Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae)

Aims: To locate and identify putative autochthonous bacteria within the grass grub gut that may have a role in symbiosis. Methods and Results: Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the grass grub larval gut. The microbial community profiles from five geographically distinct populations were compared and the influence of feeding was analysed. Bacterial community in the midgut was highly variable between locations and was affected by feeding. The hindgut contained a more diverse but stable bacterial community that was less affected by external conditions. Forty-seven distinct DGGE bands, representing different bacterial genotypes, could be distinguished from all samples, with 34 different bands occurring in the hindgut. The 22 most common bands were isolated and DNA was sequenced. Sequence analysis revealed that most bacteria (16/22) were affiliated to the Clostridiales with the predominant bacteria affiliated to the genus Clostridium. The remaining bacteria were aligned to the Betaproteobacteria, Deltaproteobacteria and Bacteroidetes. Conclusions: The grass grub larva has an autochthonous microflora with predominance of Clostridium spp. in the hindgut. Significance and Impact of the Study: Occurrence of an autocthonous microflora in the grass grub hindgut suggests a symbiotic relationship which could help explain the ability of larval scarabs to feed on recalcitrant organic matter.     

Rearing Costelytra zealandica (Coleoptera: Scarabaeidae)

Interaction between larvae can be a cause of mortality when scarabaeid larvae are concentrated in a confined volume of soil. Larvae of Costelytra zealandica (White), the New Zealand grass grub, were held for 30 days at population densities between 1 and 50 larvae per 200 ml of medium, and the effect of crowding on their survival and weight change was assessed. Larval density had no effect on survival in soil plus chopped sheep dung (3 :1), with up to 10 2nd‐instar larvae or 5 3rd‐instar larvae per 200 ml of medium. Larval survival and weight gain were increased by adding germinating ryegrass seed (Grasslands Ruanui) to the medium base, but the effect of density did not diminish. Weight gain of 3rd‐instar larvae decreased as their density increased. When the amount of germinating ryegrass seed in the medium was varied, there was no significant difference between the survival of larvae held in treatments with 5, 25, and 50 g of seed per litre of medium. Survival was significantly lower with 0 and 100 g of seed per litre of medium. It is concluded that, in rearing conditions, the final density of fully grown 3rd‐instar larvae should be a maximum of about 20 per litre of medium to prevent mortality caused by larval interaction—provided that adequate food is available.     

Feeding behaviour of the grass grub Costelytra zealandica (White) (Coleoptera: Melolonthinae)

The feeding response of 3rd‐instar Costelytra zealandica larvae to 20 amino acids tested individually at 2 concentrations was assessed. Six of these compounds had a phagostimulatory effect; in particular, the ubiquitous amino acids L‐aspartic acid, L‐glutamic acid, and L‐serine induced considerable feeding by the grubs. Ascorbic acid was also a strong feeding stimulant and, in combination with sucrose, evoked an intense behavioural response. A synergistic effect of ‘Salt Mix W on the larval response to sucrose was demonstrated.     

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.     

Electroantennogram responses of male Costelytra zealandica (Coleoptera: Scarabaeidae) to phenol and related compounds

Electroantennograms recorded from male New Zealand grass grub beetles, Costelytra zealandica (White), were used to compare the activity of phenol with that of closely related phenolics. These compounds had no masking effect on the antennal response. A comparison of activity between phenol and a crude female extract indicated that phenol is the sole pheromone.     

An iridescent virus and a rickettsia from the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae)

Natural iridescent virus and rickettsia infections of Costelytra zealandica (White) and Odontria sp. indet. larvae were studied at a site in the upper Pareora Gorge scenic reserve, S. Canterbury. By sequentially sampling the site, it was found that neither the iridescent virus nor the rickettsiae appear to give rise to host mortalities that significantly alter the population density. Many larvae were found that appeared healthy, but carried inapparent iridescent virus infections. These diseases are not suitable for biological control of the grass grub.     

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.     

Arhopalus ferus (Coleoptera: Cerambycidae): structure and function of the female reproductive system

Abstract

Arhopalus ferus (Mulsant) female pupae and adults were dissected, and both internal and external genitalia are described. The external genitalia involve segments 8 and 9. Segment 9 and its appendages are modified into a long ovipositor which telescopes with segment 8 into segment 7 when retracted. The internal genitalia include ovaries, bursa copulatrix, spermatheca, spermathecal gland, vagina, and accessory glands. The terminology applied to coleopteran genital anatomy is critically discussed.     

