Control of the bacterium Serratia marcescens in an insect host-parasite rearing program

Two occluded viruses of the Entomopoxvirus (D)/¹: ¹/¹: $\text{X}\text{X}\text{:}\text{I}\text{O}$ (= Vagoiavirus) group have been found in larvae of Dermolepida albohirtum (Scarabaeidae: Melolonthinae) and Aphodius tasmaniae (Scarabaeidae: Aphodiinae) from northern Queensland and northern Tasmania, Australia, respectively. Electron microscopical studies have been made of thin sections of occluded (mature) and nonoccluded virus particles within the fat body tissue of living diseased D. albohirtum larvae and of occluded virus particles within a dead, field-collected A. tasmaniae larva. The morphology and development of the known Australian entomopoxviruses are compared with previously known entompox or spheroidosis viruses from various insects.     

The pathogenicity of an entomopoxvirus fromOthnonius batesi [Col.: Scarabaeidae] and its possible use as a control agent

In a natural population ofOthnonius batesi Oll. at Glen Innes, N.S.W., 4.0% of larvae in 1972 and 2.2% in 1973 were exhibiting symptoms of virus infection, whilst 0.8% of pupae and adults from the same population were infected in 1972. These figures, and field observations of infected larvae, suggested that the pathogenicity of the virus was low. In laboratory experiments withO. batesi the infection had little effect on mortality, and no significant effect on duration of the first instar, food intake, or larval growth. The vast accumulation of virus in the fat body probably results in mortality prior to, or during, the pupal period. Temperature had a marked effect on both % infection (optimum 30°C) and production of viral spheroids (optimum 25°C). Very little viral development occurred below 20°C. In a host range study onlyO. batesi, Rhopaea verreauxi Blanch. andR. morbillosa Blkb. were infectedper os. The possible use of the virus as a control agent is discussed.     

Persistence of insect viruses in field populations of alfalfa insects

The persistence of viruses of five insects was observed in alfalfa fields. The insects were Autographa californica, Colias eurytheme, Pseudaletia unipuncta, Spodoptera exigua, and Trichoplusia ni. The isolated viruses were the granulosis (GV), the cytoplasmic-polyhedrosis (CPV), and the nuclear-polyhedrosis (NPV) viruses. The viruses persisted in the soil, on the alfalfa foliage, and in alternate hosts. In the soil, the viruses persisted even during the winter months when no foliage remained on the plants. Alfalfa sprouts harboring virus-infected larvae of C. eurytheme and S. exigua produced virus infections in larvae of these insects, but those with larvae of A. californica and P. unipuncta did not cause virus infection. The GVs and CPVs isolated from these insects were transmitted to nearly all of the other four species, but the NPVs appeared to be host specific.     

“Endomicrobia” and Other Bacteria Associated with the Hindgut of Dermolepida albohirtum Larvae

Symbiotic bacteria residing in the hindgut chambers of scarab beetle larvae may be useful in paratransgenic approaches to reduce larval root-feeding activities on agricultural crops. We compared the bacterial community profiles associated with the hindgut walls of individual Dermolepida albohirtum third-instar larvae over 2 years and those associated with their plant root food source among different geographic regions. Denaturing gradient gel electrophoresis analysis was used with universal and Actinobacteria-specific 16S rRNA primers to reveal a number of taxa that were found consistently in all D. albohirtum larvae but not in samples from their food source, sugarcane roots. These taxa included representatives from the “Endomicrobia,” Firmicutes, Proteobacteria, and Actinobacteria and were related to previously described bacteria from the intestines of other scarab larvae and termites. These universally distributed taxa have the potential to form vertically transmitted symbiotic associations with these insects.     

