Effects of juvenile hormone analogues on larval metamorphosis in the barnacle Elminius modestus Darwin (Crustacea: Cirripedia)

Two analogues of insect juvenile hormones (JH) have been shown to interfere with the development of Elminius modestus Darwin larvae when dispersed as acetone solutions in sea water. Stage VI nauplii metamorphosed to morphologically abnormal larvae which were intermediate in size between the nauplius and cypris stages; although these larvae were cypris-like they apparently retained some nauplius characteristics. Cyprids either metamorphosed to non-attached adults or formed larvae which were larger or morphologically abnormal. There was evidence that the effects may be related to the physiological development of the larvae at time of exposure. These observations appear to be the first report of the induction of size and morphological abnormalities in a crustacean species by analogues of insect JH and resemble those arising from the hormonal imbalance which such compounds induce during the development of holometabolous insects.     

A Madurella mycetomatis Grain Model in Galleria mellonella Larvae

Eumycetoma is a chronic granulomatous subcutaneous infectious disease, endemic in tropical and subtropical regions and most commonly caused by the fungus Madurella mycetomatis. Interestingly, although grain formation is key in mycetoma, its formation process and its susceptibility towards antifungal agents are not well understood. This is because grain formation cannot be induced in vitro; a mammalian host is necessary to induce its formation. Until now, invertebrate hosts were never used to study grain formation in M. mycetomatis. In this study we determined if larvae of the greater wax moth Galleria mellonella could be used to induce grain formation when infected with M. mycetomatis. Three different M. mycetomatis strains were selected and three different inocula for each strain were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 4 mg/larvae. Larvae were monitored for 10 days. It appeared that most larvae survived the lowest inoculum, but at the highest inoculum all larvae died within the 10 day observation period. At all inocula tested, grains were formed within 4 hours after infection. The grains produced in the larvae resembled those formed in human and in mammalian hosts. In conclusion, the M. mycetomatis grain model in G. mellonella larvae described here could serve as a useful model to study the grain formation and therapeutic responses towards antifungal agents in the future.     

Reproductive strategy of the sugi bark borer,Semanotus japonicus (Coleoptera: Cerambycidae) on Japanese cedar,Cryptomeria japonica

The inner bark of Japanese cedar,Cryptomeria japonica D. Don., is the main food of the sugi bark borer,Semanotus japonicus Lacordaire, but may also be involved in resistance to attack by the borer. I used newly hatched larvae to inoculate cedar logs that had been cut 2-weeks to 12-months earlier, and undamaged living cedar trees. On living trees, all larvae were killed by resin flow from the traumatic resin canals between the outer- and inner bark or between the inner bark and cambium. In logs that were cut more than 4 months prior to inoculation, larvae were unable to complete development. In logs cut 2 weeks prior to inoculation, almost all larvae were able to reach the adult stage but they were smaller in size than adults from living trees damaged by this borer. These results suggest that poor nutrient conditions in the inner bark of logs affected development and survival of the borer. Because of its nutritional advantage, living cedars provide a better environment for the sugi bark borer. However, all larvae are killed by resin flow, suggesting that this insect is “in between” being primary and secondary with respect to living trees, or a “weak” primary insect. The sugi bark borer seems to develop early in the season so that early instar larvae encounter reduced amounts of resin flow and so that late-instar larvae feed mostly in summer when nutrient levels in the inner bark are at their highest.     

No apparent effects of soil inoculum on green frog (Lithobates clamitans Latreille) tadpole performance

Invasions by nonnative plant species are transforming plant communities across the globe. An important challenge for ecologists is to understand how animals will respond to these changes. One way that plant invasions could affect aquatic animals is by changing the rate at which soil communities decompose litter, which could alter the flow of energy and nutrients from plant litter to aquatic communities. In this study, we measured larval amphibian responses to soil conditioned by either introduced or native genotypes of Phragmites australis L. (common reed) in northeastern North America. We collected soil from adjacent stands of introduced and native P. australis at three sites in central New York and inoculated outdoor aquatic mesocosms with soil extracts. Mesocosms contained six Lithobates clamitans Latreille (green frog) tadpoles and either low- or high-quality native P. australis americanus litter. We found that litter decomposition differed based on soil inoculum, and we observed a significant interaction between litter quality and soil inoculum; higher-quality litter tended to decompose faster when exposed to inocula from introduced P. australis, while lower-quality litter tended to decompose faster when exposed to inocula from native P. australis americanus. Tadpoles raised with high-quality litter developed faster and achieved greater body size, but soil inocula had no apparent effect on tadpoles. Our results suggest that plant invasions may alter microbial communities, causing subtle changes in litter decomposition rates, but these changes do not appear strong enough to influence larvae of a widespread amphibian.     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