Olfactory response of Costelytra zealandica (Coleoptera: Melolonthinae) to the roots of several pasture plants

The influence of natural plant odours on the locomotory behaviour of 3rd‐instar larvae of Costelytra zealandica (White) was studied by observing their movement in glass‐sided test chambers. Through an analysis of the paths followed by individual larvae in single‐option choice‐chamber tests it was possible to evaluate the responses of the larvae to different plant materials. The odour of fresh young perennial ryegrass (Loliumperenne) root was more attractive to the insects than was that of older plants. Larvae were also more strongly attracted to the root of lucerne (Medicago sativa), Lotus pedunculatus, red clover (Trifolium pratense), and white clover (T. repens) than to that of perennial ryegrass. The possible role of volatile chemical factors in plant resistance to grass grub attack is discussed.     

Influence of low temperature on pupation induction in Costelytra zealandica (Coleoptera: Scarabaeidae)

Costelytra zealandica (White) lives within the soil throughout its life cycle, except for brief, crepuscular excursions to the surface by the adults to mate and feed. Larvae collected more than 18 weeks before 50% field pupation did not pupate unless they were artificially chilled. Such chilling had no significant effect on the percentage pupation of larvae collected after this time; it is assumed that these larvae had received sufficient chilling from their environment to induce pupation. Larger larvae were more likely to pupate than smaller larvae during the period 13–7 weeks before 50% field pupation. It is postulated that larvae remain quiescent until the soil temperature increases in the spring, and pupate only then. Such a process would explain how the adults of a population emerge at about the same time.     

Two Newly Recorded Coprophagid Species from Korea (Coleoptera, Scarabaeidae and Aphodiidae)

A species of Scarabaeidae, Panelus parvulus Waterhouse and a species of Aphodiidae, Aphodius fossor (Linné) are newly recorded from Korea. The diagnosis and photographs of them are provided.     

Substituted phenols as repellents for male Costelytra zealandica (Goleoptera: Scarabaeidae) in mating flights

Methyl‐ and halogen‐substituted phenols were found to be strongly repellent to male Costelytra zealandica (White) beetles in mating flights. Phenols substituted in the 2‐position were stronger repellents than those substituted in the 3‐ or 4‐position. Some of these compounds reduced the effectiveness of a potent sex attractant source by > 90 %.     

The function and evolution of male and female genitalia in Phyllophaga Harris scarab beetles (Coleoptera: Scarabaeidae)

Genitalia diversity in insects continues to fuel investigation of the function and evolution of these dynamic structures. Whereas most studies have focused on variation in male genitalia, an increasing number of studies on female genitalia have uncovered comparable diversity among females, but often at a much finer morphological scale. In this study, we analysed the function and evolution of male and female genitalia in Phyllophaga scarab beetles, a group in which both sexes exhibit genitalic diversity. To document the interaction between male and female structures during mating, we dissected flash‐frozen mating pairs from three Phyllophaga species and investigated fine‐scale morphology using SEM. We then reconstructed ancestral character states using a species tree inferred from mitochondrial and nuclear loci to elucidate and compare the evolutionary history of male and female genitalia. Our dissections revealed an interlocking mechanism of the female pubic process and male parameres that appears to improve the mechanical fit of the copulatory position. The comparative analyses, however, did not support coevolution of male and female structures and showed more erratic evolution of the female genitalia relative to males. By studying a group that exhibits obvious female genitalic diversity, we were able to demonstrate the relevance of female reproductive morphology in studies of male genital diversity.     

中国四种蜣螂幼虫形态记述(鞘翅目:金龟科:金龟亚科)

基于3龄幼虫首次描述了中国4种蜣螂幼虫的形态特征,分别是：捷氏凯蜣螂Caccobius jessoensis Harold、短亮凯蜣螂C．brevis Waterhouse、双顶嗡蜣螂Onthophagus bivertex Heyden和小驼嗡蜣螂0．gibbulus（Pallas）。文中列出了中国凯蜣螂和嗡蜣螂属幼虫分种检索表。标本保存在沈阳农业大学昆虫博物馆。     

Behavioural and physiological responses of grass grub larvae (Costelytra zealandica) feeding on protease inhibitors