An iridescent virus fromPopillia japonica (Col.: Scarabaeidae)

A very low incidence (<0.01%) of a blue iridovirus (IV) was found in larvae of the Japanese beetle,Popillia japonica Newman, that were sampled over a two year period on Terceira Island (Azores, Portugal). In the most heavily infected larvae, a deep blue iridescence was observed, particularly in the fat body. Transmission electron microscopy revealed the characteristic crystalline arrays of the hexagonal virus particles in the cytoplasm of fat body cells, tracheal matrix, muscle, hypodermis and blood cells. Crystals of the virus particles were also observed freely circulating in the hemolymph. The average diameter of negatively stained purified virus particles was 157 nm. Similarities and differences with other IVs found in the Scarabaeidae are discussed. Considering the broad host range of some of the iridescent viruses, the relatively recent invasion of Terceira byP. japonica, and the rarity of the virus in the beetle, it is probable that the infection was the result of transmission from another species of soil-inhabiting arthropod. Its value as a potential biological control agent ofP. japonica is negligible.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

Life history and population dynamics of Ectinohoplia rufipes (Coleoptera: Scarabaeidae) on Korean golf courses

Life history and temporal distribution of Ectinohoplia rufipes larvae (Coleoptera: Scarabaeidae; Melolonthinae) were studied on multiple courses at two Korean golf clubs in Gapyeong and Anseong, Gyeonggi Provinces. Larval density of E. rufipes was higher in roughs than fairways and higher on older courses than on newly constructed ones at each club. Larval density of E. rufipes in fall reached three to four times compared with the previous year in five or six year old courses in the first opened courses, but that took two or three years to reach three to four times in the newly constructed courses in the same golf club. Monthly sampling showed that larvae were distributed 3.0 to 5.8 cm beneath the soil surface; mean larval density ranged from 0.2 to 1.2/m² at each club. Most larvae overwintered as third instars that pupated in early May. Adults were first observed in late May at each course. Adults were first seen in emergence cages installed over a grub-infested portion of rough in early June. Eggs were deposited from late June to early July. Eggs hatched in mid to late July and larvae developed to become third instars by early October. Implications of these findings for managing E. rufipes on Korean golf courses are discussed.     

Mode of transmission of nuclear-polyhedrosis virus to progeny of adult Heliothis zea

Late second-instar Heliothis armigera larvae were infected with a granulosis and a nuclear polyhedrosis virus, and all the externally visible symptoms for each virus are described. The effects of the virus infections on the feeding habits of the insects are also described, and it was found that a granulosis infection can prolong the larval period by up to 100%. The larvae continue feeding during this prolonged larval period, and can reach almost double the size and mass of normal larvae.It was further found that each of the viruses displays a distinct set of symptoms which could indicate beyond any doubt which of the two viruses induced death in the host.     

Development and Evaluation of Methods To Detect Nucleopolyhedroviruses in Larvae of the Douglas-Fir Tussock Moth, Orgyia pseudotsugata (McDunnough)

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r² = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.     

The Bacterial Symbiont Wolbachia Induces Resistance to RNA Viral Infections in Drosophila melanogaster

Wolbachia are vertically transmitted, obligatory intracellular bacteria that infect a great number of species of arthropods and nematodes. In insects, they are mainly known for disrupting the reproductive biology of their hosts in order to increase their transmission through the female germline. In Drosophila melanogaster, however, a strong and consistent effect of Wolbachia infection has not been found. Here we report that a bacterial infection renders D. melanogaster more resistant to Drosophila C virus, reducing the load of viruses in infected flies. We identify these resistance-inducing bacteria as Wolbachia. Furthermore, we show that Wolbachia also increases resistance of Drosophila to two other RNA virus infections (Nora virus and Flock House virus) but not to a DNA virus infection (Insect Iridescent Virus 6). These results identify a new major factor regulating D. melanogaster resistance to infection by RNA viruses and contribute to the idea that the response of a host to a particular pathogen also depends on its interactions with other microorganisms. This is also, to our knowledge, the first report of a strong beneficial effect of Wolbachia infection in D. melanogaster. The induced resistance to natural viral pathogens may explain Wolbachia prevalence in natural populations and represents a novel Wolbachia–host interaction.     

