Glucose-, galactose-, sucrose-, lactose-, and Sepharose-binding lectins were isolated from fruiting bodies using affinity chromatography on Sepharose-immobilized sugars or on Sepharose. The lectins were characterized biochemically and their binding specificities examined by agglutination and agglutination inhibition assays. In addition, insecticidal and anti-nutritional properties of the lectins were studied against a model organism, fruit fly (Drosophila melanogaster), and Colorado potato beetle (Leptinotarsa decemlineata). Of the several basidiomycete mushrooms screened, C. nebularis extract showed the most potent insecticidal activity. Sucrose-binding lectin showed the strongest activity against D. melanogaster, followed by lactose- and galactose-binding lectins. Feeding bioassays with Colorado potato beetle revealed that C. nebularis extract exhibited high anti-nutritional activity against the insect; and of those tested, only lactose-binding lectin, named CNL showed the effect. Mushroom C. nebularis is shown to be rich in a variety of lectins with versatile biological activities, including insecticidal and anti-nutritional effects. C. nebularis lectins could thus have potential for use as natural insecticides.

Wood Consumption by Formosan Subterranean Termites (Isoptera: Rhinotermitidae) as Affected by Wood Moisture Content and Temperature

AbstractUnderstanding foraging behaviors and food preferences of termite colonies is critical to optimizing control strategies. The relationship between the moisture content (MC) of wood blocks and the feeding preference of Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) at three ambient temperatures (19, 28, and 35°C) was investigated with multiple-choice as well as no-choice feeding bioassays. In multiple-choice tests, when four wood blocks with initial MC as dry (0–3%), low MC (22–24%), medium MC (70–90%), and high MC (125–150%) were exposed to workers and soldiers, the highest consumption was found in the high MC wood blocks at all three temperatures, although high MC wood consumption at 19°C was significantly lower than that at 28 or 35°C The preference for high MC wood blocks was detectable within the first few hours and continued for the duration of the test. In no-choice tests, the highest wood consumption also was obtained on the high MC wood blocks at all three temperatures. However, in no-choice tests, significant mortality was observed with termites exposed to dry or low MC wood blocks. Termite mortality was found to be higher with higher ambient temperature. Sustained feeding was not observed on wood blocks with ≤24% MC. When wood moisture is low, no free water exists in the cell cavities, which perhaps influenced feeding choices. Temperature played a vital and complementary role for wood consumption at all moisture levels.

Development of Mamestra brassicae and its solitary endoparasitoid Microplitis mediator on two populations of the invasive weed Bunias orientalis

AbstractThe warty cabbage Bunias orientalis is an invasive pest in much of central Europe, including much of Germany since the 1980s, whereas in other countries, such as The Netherlands, it is a less common exotic species. Here, healthy larvae of Mamestra brassicae, which has been found feeding on B. orientalis plants in Germany, and larvae parasitized by one of its major larval endoparasitoids Microplitis mediator, were reared on both herbivore‐induced and noninduced leaves of B. orientalis originating from single large populations growing in The Netherlands and central Germany. Herbivore performance was less negatively affected than parasitoid performance by differences in plant quality. Development times in both M. brassicae and Mi. mediator were shorter on Dutch than German plants and also shorter on noninduced than induced plants. Moreover, survival and body size of the parasitoid was more strongly affected by plant population and induction than survival of healthy M. brassicae. Chemical analyses of defensive secondary metabolites [glucosinolates (GS)] revealed that concentrations of the major GS sinalbin were constitutively expressed in German plants whereas they were induced in Dutch plants. However, in separate feeding bioassays in which preference for induced and noninduced leaves was compared separately, L3 instars of M. brassicae preferred noninduced German plants over Dutch plants but induced Dutch plants over German plants, revealing that changes in primary metabolites or an unidentified non‐GS compound mediates population‐related differences in plant quality. The results reveal asymmetric effects of plant quality in exotic plants on organisms in the second and third trophic level.

Pinus koraiensis twigs affect Monochamus saltuarius (Coleoptera: Cerambycidae) longevity and reproduction

This study was conducted to investigate the longevity, reproduction and ovarian development of M. saltuarius adults feeding on Pinus koraiensis twigs of three different age classes (current-year, 1-year and 2-year). Longevity and fecundity of female adults decreased with increasing twig age class (58.4, 41.9 and 35.9 days, respectively, for longevity and 43.0, 13.2 and 6.6 eggs, respectively, for fecundity). In ovarian development, the average follicle size after 1 week of feeding on current-year twigs was 3.1–3.5 times greater than those of beetles fed 1- and 2-year-old twigs. After 2 weeks of feeding, follicle size was 2.3–2.4 times greater than those of beetles fed 1- and 2-year-old twigs. These results showed that current-year twigs of P. koraiensis increase longevity and reproduction of M. saltuarius. By analyzing sugars using high performance liquid chromatography (HPLC), the quantity of fructose was found to be highest in current-year twigs and lowest in 2-year-old twigs. A sugar feeding bioassay indicated that fructose was preferred by M. saltuarius. Therefore, fructose is important to adult feeding and ovariole development in M. saltuarius.