Variation in larval properties of the Atlantic brooding coral <Emphasis Type="Italic">Porites astreoides</Emphasis> between different reef sites in Bermuda

Recent research has documented phenotypic differences among larvae released from corals with a brooding reproductive mode, both among species and within broods from a single species. We studied larvae released from the common Atlantic coral Porites astreoides in Bermuda to further evaluate phenotypic variability. Inter-site differences were investigated in larvae from conspecifics at a rim and patch reef site. Larvae were collected daily for one lunar cycle from several colonies per site each year over 5 yr. Larval volume varied with reef site of origin, with colonies from the rim reef site producing larger larvae than colonies from the patch reef site. This inter-site variation in larval size could not be explained by corallite size and may be a response to different environmental conditions at the sites. Larvae from both reef sites also varied in size depending on lunar day of release over 4 yr of study. Regardless of site of origin, smaller larvae were released earlier in the lunar cycle. Over 1 yr of study, lipid and zooxanthellae content and settlement success after 48 h covaried with larval size. However, there may be a trade-off between larger larvae and reduced fecundity. Overall, larvae released from colonies from the rim reef site were larger and had greater settlement success than those from colonies from the patch reef site. This study documents larval phenotypic variability and a distinct inter-site difference in larval ecology among conspecifics within the same geographic area, which may have implications for recruitment success, population dynamics, and resilience.     

Sheared-Root Inocula of Vesicular-Arbuscular Mycorrhizal Fungi

For efficient handling, vesicular-arbuscular mycorrhizal fungi should be processed into small and uniform inocula; however, processing can reduce the inoculum density. In this article we describe the preparation and use of sheared-root inocula of Glomus spp. in which inoculum densities were increased during processing. Our objectives were to determine inoculum viability and density after shearing and to ascertain if the sheared inocula could be pelletized or used with a gel carrier. Root samples were harvested from aeroponic cultures, blotted dry, cut into 1-cm lengths, and sheared in a food processor for up to 80 s. After shearing, the inoculum was washed over sieves, and the propagule density in each fraction was determined. Sheared inocula were also encapsulated in carrageenan or used in a gel carrier. Shearing aeroponically produced root inocula reduced particle size. Propagule density increased with decreasing size fraction down to a size of 63 μm, after which propagule density decreased. The weighted-average propagule density of the inoculum was 135,380 propagules g (dry weight) of sheared root material^-1. Sheared roots were encapsulated successfully in carrageenan, and the gel served as an effective carrier. Aeroponic root inoculum was stored dry at 4°C for 23 months without significant reduction in propagule density; however, this material was not appropriate for shearing. Moist roots, useful for shearing, began to lose propagule density after 1 month of storage. Shearing proved to be an excellent method to prepare viable root inocula of small and uniform size, allowing for more efficient and effective use of limited inoculum supplies.     

Infection of Red Clover Seedlings by Heterodera trifolii Goffart and Pratylenchus penetrans (Cobb)

Penetration of 3-day-old ''Kenland'' red clover seedlings by Heterodera trifolii Goffart and Pratylenchus penetrans (Cobb) was investigated in 50-mm petri dishes on 1% agat or discs of Miracloth® (Chicopee Mills, Inc., New York, N.Y. 10018). Penetration by both nematodes increased arithmetically with increased numbers in the inoculum. H. trifolii larvae slowly penetrated all root tissue but in relatively low numbers, 25-30% of inocula. Swelling of larvae and formation of syncytia within roots was detectable 96 hr after inoculation. Initially, adults of P. penetrans preferentially penetrated the region 3-10 mm behind the root tip, but subsequently they invaded all along roots. P. penetrans penetration efficiency was high, 75-90% of inocula, and penetration was relatively rapid. When introduced simultaneously neither nematode affected the invasiveness of the other. Prior infection by H. trifolii did not affect the invasiveness of P. penetrans.     

Substratum preference of the caddisfly Helicopsyche borealis (Hagen) (Trichoptera: Helicopsychidae)

The position of Helicopsyche borealis (Hagen) (Trichoptera: Helicopsychidae) larvae on the substratum surface is dependent on the current regime but varies with larval size. All size classes of larvae chose significantly different positions on the substratum under high versus low current velocities. All size classes preferred exposed surfaces under low current velocities. Small larvae preferred the upper surfaces of substrata under low current velocities and were physically displaced under high current velocities. Larger larvae also occurred on upper surfaces, but were more evenly dispersed over all surfaces than smaller larvae, and tended to aggregate on down-stream faces of rocks during high flow.     

