The effect of a seasonal time constraint on development time, body size, condition, and morph determination in the horned beetle Allomyrina dichotoma L. (Coleoptera: Scarabaeidae)

Abstract.  1. In horned beetles selection favours males that adjust their investment in horn development in relation to cues that predict adult body size. Here it is shown that in the Japanese horned beetle, Allomyrina dichotoma . There is a significant discontinuity in the horn length body size allometry. This can be described as a linear relationship that is shifted towards an increased horn length to body length ratio in males with horns longer than 16 mm.
2. Larval nutrition explains morph determination in A. dichotoma . However, unlike other species, variation in larval nutrition was the result of a seasonal time constraint that limits the time available for feeding prior to the onset of winter diapause.
3. Even when eggs were reared with an ad libitum food supply, minor morphs were still observed. Individuals that were oviposited later in the season had less time to feed, shorter development times, eclosed as smaller individuals and, in the case of males, were more likely to be hornless. Major morphs, minor morphs, and females all reduced their body size in response to seasonal time constraints in the same way. However, males that were laid later in the season had faster development times than females laid at the same time, but showed no reduction in their size relative to females, suggesting seasonal time constraints increase growth rates in males but not in females.
4. No evidence was found that seasonal time constraints resulted in a reduction of size-corrected fat reserves at eclosion, or that minor morphs gained any developmental advantage by reducing investment in horn length.     

Size dependent predatory pressure in the Japanese horned beetle,Allomyrina dichotoma L. (Coleoptera; Scarabaeidae)

We surveyed male survival and reproductive performances associated with dimorphism in the Japanese horned beetleAllomyrina dichotoma L. in a secondary forest in 1996. Morphological comparisons between living marked and prey individuals indicated that the larger horned males suffered higher predatory pressure than the smaller ones. The dominant predators of the beetles were suspected to be 2 crow species. The small-horned males showed lower recapture rates than the large-horned ones. This suggested that the former was more sensitive to disturbance, and/or dispersed more than the latter. Fighting behavior was rarely seen because of the low population density of the beetles in the study area. These results suggested that the large-horned males suffer not only the injury risk of intrasexual competition but also more predatory risk than the small-horned ones.     

The first report on the characterization of a virus isolated from Allomyrina dichotoma in the Republic of Korea

This report reveals the structure of a virus extracted from the Korean horn beetle Allomyrina dichotoma. The purified virus particle was 100% identical to Allomyrina virus lef‐8 sequence registered as KM_233709.1. The structure of this virus was confirmed to be closely related to that of the Nudiviridae family, and it was rod shaped and enveloped, and observed to be of approximately the mean length of a single viral nucleocapsid of 200–210 nm and mean diameter of 100–110 nm. These results provide an insight into the structural characteristics of the Nudiviridae family that can be used for nudiviral identification.     

Establishment of a loop‐mediated isothermal amplification system for on‐site diagnosis of Oryctes rhinoceros nudivirus in Allomyrina dichotoma (Coleoptera: Scarabaeidae)

The Korean insect industry is rapidly developing, with the Korean horn beetle Allomyrina dichotoma as one of the most popular insects kept as pets. Korean horn beetles reared in local farms are suffering from Oryctes rhinoceros nudivirus (OrNV). In a nationwide investigation on the early death of young larvae due to this virus, 70 to 77% mortality was found. It was also confirmed that several larvae collected from some wild horn beetle habitats were infected with the virus. Thus, it was concluded that the virus could be transmitted vertically from an infected adult to the offspring and that the OrNV disease is a very serious threat to the wild horn beetle in Korea. With local farmers expecting that an early detection technique would be helpful for the quick removal of infected larvae, an on‐site diagnosis method for the viral disease using loop‐mediated isothermal amplification (LAMP) assay was thus tested. To avoid the DNA extraction process, the LAMP assay used a 50‐fold diluted hemolymph was set for diagnosis standardization and convenient on‐site application to infected larvae from local farms. A. dichotoma larvae were assayed for the OrNV infectivity with LAMP primers targeting the OrNV_gp102 gene. To evaluate the LAMP specificity, two bacterial pathogens, Bacillus thuringiensis and Serratia marcescens, causing disease in A. dichotoma were tested along with OrNV. The positive results were detected only from the OrNV‐infected larvae.     

