Mobilization of soil phosphorus during passage through the gut of larvae of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

Soil macrofauna play important roles in the mobilization of soil nutrients. The influence of gut passage on soil phosphorus availability was investigated using the larvae of the scarabaeid beetle Pachnoda ephippiata. Gut passage significantly increased the levels of available P in the feces over that of the soil consumed. ³¹P-NMR recorded the changes in the nature of organic P in alkaline extracts from gut content. Alkaline phosphatase activity was high in the alkaline midgut, but low in the hindgut and soil. Ferric iron (Fe³⁺) in the soil was reduced to ferrous iron (Fe²⁺) in the gut. The amount of Ca²⁺ was lower in the gut than in the soil. Iron reduction and the decrease of Ca²⁺ concentration in the gut would reduce P sorption. We think that the following effects are responsible for increased availability of P during the gut passage: alkaline extraction of soil organic matter; hydrolysis of organic phosphate esters with alkaline phosphatase; digestion by the resident microorganisms; and changes in the concentration of metal ions during gut passage. This study suggests that the feeding activities of humivorous larvae would affect the amount of soil P available to plant growth.     

Transformation and mineralization of soil organic nitrogen by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In common bean (Phaseolus vulgaris L.), Fusarium root rot (caused by Fusarium solani f. sp. phaseoli) disease severity is increased by environmental factors that stress the plant. The current study used reciprocal grafting techniques with the resistant cultivar FR266 and the susceptible cultivar Montcalm to determine if the genetic control of resistance is conferred by the rootstock (root genotype) or the scion (shoot genotype) and if root vigor played a role in resistance. The influence of a compacted layer on root and shoot genotype response and root rot resistance was studied. Root rot resistance was found to be controlled by the root genotype, such that on a scale of 1 to 7 (severe disease) the FR266 root had an average score of 2.3 and the Montcalm root had an average score of 4.4. However, when grafted plants were grown in the presence of a compacted layer, the FR266 root and/or shoot genotype in any graft combination with the susceptible Montcalm had reduced root rot (score = 2.4 average) than the Montcalm self graft (score = 4.5). Root mass was shown to be controlled by the root genotype in the absence of compaction such that the FR266 root was 26% larger that the Montcalm root when grafted onto a FR266 shoot or a Montcalm shoot. When a compacted layer was present the root and shoot genotype both contributed to root mass. Average root diameter was controlled by the shoot genotype, as the FR266 shoot grafted to Montcalm or FR266 roots had thicker roots (average diameter 0.455 mm) than the Montcalm shoot (average diameter 0.418 mm). This study shows evidence that root vigor in the presence of Fusarium disease pressure should be evaluated to effectively develop common bean lines resistant to Fusarium root rot across a range of environments.     

Promicromonospora pachnodae sp. nov., a member of the (hemi)cellulolytic hindgut flora of larvae of the scarab beetle Pachnoda marginata

Intestinal microorganisms play an important role in plant fiber degradation by larvae of the rose chafer Pachnoda marginata. In the hindgut of the larvae 2.5 to 7.4 × 10⁸ bacteria per ml of gut content with xylanase or endoglucanase activity were found. Bacteria in the midgut were not (hemi)cellulolytic, but the alkaline environment in this part of the intestinal tract functions as a precellulolytic phase, solubilizing part of the lignocellulosic material. Accordingly, the degradation of lignocellulose-rich material in Pachnoda marginata larvae appeared to be a combination of a physico-chemical and microbiological process. A number of different facultative anaerobic and strictly anaerobic bacteria with (hemi)cellulolytic activity were isolated from the hindgut. A dominant (hemi)cellulolytic species was a Gram positive, irregular shaped, facultative aerobic bacterium. Further physiological identification placed the isolate in the genus Promicromonospora. Comparative 16S rDNA analysis and phenotypic features revealed that the isolate represented a new species for which the name Promicromonospora pachnodae is proposed. P. pachnodae produced xylanases and endoglucanases on several plant derived polymers, both under aerobic and anaerobic conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Reproductive properties of two sympatric dung beetles,Aphodius haroldianus andA. Elegans (Coleoptera: Scarabaeidae)