Abstract

Growth of first instar Costelytra zealandica larvae was significantly reduced after 6 weeks when reared on an artificial diet containing 0.3 and 1% soybean trypsin inhibitor (SBTI), 0.1% and 0.3% potato inhibitor II, and 0.3% potato protease inhibitor I and cowpea trypsin inhibitor. Limabean trypsin inhibitor at 1% significantly stimulated growth compared with that on diet with corresponding levels of added casein. A direct relationship between increased free-trypsin activity and decreased larval growth was observed. Sequential measurement of enzyme activity in third instar larvae feeding on SBTI was compared with that of larvae feeding on casein. The increase in enzyme activity was observed after 14 days in larvae feeding on SBTI. Larvae preferred to feed on SBTI-free diet when given a choice between diet containing this inhibitor at 0.3% and added casein at 0.3%.     

The biology and life history of currant clearwing,Synanthedon tipuliformis (Lepidoptera: Sesiidae), in Canterbury

Abstract

Details of the life cycle and biology of currant clearwing, Synanthedon tipuliformis (Clerck), in Canterbury, New Zealand, are discussed. The adults live for about 7 days, and are found from mid November to early January. The sex ratio approximates 1:1. Adults are active only when the air temperature exceeds 15°c, and peak flight activity is observed on cloudy days at 20-23°c. Males are attracted to females between 1400 h and 1600 h, and mating lasts for over 2 h. Fecundity averaged about 100 eggs per female, and fertility exceeded 98%. Egg incubation required an average of 19 days in the field; the thermal constant is 91 day degrees. The mean number of eggs per blackcurrant cane varied in different years from 2.68 to 13.54; there was no significant difference between blackcurrant varieties in the same year. Larvae entered the cane through openings or axillary buds and then fed on the pith until late April or May. Quiescence occurred during winter, and feeding resumed in late August or September. Larvae tunnelled 15.20 ± 0.74 cm of pith. It is suggested that there are six larval instars. Mean numbers of larvae per cane varied in the early stage from 1.91 to 7.36, and at the post-winter stage from 1.23 to 3.10. Pupation occurred in mid October, and the pupal stage lasted for up to 6 weeks; the thermal constant is 216 day degrees. Mortality factors discussed include establishment failure, parasites, pathogens, predators, larval combat, winter pruning, loss of pupae at cane breaks, and pupal eclosion failure. The first record of the fungus Cordyceps sp. attacking currant clearwing is reported.     

Adhesion of bacteria (Serratia spp.) to the foregut of grass grub (Costelytra zealandica (white)) larvae and its relationship to the development of amber disease

Adhesion of pathogenic strains of Serratia spp. to the foregut tissue of the New Zealand grass grub (Costelytra zealandica) was shown to be associated with the development of amber disease. Bacteria were always found adhering to the crop in the region of the cardiac valve in larvae showing disease symptoms after in vivo treatment with pathogenic bacteria while no significant colonization was observed in larvae treated with wild‐type, non‐pathogenic strains. The in vitro inoculation of excised crops with pathogenic and non‐pathogenic strains resulted in a similar pattern of adhesion. It is suggested that adhesion is an early step in pathogenesis and that farther bacterial mediated factors could be required for fall expression of amber disease.     

Nesting of Afrotropical Oniticellus (Coleoptera, Scarabaeidae) and its evolutionary trend from soil to dung

Abstract. 1. Oniticellus egregius Klug constructs brood ovoids of dung in the soil immediately under the edge of animal droppings. Each successive brood ovoid is enveloped within a soil shell. After completion of brood construction, loose earth is cleared from around the broods to produce a brood chamber. The immatures are then abandoned as eggs or first instar larvae.
2. O.planatus Castelnau and O.formosus Chevrolat usually construct brood balls of dung within animal droppings. Each brood is progressively enlarged by the addition of further dung after egg-laying. This enlargement is slight in O.planatus and marked in O.formosus. Parental females of both species remain in the brood chambers during development of the immatures which are abandoned principally as pupae.
3. Under very moist experimental conditions, O.planatus buries dung and constructs broods shallowly in the soil. Such nests are frequently connected to the pad by a short tunnel.
4. From a consideration of behavioural patterns it is suggested that the specialized nesting habits of these species have been derived from those of dung-burying ancestors similar to the modern genus, Euoniticellus, through reduction and loss of tunnelling in the soil.