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 γ-Proteobacteria were restricted to the lumen, whereas clones related to the β- and δ-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.     

Characterization of the proteolytic enzymes in the midgut of the European Cockchafer,Melolontha melolontha (Coleoptera: Scarabaeidae)

In previous studies we showed that the resistance of the European Cockchafer, Melolontha melolontha, towards the Scarab specific Cry8C toxin of Bacillus thuringiensis japonensis strain Buibui is due to the complexity of proteinases in the midgut of the pest insect. In this study these proteinases were identified and characterized using a combination of synthetic substrates and specific inhibitors in zymograms, activity blots, and photometric/fluorometric assays. In the midgut juice three trypsin-like and three elastase-like serine proteinases are predominantly present. In addition, two metalloendoproteinases were detected. At least one of them is most likely to belong to the astacin family, proteinases which normally do not play a role in general protein digestion outside the decapod crustacean. Furthermore, a free aminopeptidase as well as a membrane-associated aminopeptidase, isolated from the brush boarder membrane vesicles (BBMV) of the midgut epithelium, were characterized.     

Specialized ommatidia for polarization vision in the compound eye of cockchafers,Melolontha melolontha (Coleoptera,Scarabaeidae)

Summary The superposition eye of the cockchafer, Melolontha melolontha, exhibits the typical features of many nocturnal and crepuscular scarabaeid beetles: the dioptric apparatus of each ommatidium consists of a thick corneal lens with a strong inner convexity attached to a crystalline cone, that is surrounded by two primary and 9–11 secondary pigment cells. The clear zone contains the unpigmented extensions of the secondary pigment cells, which surround the cell bodies of seven retinula (receptor) cells per ommatidium and a retinular tract formed by them. The seven-lobed fused rhabdoms are composed by the rhabdomeres of the receptor cells 1–7. The rhabdoms are optically separated from each other by a tracheal sheath around the retinulae. The orientation of the microvilli diverges in a fan-like fashion within each rhabdomere. The proximally situated retinula cell 8 does not form a rhabdomere. This standard form of ommatidium stands in contrast to another type of ommatidium found in the dorsal rim area of the eye. The dorsal rim ommatidia are characterized by the following anatomical specializations: (1) The corneal lenses are not clear but contain light-scattering, bubble-like inclusions. (2) The rhabdom length is increased approximately by a factor of two. (3) The rhabdoms have unlobed shapes. (4) Within each rhabdomere the microvilli are parallel to each other. The microvilli of receptor 1 are oriented 90° to those of receptors 2–7. (5) The tracheal sheaths around the retinulae are missing. These findings indicate that the photoreceptors of the dorsal rim area are strongly polarization sensitive and have large visual fields. In the dorsal rim ommatidia of other insects, functionally similar anatomical specializations have been found. In these species, the dorsal rim area of the eye was demonstrated to be the eye region that is responsible for the detection of polarized light. We suggest that the dorsal rim area of the cockchafer eye subserves the same function and that the beetles use the polarization pattern of the sky for orientation during their migrations.     

Pre-copulatory isolation in sympatric Melolontha species (Coleoptera: Scarabaeidae)

1 The two most abundant cockchafer species in Europe, the forest cockchafer Melolontha hippocastani Fabr. and the European cockchafer Melolontha melolontha L., tend to form calamitous mass breedings with casual reports on sympatric and simultaneous occurrence. 2 Both species are known to use feeding‐induced green leaf volatiles (GLV) as primary attractants (sexual kairomones) for mate finding. The attractiveness of GLV is enhanced by the sex pheromones 1,4‐benzoquinone in M. hippocastani and toluquinone in M. melolontha. Phenol attracts males from both species. All three compounds are present in females of both species. 3 In the present study, it is confirmed that only male M. melolontha perform the typical swarming flight at dusk, as has already been shown for M. hippocastani. Furthermore, whether swarming Melolontha males were cross‐attracted to heterospecific females, and whether males could discriminate olfactorily between conspecific and heterospecific females, was tested in the field. 4 Males of both species preferred females when given the choice between females and males of the other species. However, they preferred conspecific females when females from both species were offered simultaneously. 5 The results suggest that species‐specific pheromone blends contribute to precopulatory reproductive isolation in sympatric populations of M. melolontha and M. hippocastani, but are not mutually exclusive or indispensable prerequisites for mate finding as in other insects.     