Anisakis,Phocanema, Contracaecum, and Sulcascaris Spp.: Electrophoresis and thermostability of alcohol and malate dehydrogenases from larvae

Electrophoretic surveys were conducted on individual larvae of four anisakine nematode genera: Anisakis, Phocanema, Contracaecum, and Sulcascaris. The larval worms were obtained from a variety of fish and molluscan hosts from widely dispersed geographic regions. Of several enzymes detected, constant and apparently species-specific electrophoretic patterns were obtained for alcohol dehydrogenase (ADH, alcohol:NAD oxidoreductase, EC 1.1.1.1) and malate dehydrogenase (MDH, l-malate: NAD oxidoreductase, EC 1.1.1.37). ADH, in all but Sulcascaris sp., possessed two isozymes, the slower of which was sensitive to temperature and inhibitors. Failure of preelectrophoretic treatment with NAD to cause interconversion of these isozymes suggests that they are products of separate genetic loci. Both isozymes were maximally active with isopropanol, sec-butanol, and amyl alcohol. Within a given species, ADH showed negligible variation (i.e., apparent genetic polymorphism) with respect to individual larvae, site of larvae in the host, or geographical origin of the host. MDH from Anisakis, Sulcascaris, and Phocanema spp. possessed one, two, and three bands of activity, respectively; MDH is highly thermostable in Anisakis sp. but not in the other species.     

Laboratory studies of the entomopathogenic fungus Nomuraea rileyi: Soil-borne contamination of soybean seedlings and dispersal of diseased larvae of Trichoplusia ni

Germinating soybean seedlings were contaminated by soil-borne conidia of the entomogenous fungus Nomuraea rileyi. Although N. rileyi was detected on the unifoliate and trifoliate leaflets, most of the inoculum was found on the cotyledons. Diseased larvae of Trichoplusia ni released on the first trifoliate leaves of soybean plants dispersed to all trifoliates and died. In this way, inoculum is produced that infects other larvae feeding on these leaves.     

Optimization of Inocula Conditions for Enhanced Biosurfactant Production by Bacillus subtilis SPB1, in Submerged Culture, Using Box–Behnken Design

The inoculum age and density can influence considerably the production yield and cost of the fermentation process. Some literature studies report the use of two-stage inocula to enhance metabolite production. In the present study, optimization studies were done in order to define the best inocula conditions supporting a maximum biosurfactant production by Bacillus subtilis SPB1. Hence, by adjusting the levels of the two-stage inocula strategy, lipopeptide production was effectively enhanced to almost 3.4 g/l as estimated gravimetrically. The new defined parameters were as follows; a first inoculum age of 23 h followed by a second inoculum age and size of 4 h and 0.01, respectively. Thereby, we note an improved production as compared to the production yield described under non-optimized inocula conditions reported in our previous work.     

The phospholipase B content of the intestines of rats infected with varied larvae doses of Nippostrongylus brasiliensis.

Phospholipase B activity, bone marrow eosinophilia and worm burden were measured in separate groups of rats that received initial infections of 100, 400, 800, 1200, 2400, and 4800 Nippostrongylus brasiliensis larvae. The results indicated that as the numbers of larvae in the inocula were increased, the resulting higher worm burdens caused the development of earlier and more intense bone marrow eosinophilias and earlier and greater phospholipase B activities. The expulsion of the adult worms from the host was followed by equally rapid decreases in bone marrow eosinophilia and enzyme activity. It was found that as the number of intestinal adult worms was increased from low to large numbers, the degree of the inflammatory response changed from mild to severe. This suggests that the inflammatory response was responsible for the elevated phospholipase B levels.     

Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients.

Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis.     

Some beneficial effects of aggregation in young larvae ofPryeria sinica moore (Lepidoptera: Zygaenidae)

The effects of group size on the survival and development of young larvae of Pryeria sinicaMoore were investigated by laboratory and field experiments. Under laboratory conditions, about 20% of isolated larvae died of unsuccessful feeding in the first instar, however, larvae survived successfully in aggregations of four or more individuals. In the field, larvae emerge in early spring and wait for new leaves to open before feeding. In this period, the larger the group size of hatchlings the survival rate became higher. The nest-web spun by hatchlings was considered to play an important role in protecting them from desiccation. In the period that larvae began to feed on leaves, more than 36 larvae are necessary to aggregate for the successful establishment of feeding groups. The nest-web played an important role also in the establishment of feeding group. However, the natural group size of the first instar larvae was larger than the minimum group size to spin a sound nest-web in the field experiment. On the other hand, in later stage, larvae in a large group did not have an excess advantage in survival or developmental rate over larvae in a small group. It was found that the experiments on survival and developmental rates could not explain the reason that this species maintain large compact groups in the most part of larval period.     