Plant invasions, generalist herbivores, and novel defense weapons

One commonly accepted mechanism for biological invasions is that species, after introduction to a new region, leave behind their natural enemies and therefore increase in distribution and abundance. However, which enemies are escaped remains unclear. Escape from specialist invertebrate herbivores has been examined in detail, but despite the profound effects of generalist herbivores in natural communities their potential to control invasive species is poorly understood. We carried out parallel laboratory feeding bioassays with generalist invertebrate herbivores from the native (Europe) and from the introduced (North America) range using native and nonnative tetraploid populations of the invasive spotted knapweed, Centaurea stoebe. We found that the growth of North American generalist herbivores was far lower when feeding on C. stoebe than the growth of European generalists. In contrast, North American and European generalists grew equally well on European and North American tetraploid C. stoebe plants, lending no support for an evolutionary change in resistance of North American tetraploid C. stoebe populations against generalist herbivores. These results suggest that biogeographical differences in the response of generalist herbivores to novel plant species have the potential to affect plant invasions.

Antixenosis in Maize Reduces Feeding by Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae)

SUM2162 is the first known example of a naturally occurring maize, Zea mays L., genotype with antixenosis (nonpreference) resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding. Behavioral responses of neonate western corn rootworm larvae were evaluated in laboratory bioassays with seven maize genotypes selected for native resistance to rootworm feeding damage. Two susceptible maize genotypes and one transgenic (Bacillus thuringiensis) maize genotype were included as controls. In soil bioassays with cut roots, no larvae entered the roots of the resistant variety SUM2162, but at least 75% of the larvae entered the roots of every other maize type. Larvae made significantly fewer feeding holes in the roots of SUM2162 than in all the other maize genotypes, except the isoline control. In feeding bioassays, larval feeding varied significantly among maize genotypes, but there was no significant difference between the resistant varieties and the susceptible controls. There were no significant differences among any of the genotypes in host recognition (search) behavior of larvae after exposure to the roots. Little variation in feeding stimulant blends was observed among maize genotypes, indicating minimal contribution to the observed antixenosis.

Growth and development of melon fruit fly larvae under the influence of soybean trypsin inhibitors

Protease inhibitors (PIs) retard growth and development and cause mortality in a range of insect pests belonging to various orders. Some transgenic plants expressing PI genes have also been effective against insect pest attack. The effects of two purified soybean PIs, i.e. trypsin inhibitor (Kunitz type; SBTI) and trypsin-chymotrypsin inhibitor (Bowman-Birk type; SBBI), were investigated on first and third instar larvae of the melon fruit fly Bactrocera Cucurbitae (Coquillett) in laboratory feeding bioassays. The larval period of both the first and third instar larvae was prolonged when the larvae were treated with various concentrations of SBTI and SBBI. The total development period prolonged significantly in both the instars of larvae treated with SBTI, but in SBBI-treated larvae, the development period was delayed significantly only in the third instar larvae. A decrease in percentage pupation and adult emergence was observed in the first and third instar larvae treated with different concentrations of both SBTI and SBBI. The present studies showed that SBTI and SBBI have a potential in inhibiting the growth and development of melon fruit fly, but further detailed and molecular level studies are required before their use in transgenics.

Development of strategies for monitoring indoxacarb and gel bait susceptibility in the German cockroach (Blattodea: Blattellidae)

Advion(®) cockroach gel bait (6 g kg(-1) indoxacarb) is in widespread use for Blattella germanica (L.) control in the United States. However, baseline susceptibility levels to indoxacarb in German cockroach field populations are not known. Hence, this research sought to develop monitoring strategies to estimate the susceptibility of German cockroach populations to indoxacarb. Four bioassays were evaluated: topical lethal dose (LD), formulated gel bait lethal time (LT), vial lethal concentration (LC) and gel bait matrix LD. Of these methods, the vial LC and gel bait matrix LD bioassays were the most economical and relevant assessment strategies. For indoxacarb susceptibility monitoring, a two-tiered approach was developed that utilizes diagnostic concentrations and doses in vial LC (30 and 60 µg vial(-1) ) and gel bait matrix LD (1.0, 1.5 and 2.5 µg insect(-1) ) formats. A two-tiered susceptibility monitoring strategy was developed that includes testing field populations at diagnostic concentrations and doses in first-tier vial LC bioassays and second-tier gel bait matrix LD bioassays. The vial method facilitates rapid identification of field strains with reduced susceptibility. The feeding bioassay effectively simulates field exposure to Advion(®) and therefore has utility for secondary confirmation of susceptibility shifts and identification of behavioral resistance (i.e. bait aversion).