Dose response of coconut rhinoceros beetle (Coleoptera: Scarabaeidae) to 92 kV x-ray irradiation

To evaluate the potential suitability of x-ray irradiation as a physical control method for invasive Coconut Rhinoceros Beetle (CRB) (Oryctes rhinoceros Linnaeus 1758; Coleoptera: Scarabaeidae), either through sterile insect technique (SIT) or through direct irradiation of naturally infested materials in the field, we recorded longevity of eggs, larval and adult life stages of CRB irradiated with different doses of x-rays emitted from a tube energized at 92 kV. Eggs and larvae were highly susceptible to radiation at all tested doses (down to about 2–5 Gy), though adults required larger doses (at least 50 Gy) to render them incapable of reproducing. At exposures near 50 Gy sterilized adults nevertheless were observed to survive for more than a month, suggesting SIT may be a viable control approach for this beetle. Similarly, these results may facilitate the discovery of hidden breeding sites in the wild by tagging and releasing sterilized adults. While larvae were highly susceptible to x-ray irradiation, irradiation would probably not be an effective tool for field control due to rapid attenuation of radiation energy (exponential decay coefficients ranging from about 0.3–0.6 cm⁻¹) in the organic nesting materials we tested, and the tendency for CRB adults to burrow and oviposit deep in mulch (1 m or more).     

Mate securing tactics and the cost of fighting in the Japanese horned beetle,Allomyrina dichotoma L. (Scarabaeidae)

Males of the horned beetleAllomyrina dichotoma L. show a bimodal frequency distribution with respect to horn size. The 2 morphs distinguished by this criteria showed different mate-securing tactics. Major males fought for possession of areas on oak trees that exuded sap. Fights escalated through a series of stereotyped encounters before entering the potentially damaging phase of close-quarter combat when the largest males in particular risked serious damage. Minor males, on the other hand, were never observed to fight with conspecific males, but retreated after making contact with them. Minor males arrived at sap sites earlier in the diurnal cycle than major males and so avoided them temporally as well as behaviourly. Minors appeared to be relatively as successful at gaining copulations as majors, but did so earlier in the diurnal cycle. Since females showed a slight tendency to remate on the same night, minors may lose fertilization opportunities if last male sperm precedence is high. Actual sperm precedence values are not know so the reproductive payoffs for the 2 morphs could not be assessed.     

Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms

Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing.     

Phylogeny and biogeography of the Japanese rhinoceros beetle,Trypoxylus dichotomus (Coleoptera: Scarabaeidae) based on SNP markers

The Japanese rhinoceros beetle Trypoxylus dichotomus is one of the largest beetle species in the world and is commonly used in traditional Chinese medicine. Ten subspecies of T. dichotomus and a related Trypoxylus species (T. kanamorii) have been described throughout Asia, but their taxonomic delimitations remain problematic. To clarify issues such as taxonomy, and the degree of genetic differentiation of Trypoxylus populations, we investigated the genetic structure, genetic variability, and phylogeography of 53 specimens of Trypoxylus species from 44 locations in five Asian countries (China, Japan, Korea, Thailand, and Myanmar). Using specific‐locus amplified fragment sequencing (SLAF‐seq) techniques, we developed 330,799 SLAFs over 114.16M reads, in turn yielding 46,939 high‐resolution single nucleotide polymorphisms (SNPs) for genotyping. Phylogenetic analysis of SNPs indicated the presence of three distinct genetic groups, suggesting that the various subspecies could be treated as three groups of populations. PCA and ADMIXTURE analysis also identified three genetic clusters (North, South, West), which corresponded to their locations, suggesting that geographic factors were important in maintaining within population homogeneity and between population divergence. Analyses of SNP data confirmed the monophyly of certain subspecies on islands, while other subspecies (e.g., T. d. septentrionalis) were found to be polyphyletic and nested in more than one lineage. AMOVA demonstrated high level of differentiation among populations/groups. Also, pairwise F_ST values revealed high differentiation, particularly between South and West, as well as between North and South. Despite the differentiation, measurable gene flow was inferred between genetic clusters but at varying rates and directions. Our study demonstrated that SLAF‐seq derived markers outperformed 16S and COII sequences and provided improved resolution of the genetic differentiation of rhinoceros beetle populations from a large part of the species’ range.     