1. Reproductive properties of two sympatric dung beetles, Aphodius haroldianus and A. elegans, were studied at a pasture in the central Japan.
2. Overwintered adults of A. haroldianus came flying to dung pats from May to early August and bred from June to July. A. elegans overwintered as larvae, new adults came to dung pats from mid May to mid June. After aestivation, they reproduced from October to November.
3. A. haroldianus was a species with low fecundity and large food reserve for larvae. Female of this species had short duration of residence and laid fewer number of eggs in one dung pat. On the other hand, A. elegans was a species with high fecundity and small food reserve for larvae. Female of this species had long duration of residence and laid larger number of eggs in one dung pat.
4. The daily egg production in A. haroldianus reached a peak (0.40 eggs/female/day) at middle of oviposition period, but, that in A. elegans reached a peak (6.49 eggs/female/ day) at the beginning of oviposition period. The mortality of A. haroldianus female occurred after the daily egg production reached a peak and then rapidly increased, whereas that of A. elegans began soon after the beginning of oviposition and then gradually increased.

     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

The influence of amino acids and inorganic salts on feeding by larvae of Heteronychus arator (Coleoptera: Scarabaeidae)

Abstract

Twenty amino acids were offered at 2 concentrations to Heteronychus arator F. larvae in an agar/cellulose powder medium. In comparison with the plain medium, only 0.1M l-alanine, 0.01M l-aspartic acid, and 0.01M l-glutamic acid significantly stimulated feeding. A mixture of inorganic salts did not induce feeding when presented in the medium alone, nor did it affect the vigorous feeding response of larvae to 0.1M maltose.     

Structure and topology of microbial communities in the major gut compartments of Melolontha melolontha larvae (Coleoptera: Scarabaeidae)

Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O2, H2, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut. Methanobrevibacter spp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to the Clostridiales. Clones related to the Actinobacteria, Bacillales, Lactobacillales, and gamma-Proteobacteria were restricted to the lumen, whereas clones related to the beta- and delta-Proteobacteria were found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented that Desulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5'-phosphosulfate reductase gene apsA to DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.     

Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae

Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with specific toxicity is of great importance to biological control of insect pests. In this study, by screening 66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene from Bt strain B-JJX was identified via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73^?. The open reading frame of the cry8Ab1 gene consists of 3543 bp with a G + C content of 37.99% and encodes a protein of 1180 amino acids with a putative MW of 133.3 kDa which was confirmed by SDS-PAGE analysis. The Cry8Ab1 protein was expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73^? along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid pests, Holotrichia oblita and H. parallela, with an LC₅₀ of 5.72 and 2.00 μg toxin g^?1 soil, respectively. The results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC₅₀ of 1.72 and 0.96 μg toxin g^?1 soil against H. oblita and H. parallela, respectively.     

Rate of soil egestion by larvae of Hylamorpha elegans (Burm.) and Phytoloema hermanni Germ. (Coleoptera: Scarabaeidae)

Larvae of Hylamorpha elegans (Burm.) and Phytoloema herrmanni Germ. feed on roots, although many Scarabaeidae species are able to feed and survive on soil without living roots. In this study we evaluated the potential of H. elegans and P. herrmanni to ingest soil by estimating the rate of soil egestion. In the laboratory, the rate of soil egestion was determined from gut content and gut transit time of 3rd-instar larvae feeding on soil without roots. The soil egestion rate was 14-21 mg g(-1) d(-1) for H. elegans and 11-16 mg g(-1) d(-1) for P. herrmanni. The gut transit time (time of soil passage from mouth to anus) was 2-3 d for both species and the gut content was 41±2 mg g(-1) for H. elegans and 32±2 mg g(-1) for P. herrmanni. The quantitative importance of feeding activity on soil depends upon the population density of larvae in the field, which ranges from 1 to 25 larvae m(-2), but in severe outbreaks can reach 300 larvae m(-2). High population densities could result in soil egestion rates reaching 20 kg d(-1) ha(-1) for P. herrmanni and 30 kg d(-1) ha(-1) for H. elegans.     