Potential risks of systemic imidacloprid to parasitoid natural enemies of a cerambycid attacking Eucalyptus

A community of insect herbivores has established on eucalyptus species in California following their introduction from Australia. A number of the species are under complete or partial biological control. A response to introduction of additional pest species into the complex has been the application of systemic insecticides to infested trees. Natural enemies that have been introduced to control the various pest species feed on the nectar of treated trees, and thus may be affected by these pesticides. In this study, Eucalyptus rudis trees were treated at label rates with the neonicotinoid systemic insecticide imidacloprid and nectar sampled at 5 months post-treatment during the spring bloom. The concentration of imidacloprid and its toxic metabolites in nectar was measured by ELISA at 660 ppb. Adults of the encyrtid egg parasitoid Avetianella longoi that were fed floral nectar collected from treated trees had significantly lower survival and reproductive fitness than adults fed nectar from untreated trees. In feeding bioassays, in which the adults were fed a range of concentrations of imidacloprid in sugar water, the LC 50 for A. longoi was 212 ppb imidacloprid. Bioassays were also conducted with the braconid larval parasitoid, Syngaster lepidus. The LC 50 for S. lepidus was 288 ppb imidacloprid. The insecticide appears to be concentrating in the nectar at higher levels than reported from other plant species and at concentrations exceeding the LC 50 for two important parasitoids. If tree treatments become widespread as a result of continual introductions of new eucalypt herbivores, established biological control programs could be at significant risk.

First insights into the chemical defensive system of the erotylid beetle, Tritoma bipustulata

The present study provides the first insights into the chemical defensive system of the erotylid beetle, Tritoma bipustulata, and furthermore reports the previously hardly known ability of abdominal reflex bleeding in this coleopteran family. The defensive chemistry of the secretion of pronotal glands, abdominal reflex blood as well as of the haemolymph were analysed by GC-MS. The different secretions were dominated by aromatic compounds; in addition, we detected alkenes, ketones, organic acids as well as a single sesquiterpene. The majority of these detected compounds had strong antimicrobial properties in microbiological assays with entomopathogenic micro-organisms. In feeding bioassays with ants, only benzyl alcohol, benzothiazole, indole and 3-methylindole, detected in the abdominal reflex blood, were significantly deterrent.

Suppression of hypernodulation in soybean by a leaf‐extracted, NARK‐ and Nod factor‐dependent, low molecular mass fraction

*Legumes regulate the number of nodules they form via a process called autoregulation of nodulation (AON). This involves a shoot-derived inhibitor (SDI) molecule that is synthesized in the shoots and is transported down to the roots where it inhibits further nodule development. *To characterize SDI, we developed a novel feeding bioassay. This involved feeding aqueous leaf extracts directly into the petiole of hypernodulating and supernodulating nark mutant plants of Glycine max (soybean). These mutants normally exhibit an increased nodulation phenotype because SDI is not produced and thus AON is nonfunctional. *Feeding wild-type leaf extracts presumed to contain SDI was successful in suppressing the increased nodulation phenotype, whereas feeding with Gmnark leaf extracts did not. Suppression activity was inoculation-dependent, Nod factor-dependent, required GmNARK activity, and was heat-, Proteinase K- and ribonuclease A-resistant. Wild-type extracts maintained suppressive activity even at a ninefold dilution. Sinorhizobium meliloti-inoculated Medicago truncatula leaf extracts from wild-type, but not from supernodulating mutant Mtsunn, suppressed hypernodulation in soybean. *Our results demonstrate that the petiole feeding bioassay is an efficient and effective technique to introduce aqueous extracts into plants. They also demonstrate that SDI is a small compound with an apparent molecular mass of < 1000 Da and is unlikely to be a protein or an RNA molecule.

An evaluation of sponge-associated amphipods from the Antarctic Peninsula

AbstractNearshore marine benthic algal communities along the western Antarctic Peninsula harbour extremely high densities of amphipods that probably play important roles in nutrient and energy flow. This study extends our evaluation of the importance of amphipods in the nearshore Antarctic Peninsular benthic communities and focuses on sponge associations. We found a mean density of 542 amphipods per litre (L) sponge for twelve species of ecologically dominant sponges. The highest mean density (1295 amphipods per L sponge) occurred withDendrilla membranosaPallas. The amphipod community associated with the 12 sponges was diverse (38 species), with mean species richness values ranging from two to eight species. Mean Shannon diversity indices (H’) ranged from 0.52 to 1.49. Amphipods did not appear to have obligate host relationships. Qualitative gut content analyses indicated that 12 of the 38 amphipod species were found with sponge spicules in their guts. However, only one of the amphipods,Echiniphimedia hodgsoniWalker, had considerable amounts of spicules in the gut. Organic lipophilic and hydrophilic extracts of the twelve sponges were presented in alginate food disks to a sympatric omnivorous amphipod in feeding bioassays and extracts of only two sponges deterred feeding.