A method for rearing and assessing the health of the sugarcane pest Dermolepida albohirtum (Coleoptera: Scarabaeidae)

Rearing insects in controlled conditions is a prerequisite to supply high-quality specimens for bioassays. However, while artificial diets and standardized rearing methods have been developed for many phytophagous insects, especially Lepidoptera, there are limited published diets for root-feeding Coleoptera which are commonly fed either grass roots or pieces of vegetables as a simple alternative to artificial diets during bioassays. These feeding options, while convenient, can be considered suboptimal as they do not maximise the insects' development and health. Additionally, it is also important to develop standardised screening methods designed to test sublethal effects of control agents which may have repellent, antifeedant, antimetabolic and/or delayed mortality effects. The greyback canegrub (Dermolepida albohirtum, Waterhouse) is the most damaging native pest of Australian sugarcane, but no rearing method or artificial diet has ever been developed for this species. Our objectives were to improve bioassay methodology for D. albohirtum by describing and developing standard rearing and health assessment protocols. We describe a successful rearing method to raise healthy D. albohirtum larvae with a total of 48.8% of first instars successfully moulting to the second instar. We also tested a modified artificial diet which increased the weight, size and food uptake of larvae compared to traditional methods (i.e., pieces of carrots). For example, the average weight increase of larvae fed with the modified diet was 3.4 times higher than for carrot-fed larvae while modified diet-fed larvae were 2.1 times wider than if they were fed with carrots. Finally, we developed a method to measure larval activity which can be used to identify sublethal effects of control agents such as effects on activity level. Our methods may also be applied to improve bioassay methodology for other root-feeding Coleoptera.     

Infestation of the white grub Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) in hayfields on Miyako and Ishigaki Islands, Japan

To examine the local distribution of Dasylepida ishigakiensis Niijima et Kinoshita larvae (Coleoptera: Scarabaeidae) on Miyako Island and Ishigaki Island, a survey was conducted in hayfields on the two Islands in February and March 2011. Symptoms of white grub infestation, such as patches of wilted grass and thin, yellow grass stands, were observed in some of the 20 hayfields surveyed on Miyako Island. Three species of white grub were observed there: D. ishigakiensis, Anomala cpustulata cpustulata Matsumura in Hirayama, and Anomala albopilosa (Hope), in the ratio 92:7:1, respectively. The mean population density of D. ishigakiensis in the hayfields was 54.3 m^?2 and the maximum was 225 m^?2. No significant relationship was found between the damage levels of hay grass and the D. ishigakiensis larval densities. In contrast, no damage was observed in the 14 hayfields surveyed on Ishigaki Island, although some D. ishigakiensis larvae were found at a low density in 4 hayfields. In 2006, no D. ishigakiensis larvae were found in sugarcane fields on this island in a separately conducted intensive survey. These observations may suggest that D. ishigakiensis larvae are present in hayfields at a low density but that few individuals occur in sugarcane fields on Ishigaki Island.     

Sur la spécificité deVagoiavirus melolonthae

Summary The activity ofVagoiavirus melolonthae towards 16 species ofScarabaeidae, 3 Coleoptera, 3 Lepidoptera and 2 Diptera was studied by injection into the hemocoel and contamination of the substratum or of the rearing host plant. Only insects belonging to 3 sub-families ofScarabaeidae: Hopliinae, Melolonthinae, Rutelinae, are susceptible to the disease.      

Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.     

Review on microbial control of insect pests in forests in Japan

In Japan, two different serious defoliators were controlled by viruses:Dendrolimus spectabilis using cytoplasmic polyhedrosis virus andLymantria fumida using cytoplasmic and nuclear polyhedrosis viruses. For controllingD. spectabilis, good results were obtained when the spraying was done against old larvae of intermediate population density. AgainstL. fumida, a mixed suspension of native nuclear and cytoplasmic polyhedrosis viruses was sprayed at the early stage. The epizootic was initiated earlier than being supposed, and the population collapsed.     