Partial purification and characterization of the major midgut proteases of grass grub larvae (Costelytra zealandica,Coleoptera: Scarabaeidae)

The major proteases of the grass grub (Costelytra zealandica) larval midgut have been identified, partially purified and characterized. Identification was made initially on the basis of hydrolysis of synthetic substrates (blocked and partially blocked esters and amides of specific amino acids), thus classifying the activities into different classes of endo- and exopeptidases. A range of inhibitors specific to different classes of proteases were used to confirm the presence of trypsin, chymotrypsin, elastase, leucine aminopeptidase and carboxypeptidases A and B and to establish the absence of thiol- and metallo-endopeptidases. The dominant endopeptidase in the midgut is trypsin, which is present in four forms, distinguishable by net charge, but indistinguishable either in terms of Michaelis-Menten parameters (K_m and k_cat) or in molecular weight (23,000). The pH optimum lies between pH 9–10. Leucine aminopeptidase has a molecular weight of 91,000 and a pH optimum at pH 8.0. Carboxypeptidase A has a molecular weight of 43,000 and a pH optimum at pH 8.5. All enzymes retained substantial activity at pH 7.0–7.1, the pH of the midgut lumen, where the bulk of the activity was located. Protease levels in the hindgut (or fermentation sac) were 1–5% of those in the midgut. The range of enzymes appears sufficient for complete breakdown of ingested protein.     

Sibling and non-sibling cannibalism by larvae of a lady beetleHarmonia axyridis Pallas (Coleoptera: Coccinellidae) in the field

Sibling (cannibalism among siblings) and non-sibling cannibalism (cannibalism among non-siblings) were studied in a natural population of a lady beetle, Harmonia axyridisPallas . Of all the eggs laid (n=2269), 24.76% (n=562) were killed by sibling cannibalism and 36.10% (n=819) were killed by non-sibling cannibalism. Sibling cannibalism occurred constantly and intensively in most egg batches throughout the entire oviposition period. On the other hand, non-sibling cannibalism was more intense in the middle and late oviposition periods, and when the egg batches were close to an aphid colony. This may be due to the high density of H. axyridis larvae relative to aphid density in the middle and late oviposition periods and also the larvae searching intensively near an aphid colony.     

Effect of population density on reproduction of two sympatric dung beetle species,Aphodius haroldianus andA. Elegans (Coleoptera: Scarabaeidae)

The effect of population density on reproduction as well as the oviposition modes of two sympatric dung beetle species, Aphodius haroldianus (a low fecundity/high parental effort species) and A. elegans (a high fecundity/low parental effort species) was studied at a pasture in central Japan from 1982 to 1986.

1. The adult population density of A. haroldianus was high (>40 per dung pat). The density of A. elegans was low (<5 per dung pat). Oviposition of A. haroldianus was suppressed above the density of about 10 adults of conspecifics but not affected by the density of other species. The intra- and interspecific density effects on oviposition were not detected in A. elegans.
2. The number of A. haroldianus adults per pat decreased with dung age, while that of A. elegans increased until sixth day after deposition and then decreased. The number of eggs laid per pat was not different between 1-day-and 3-day-old pats for A. haroldianus. However, more eggs of A. elegans were found in 3-day-old pats than in 1-day-old ones.
3. In the both species, the amplitude of population fluctuations was not remarkable; the maximum/minimum ratio for five years being 2.3 for A. haroldianus and 2.9 for A. elegans. Different density dependent processes were suggested to function for the small fluctuation of population density between the two species, i.e. intraspecific density effect on oviposition for A. haroldianus and contest type competition for food among larvae of A. elegans.

     

Site‐specific profiles of biochemical properties in the larval digestive tract of Japanese rhinoceros beetle,Trypoxylus dichotomus (Coleoptera: Scarabaeidae)

The larvae of Japanese rhinoceros beetle, Trypoxylus dichotomus, feed on dead plant material in forest soils that are derived from fallen leaves broken down by basidiomycete fungi. Our previous work provided an understanding of the degradation of polysaccharides in dead plant material by T. dichotomus larvae and reported the complexity of the physicochemical and biochemical environment of the larval gut. Here, we examined ten divisions of the digestive tract of T. dichotomus larvae for physicochemical and biochemical conditions to elucidate site‐specifically functional properties along the tract. The distribution of potassium ions, pH, and acetic acid differed markedly along the length of the digestive tract with the potassium ion concentration profile closely reflecting that of pH along the length of the digestive tract. Distinct physicochemical environments were maintained in the digestive tract along with site‐specific polysaccharide degradation. Based on these findings, we suggest that there are metabolic relationships between the activities of the enzymes involved in polysaccharide degradation, the presence of intermediate metabolites and location along the digestive tract. Furthermore, we revealed that the anterior region of the gut plays an important role in the degradation of polysaccharides in the digestive tract of T. dichotomus larvae.     