An Iridovirus from the black beetle,Heteronychus arator (Coleoptera : Scarabaeidae)

Abstract

Iridescent virus type 23 from the black beetle (Heteronychus arator) replicated in Spodoptera frugiperda cells, producing some cell vacuolation after 3 days and extensive vacuolation and detachment of cells 7 days after infection. The virus also replicated in Aedes albopictus cells, and in the larvae and pupae of Galleria mellonella.     

The Oryctes virus: its detection, identification, and implementation in biological control of the coconut palm rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae)

In view of the increasing and devastating damage by rhinoceros beetle (Oryctes rhinoceros) to coconut palms in the middle of last century, many efforts were made to find an efficient natural control factor against this pest, which could not be controlled by pesticides. The basic procedures of these monitoring programmes are outlined together with the final detection of a virus disease in oil palm estates in Malaysia in 1963. In extensive laboratory studies, the virus was isolated and identified as the first non-occluded, rod-shaped insect virus, morphologically resembling the baculoviruses. Infection experiments clarified the pathology, histopathology, and virulence of the virus and demonstrated that the virus was extremely virulent to larvae after peroral application. These findings encouraged the first pilot release of virus in 1967 in coconut plantations of Western Samoa where breeding sites were contaminated with virus. Surprisingly, the virus became established in the Samoan rhinoceros beetle populations and spread autonomously throughout the Western Samoan islands. As a consequence, there was a drastic decline of the beetle populations followed by a conspicuous recovery of the badly damaged coconut stands. This unexpected phenomenon could only be explained after it was shown that the adult beetle itself is a very active virus vector and thus was responsible for the efficient autodissemination of the virus. The functioning of the beetle as a 'flying virus factory' is due to the unique cytopathic process developing in the midgut after peroral virus infection. Pathological details of this process are presented. Because of the long-term persistence of the virus in the populations, rhinoceros beetle control is maintained. Incorporation of virus into integrated control measures and successful virus releases in many other countries are recorded.     

椰扁甲啮小蜂寄生后椰心叶甲蛹血淋巴蛋白质双向电泳图谱分析

分析昆虫被寄生蜂寄生后血淋巴蛋白质组分及组分含量变化,从蛋白质水平分析寄生蜂寄生引起寄主昆虫的生理变化,有利于探索寄生蜂调控寄主的机理。以椰心叶甲Brontispa longissima和椰扁甲啮小蜂Tetrastichus brontispae为研究材料,采用蛋白质双向电泳技术及图像分析技术分析了椰心叶甲蛹被椰扁甲啮小蜂寄生后不同时期血淋巴蛋白质图谱及其变化情况。结果表明:寄生后0.5, 1, 2, 3和4 d,被寄生蛹血淋巴共检测到的蛋白斑点数分别为982, 926, 712, 636和680个,其中特异性蛋白斑点的数量分别为650, 400, 336, 229和150个;同期未被寄生蛹血淋巴共检测到的到的蛋白斑点数分别为645, 817, 640, 873和940个,其中特异性蛋白斑点的数量分别为313, 291, 264, 466和410个;被寄生蛹和同期未被寄生蛹能匹配的蛋白斑点分别为332, 526, 376, 407和530对。被寄生蛹血淋巴蛋白斑点数量的异常变化表明寄主的生理功能受到了严重的影响。     

Ecological methods for adult populations of Oryctes rhinoceros (Coleoptera, Scarabaeidae)