A pathogenic nonoccluded virus in hemocytes of the navel orangeworm,Amyelois transitella (Pyralidae: Lepidoptera)

A small, nonoccluded virus was isolated from the granular hemocytes (=adipocytes) of moribund larvae of the navel orangeworm, Amyelois transitella, collected from almonds in northern California. The virus was readily transmitted perorally in laboratory tests and was highly pathogenic to neonate larvae; however, third- and fourth-stage larvae usually acquired attenuated infections that retarded growth and delayed mortality. Large paracrystalline viral arrays filled the cytoplasm of granular hemocytes of diseased larvae. Attempts to transmit the virus perorally to larvae of nine other species of moths were unsuccessful. The virus is isometric, about 25 nm in diameter, and contains single-stranded RNA. It has been designated “chronic stunt virus” (CSV) and tentatively assigned to the picorna-like viruses.     

Hidden burrow plugs and their function in the tiger beetle, Cosmodela batesi (Coleoptera, Cicindelidae)

Tiger beetle larvae excavate and live in underground burrows, whose openings they sometimes plug with soil. This study documents the burrow plugging behavior of the tiger beetle, Cosmodela batesi (Fleutiaux), in the field. We also tested the function of burrow plugs in the laboratory. In the field, C. batesi more frequently made a plug when it rained. Most larvae made plugs inside their burrows (rather than at the soil surface), and the use of an endoscope was necessary to detect these sub-surface plugs. In the laboratory, flooding was simulated by artificially introducing water into specially-made arenas. Water filled the entire burrow when there was no plug, whereas plugged burrows maintained air chambers inside. When a plug was broken with a wire, burrows filled up with water. The burrowing and plugging behavior described in this study is likely an important adaptation of C. batesi to its habitat.     

Larvae of the tachinid fly,Drino inconspicuoides (Diptera: Tachinidae), suppress melanization in host lepidopteran insects

The ovoviviparous parasitoid, Drino inconspicuoides (Diptera: Tachinidae) parasitizes a wide range of lepidopteran insects; larval period progresses in the host hemocoel. Here, we examined how D. inconspicuoides responds to melanization, which involves the activation of prophenoloxidases and is the first immune reaction induced by the host against invading organisms. We found that the larvae of D. inconspicuoides suppressed the activation of prophenoloxidases in its natural hosts, Mythimna separata (Lepidoptera: Noctuidae) and Bombyx mori (Lepidoptera: Bombycidae). The suppression of melanization starts immediately after invasion and is maintained for at least 24 h. We did not detect a drastic degradation of prophenoloxidases, suggesting that the presence of other molecules targeted by D. inconspicuoides suppresses melanization. D. inconspicuoides does not inhibit a cellular immune reaction, encapsulation, and thus, it is likely that the tachinids survive secondary infections of the host by partially retaining the host immune function.     

Transmission of a baculovirus in populations of Oryctes rhinoceros

The transmission of the baculovirus of Oryctes rhinoceros, previously called Rhabdionvirus oryctes, was studied. O. rhinoceros adults became infected with the virus when kept in a mixture of sawdust and ground-up virus-killed larvae or together with other virus-infected adults. In the field, mated females were more frequently infected than unmated females. Adults developing from larvae that had survived exposure to various dosages of the virus were not infected. No virus infections occurred in larvae hatching from eggs surface-contaminated with the virus. Larvae hatching from eggs laid by virus-infected females very rarely were infected.In the O. rhinoceros population the virus is transmitted most frequently during mating, possibly when the uninfected partner contacts virus material excreted by the infected partner. The virus can be transmitted in a similar way when infected and healthy beetles feed together in palm trees. Beetles visiting larval breeding sites containing freshly virus-killed larvae can become infected. Virus-infected beetles can pass the infection to healthy larvae when visiting a breeding site.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.