The anatomy and ultrastructure of the antennal circulatory organs in the cockchafer beetle Melolontha melolontha L. (Coleoptera,Scarabaeidae)

Summary The antennal circulatory organs of Melolontha are described for the first time. They consist of small sac-like ampullae located near the base of each antenna and connected to a long non-muscular antennal blood vessel. Small branches of this vessel extend into each lamella of the antennal club and open out distally. The membranous wall of the ampulla provides no contractile structures. An outer adjacent compressor muscle is responsible for the pumping movements of the ampulla and antagonist to it is an obviously elastic connective tissue band. The position of this elastic band causes the uncontracted muscle to be pulled away from the ampulla. As a consequence blood can enter the dilated ampulla through a valvular ostium. The functional type of the antennal circulatory organs in Melolontha is compared to that found in other insects and their histologic structure is interpreted in relation to mechanical and hemodynamical aspects. Furthermore the possible function of the antennal hearts in connection with the spreading of its lamellate antennal club is discussed.Dedicated to Prof. Dr. M. Gersch's 70th birthday.Supported by project 3,106 of the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich     

Sensing the Underground - Ultrastructure and Function of Sensory Organs in Root-Feeding Melolontha melolontha (Coleoptera: Scarabaeinae) Larvae

Introduction

Below ground orientation in insects relies mainly on olfaction and taste. The economic impact of plant root feeding scarab beetle larvae gave rise to numerous phylogenetic and ecological studies. Detailed knowledge of the sensory capacities of these larvae is nevertheless lacking. Here, we present an atlas of the sensory organs on larval head appendages of Melolontha melolontha. Our ultrastructural and electrophysiological investigations allow annotation of functions to various sensory structures.

Results

Three out of 17 ascertained sensillum types have olfactory, and 7 gustatory function. These sensillum types are unevenly distributed between antennae and palps. The most prominent chemosensory organs are antennal pore plates that in total are innervated by approximately one thousand olfactory sensory neurons grouped into functional units of three-to-four. In contrast, only two olfactory sensory neurons innervate one sensillum basiconicum on each of the palps. Gustatory sensilla chaetica dominate the apices of all head appendages, while only the palps bear thermo-/hygroreceptors. Electrophysiological responses to CO₂, an attractant for many root feeders, are exclusively observed in the antennae. Out of 54 relevant volatile compounds, various alcohols, acids, amines, esters, aldehydes, ketones and monoterpenes elicit responses in antennae and palps. All head appendages are characterized by distinct olfactory response profiles that are even enantiomer specific for some compounds.

Conclusions

Chemosensory capacities in M. melolontha larvae are as highly developed as in many adult insects. We interpret the functional sensory units underneath the antennal pore plates as cryptic sensilla placodea and suggest that these perceive a broad range of secondary plant metabolites together with CO₂. Responses to olfactory stimulation of the labial and maxillary palps indicate that typical contact chemo-sensilla have a dual gustatory and olfactory function.     

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.     

Spiracle structure and function in Costelytra zealandica larvae (Coleoptera: Scarabaeidae)

The larvae of Costelytra zealandica have cribriform spiracles with a smooth spiracular plate pierced by aeropyles measuring about 5×0.2 μm. The contact angle of water on the sclerotised cuticle of the spiracular plate is sufficiently high to prevent the aeropyles from filling with water even under the wettest conditions. The spiracles have no closing apparatus, but this has little effect on transpiration rate.     

Physicochemical conditions and microbial activities in the highly alkaline gut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

The soil macrofauna plays an important role in the carbon and nitrogen cycle of terrestrial ecosystems. In order to gain more insight into the role of the intestinal microbiota in transformation and mineralization of organic matter during gut passage, we characterized the physicochemical conditions, microbial activities, and community structure in the gut of our model organism, the humus-feeding larva of the cetoniid beetle Pachnoda ephippiata. Microsensor measurements revealed an extreme alkalinity in the midgut, with highest values (pH > 10) between the second and third crown of midgut ceca. Both midgut and hindgut were largely anoxic, but despite the high pH, the redox potential of the midgut content was surprisingly high even in the largest instar. However, reducing conditions prevailed in the hindgut paunch of all instars (E(h) approximately -100 mV). Both gut compartments possessed a pronounced gut microbiota, with highest numbers in the hindgut, and microbial fermentation products were present in high concentrations. The stimulation of hindgut methanogenesis by exogenous electron donors, such as H(2), formate, and methanol, together with considerable concentrations of formate in midgut and hemolymph, suggests that midgut fermentations are coupled to methanogenesis in the hindgut by an intercompartmental transfer of reducing equivalents via the hemolymph. The results of a cultivation-based enumeration of the major metabolic groups in midgut and hindgut, which yielded high titers of lactogenic, propionigenic, and acetogenic bacteria, are in good agreement not only with the accumulation of microbial fermentation products in the respective compartments but also with the results of a cultivation-independent characterization of the bacterial communities reported in the companion paper (M. Egert, B. Wagner, T. Lemke, A. Brune, and M. W. Friedrich, Appl. Environ. Microbiol. 69:6659-6668, 2003).     