ABSTRACT. 1. For comparing assessment methods, O.rhinoceros (L.) populations were monitored in five 30–50 ha plots in southern Luzon, Philippines. No consistent correlations were found between number of beetles caught in traps, amount of palm damage and number of breeding sites. Apparently, plots were too small to account for fast dispersal of beetles.
2. Coconut cap traps baited with ethylchrysanthemumate attracted O.rhinoceros adults searching for breeding sites. Several factors were identified influencing catches but trapping was found to be unsuitable for accurately assessing pest populations.
3. A new method which gives estimates on the monthly number of O.rhinoceros attacks on coconut palms, was tested in the Maldives. Palms are climbed about once a year and the sequence of fronds, the number and the position of beetle cuts are recorded.
4. Reduction with age of the length of three teeth on the fore tibia of O. rhinoceros adults was studied in the Philippines. The data was insufficient to obtain a clear correlation between age and length of teeth.
5. The observations indicated that in the Philippines after leaving their site of pupation, O.rhinoceros adults spend about 5 weeks feeding on coconut palms. This is followed by a period of about 7 weeks in breeding sites and, on occasion, additional visits to palms. With these estimates it was possible to relate palm damage records with numbers of feeding adults.     

Genetic and electron-microscopic characterization of Rickettsiella bacteria from the manuka beetle, Pyronota setosa (Coleoptera: Scarabaeidae)

Larvae of manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae) cause pasture damage in New Zealand by feeding on the roots of grasses. Surveys for potential biocontrol agents revealed a putative disease, expressed as whitened larvae of one of the outbreak species, Pyronota setosa. Microbial diagnosis indicated an intracoelomic, intracellular infection, and intracellular bacteria have been identified with subcellular structures characteristic of infection by Rickettsiella-like microorganisms. These bacteria were rod-shaped, often slightly bent with a mean of 628 nm in length and 220 nm in width. Numerous associated protein crystals of variable size and shape occurred within round to oval shaped “giant bodies” either singly or as clusters of smaller crystals. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the manuka beetle pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the pathotype designation ‘Rickettsiella pyronotae’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that - on the basis of the currently accepted organization of the genus Rickettsiella - this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.     

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.     

Potential resistance of crape myrtle cultivars to flea beetle (Coleoptera: Chrysomelidae) and Japanese beetle (Coleoptera: Scarabaeidae) damage

Field and laboratory studies were conducted to identify potential resistance among crape myrtles, Lagerstroemia spp., to Japanese beetle, Popillia japonica Newman and to flea beetles, Altica spp. Damage ratings revealed variation among cultivars in susceptibility to beetle feeding. Cultivars with Lagerstroemia fauriei Koehne in their parentage exhibited the least amount of damage in choice and no-choice experiments, with few exceptions. The data indicate that both beetle species cause more feeding damage on certain cultivars of Lagerstroemia indica L., such as 'Country Red', 'Twilight', and 'Carolina Beauty' than interspecific cultivars with L. fauriei in their parentage, such as 'Natchez', 'Tonto', and 'Muskogee'. When comparing the effect of parentage on all of the major pests of crape myrtle, L. faurei confers resistance to all pests except crape myrtle aphid. No correlation was found between leaf toughness, leaf color, and leaf nutrients in estimating flea beetle cultivar preference. With this information, growers can more effectively target scouting measures to the most susceptible cultivars. and breeders can select plants that will require the fewest chemical inputs.     

A virus of black beetle,Heteronychus arator (F.) (Coleoptera: Scarabaeidae)

During a survey for pathogens of black beetle, high mortality was observed in a population in kikuyu pasture at Wharepapa, near Helensville. Several pathogens were isolated from larvae and adults, including a protozoan (possibly Adelina sp.), a rickettsia, a fungus (Beauvaria sp.), a neoaplectanid nematode, and a small RNA virus. The virus was transmitted to larvae of Pseudaletia separata, Galleria mellonella, Aphodius tasmaniae, and Pericoptus truncatus, and a fatal paralysis ensued after 14–37 days. The potential of the neoaplectanid nematode and the virus in control is discussed.     

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.