Preference of temperate Chinese elms (Ulmus spp.) for the adult Japanese beetle (Coleoptera: Scarabaeidae)

Trees of newly acquired Ulmus species originating from the temperate regions of China and growing at The Morton Arboretum, Lisle, IL, were evaluated in no-choice and multiple-choice laboratory bioassays and cage-studies for feeding preference for adults of the Japanese beetle, Popillia japonica Newman. Ulmus lanceaefolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis were less preferred for feeding by adult Japanese beetles. No-choice, multiple-choice, and cage feeding studies revealed that Ulmus wallichiana was moderately preferred, and U. procera and U. americana were highly preferred for feeding. Dry fecal pellet weights were strongly correlated with the percent of leaf tissue removed. Feeding preference in the multiple-choice study was strongly correlated with feeding preference in the cage study and in the no-choice study. Likewise, adult feeding preference in the cage study was strongly correlated with preference in the multiple-choice and no-choice studies. The less preferred species of U. lanceafolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis show promise for future elm breeding programs.     

Diel pattern in the structure and function of the gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae

In the present study, diel pattern in gut microbial communities in insects were evaluated. Lymantria dispar asiatica fourth instar larvae (72 ± 2 hr after molting) at noon (LdD) and midnight (LdN) were used for a comparative analysis of the gut microbial community. Ten bacterial operational taxonomic units (OTUs) were shared between LdD and LdN samples. One bacterial OTU was specific to LdD. The dominant gut microbes were OTU72 in LdD and OTU75 in LdN. A linear discriminant analysis effect size cladogram suggested that ten bacterial OTUs maintain significant differences in relative abundances between LdD and LdN. These results agreed with the discrete ellipses between LdD and LdN in principal coordinates analysis plots. Additionally, using phylogenetic investigation of communities by reconstruction of unobserved states, the gut microbial community was assigned to 23 functional terms, among which 22 exhibited significant differences between LdD and LdN. To conclude, the present study documented a diel pattern in the gut microbial community of L. dispar asiatica larvae.     

Characterization of cellulose degrading bacteria from the larval gut of the white grub beetle Lepidiota mansueta (Coleoptera: Scarabaeidae)

The goal of this study is to identify and characterize the cellulose degrading microorganisms in the larval gut of the white grub beetle, Lepidiota mansueta. Thirty bacterial strains were isolated and tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays. Of these strains, five (FGB1, FB2, MB1, MB2, and HB1) degrade cellulose. Cellulolytic activity was determined based on formation of clear zone and cellulolytic index on CMC plate media. The highest cellulolytic index (2.14) was found in FGB1. Partial 16S rDNA sequencing, morphological, and biochemical tests were used to identify and characterize the five isolates, all Citrobacter sp. (Enterobacteriaceae). This study identifies new cellulose degrading microorganisms from the larval gut of L. mansueta. The significance of identifying these strains lies in possible application in cellulose degradation.     

Interaction between halofenozide and the entomopathogenic nematode Heterorhabditis marelatus for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae

Japanese beetle, Popillia japonica Newman is a major pest of turf and ornamentals. Laboratory bioassays were conducted to evaluate the potential interactions between a biological control agent, Heterorhabditis marelatus (Nematoda: Heterorhabditidae) IN strain and the insecticide halofenozide against both overwintered and nonoverwintered 3rd instars of Japanese beetle. Treatments consisted of all combinations of 2 rates of halofenozid with H. marelatus nematodes Imidacloprid was used as a standard. Percentage larval mortality was evaluated at 7, 14, and 21 d after treatment. No deleterious effects were observed. The nematode treatments generally produced significantly greater larval mortality relative to both chemical treatments. Twenty-one days after treatment, both rates of nematodes resulted in 100% mortality, whereas insecticide treatments did not surpass 60% mortality. No synergism was detected in any of the combination treatments. There were no significant differences in nematode reproduction in larvae exposed to halofenozide and nematodes versus larvae exposed to only nematodes.     

Nosema takapauensis n.sp., a microsporidan parasite of larvae of Costelytra zealandica (Coleoptera: Scarabaeidae) in New Zealand

Morphological and biological features of a microsporidan protozoan parasite of larvae of Costelytra zealandica collected at Takapau, southern Hawkes Bay, are described and evaluated taxonomically. The parasite multiplies in the fat body of all larval instars, causing massive tissue disintegration in advanced infection resulting in the retardation of development and ultimately death before pupation. The microsporidan forms one spore per sporont, and therefore belongs to genus Nosema’, it is considered to be specifically distinct from its nearest congener, N. melolonthae.     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.