Chemical attractants for larvae of Costelytra zealandica (Coleoptera: Scarabaeidae)

In controlled laboratory tests, using shallow glass chambers containing a coarse river sand substrate between ‘blank’ and ‘test’ sample cages, 3rd‐instar larvae of Costelytra zealandica (White) were attracted to parsnip and carrot root, freshly ground nutmeg, and ß‐pinene (a constituent common to these materials). The terpenol a‐fenchol was strongly attractive to the larvae. Ryegrass root, α‐pinene, and limonene were not significantly attractive, and limonene may indeed have some repellent effect.     

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.     

Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae)

Humivorous scarab beetle larvae can thrive exclusively on soil organic matter. Feeding experiments have revealed that the larva of Pachnoda ephippiata mineralizes all major humus components except the polyphenolic fraction. High proteolytic activity in the alkaline midgut fluid and an enormous ammonia production during gut passage suggested that peptidic soil components are an important dietary resource for the larva. By comparing acid-hydrolyzable amino acids in food soil and feces, we showed that a significant fraction of the peptides in soil are removed during gut passage. This agrees well with the high concentrations of free amino acids found the midgut section. Incubation experiments revealed the presence of substantial particle-associated proteolytic activity also in the hindgut, most probably due to microbial activity. High rates of ammonia formation in hindgut homogenates and the conversion of radiolabeled amino acids to acetate and propionate indicated that microbial fermentations of soil peptides play an important role in the hindgut. This was corroborated by viable counts of amino-acid-fermenting bacteria, which formed a substantial fraction of the hindgut microbiota. A complete inventory of organic and inorganic nitrogen species before, during, and after gut passage revealed the formation of nitrite and nitrate in midgut and hindgut, and a substantial nitrogen deficit in the feces, suggesting that part of the ammonia formed by mineralization is subjected to oxidation and subsequent denitrification to N2. Together, the results strongly support the hypothesis that peptidic soil components form a major energy and nutrient source for humivorous insects, supplying the animal with microbial fermentation products and essential amino acids.     

Control decision rule for European chafer (Coleoptera: Scarabaeidae) larvae in field corn

After greenhouse and outdoor microplot experiments, a critical density of two third instars per microplot for third instars of European chafer, Rhizotrogus (Amphimallon) majalis (Razoumowsky), in corn, Zea mays L., was derived. On average, the number of missing or damaged plants increased approximately 8% from zero to two larvae per 900 cm2. Furthermore, 23 fields in 2 yr were sampled for larvae along transects by using a golf cup cutter as the sampling tool and the critical density of 0.2 larva per sampling unit as the critical density. The sampling unit was one golf cup cutter with a diameter of 10.8 cm or 91.4 cm2 (10 sampling units approximately 900 cm2 approximately 1 foot2). Fieldwide means and variation were modeled to Taylor's power law, a = 1.42 and b = 1.47, and 20 of 23 fields fit the negative binomial probability distribution. Wald's formula for a sequential sampling plan was most accurate and least time-consuming, according to the operating characteristic and the average sample number function, relative to Iwao's and converging lines formulae. Percentage of sand, topography, soil bulk density, and proximity to trees were measured as potential predictors of areas with high larval density. Percentage of sand and soil bulk density were significant predictors, and topography and proximity to trees were not significant predictors. Field areas where the percentage of sand is high and the soil bulk density is low to moderate or where the percentage of sand is moderate and the soil bulk density is low should be chosen as sampling locations.     

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae)

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.     

Growth energetics in relation to temperature of the larvae of Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Summary Instantaneous energy budgets were constructed at a range of constant temperatures (7.5°–27.5°C) for the larval stages of the scarabaeid Rhopaea verreauxi. It was found that as larvae increased in size the temperature optima/maxima for the components of the energy budget shifted to lower temperatures. Also, as larvae increased in size the instantaneous assimilation efficiency (A/C) decreased and the temperature range over which energy could be assimilated narrowed. Within this narrowing range, temperature was found to have an increasingly greater influence upon A/C. This was attributed to its influence upon the post-consumption energetics processes rather than upon consumption itself. The instantaneous net production efficiency (P/A) also decreased with increasing body size. Also, the temperature range over which assimilated energy could be partitioned to growth production became narrower as body size increased. These findings are discussed in relation to those of other energy budget studies. Some comment is made on the importance of temperature acclimation in studies such as this, and on the relation of energetics conversion efficiencies to ectothermy and endothermy and to trophic status. It was concluded that in terms of instantaneous conversion efficiences R. verreauxi could be described as a typical ectothermic herbivore, a moderately efficient converter.