Co-Occlusion and Persistence of a Baculovirus Mutant Lacking the Polyhedrin Gene

A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. Coinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Autographa californica nuclear polyhedrosis virus (AcMNPV) and an AcMNPV mutant (Ac-E10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. The amount of occluded Ac-E10 virions in progeny polyhedra populations during serial passage in Trichoplusia ni larvae was evaluated. Maintenance of the mutant in progeny polyhedra required polyhedra inocula containing equal numbers of the two virus types at a high dose. A significant reduction in occluded mutant nucleocapsids occurs with inoculum levels below a 100% lethal dose. At inoculum levels below a 30% lethal dose, the majority of fourth-instar larvae were infected with only one type of virus. The commercial application and ecological advantages of the co-occlusion process are discussed.     

Effects of Inoculum Size on Solid-Phase Fermentation of Fodder Beets for Fuel Ethanol Production

This fuel ethanol study examined the effects of Saccharomyces cerevisiae inoculum size on solid-phase fermentation of fodder beet pulp. A 5% inoculum (wt/wt) resulted in rapid yeast and ethanol (9.1% [vol/vol]) production. Higher inocula showed no advantages. Lower inocula resulted in lowered final yeast populations and increased fermentation times.     

Status and importance ofRhyzobius forestieri (Col.: Coccinellidae) on citrus at chios Island, Greece, nine years after its introduction

The originally Australian coccidophagous predatorRhyzobius forestieri (Coleoptera: Coccinellidae), imported from California into Greece in 1981, was first released on citrus at Cambos, Chios, in 1983 against soft scalesCoccus pseudomagnoliarum andSaissetia oleae (Homoptera: Coccidae). It was successfully established in two orchards there. During the following decade,R. forestieri spontaneously dispersed throughout the Cambos coastal plain. In July 1992, a survey of coccidophagous coccinellids was carried out, to follow up on the dispersal ofR. forestieri on citrus and to compare its population size with that of the main native coccidophagous coccinellids. The samplings were made in 4 selected scale-infested orchards of Cambos spaced 1–2 km apart. Of a total of 337 adults and 237 larvae of coccidophagous coccinellids found,R. forestieri comprised 84.0% and 75.1%, respectively. It was present in all 4 localities; in 2,R. forestieri adults and larvae were by far the most abundant coccidophagous coccinellids.     

Larval gregariousness and neonate establishment of the eucalypt-feeding beetle Chrysophtharta agricola (Coleoptera: Chrysomelidae: Paropsini)

The selective advantage offered to individuals living within groups may relate to natural enemy defence, but in leaf feeding insects may also relate to overcoming plant defences, especially with respect to feeding establishment. We conducted a series of experiments focusing on neonate larval survival, examining the effect of group size and leaf age on the survival of a eucalypt-feeding beetle, Chrysophtharta agricola , which formed groups of up to 43 larvae on the foliage of Eucalyptus nitens in the field. In the laboratory, in the absence of natural enemies, we found that initial density, leaf age and damage to the leaf margin significantly affected larval survival. Survival of solitary first-instar larvae on young foliage was around 80% whereas on older foliage it was around 11%. Prior damage to the leaf margin significantly increased survival on older leaves to around 61%. Initial larval density also affected survival, although mortality was always significantly higher on older leaves. On older leaves the larval group size above which mortality increased no further was over two-fold that on young leaves. Observations of group feeding behaviour at each instar showed that the majority of larvae (75.7%) were aligned facing away from the feeding site and that only around 7.5%, or just 1–2 larvae per group, fed at any one time. Feeding larvae chewed the leaf edge by straddling the leaf margin. Measurements of leaf margins showed that older leaves had significantly thicker leaf margins and 'thickness' ratios (leaf margin to leaf lamina proper). In the field, approximately 85% of all larvae occurred on the first two expanded leaf pairs, and larval mortality was highest between eclosion and establishment of the first instar. However, beetles apparently did not adjust clutch size according to leaf age.     

Estimation of the numbers and infectivity of Nippostrongylus larvae

Experimental investigations of the subcutaneous infection of third-stage larvae of Nippostrongylus brasiliensis reveal discrepancies between the actual and intended inoculum, and also variability between replicate inocula. Emphasis is thus placed on the importance of obtaining accurate estimates of the level of confidence associated with inoculum size. Under specified laboratory conditions, the retention of infectivity of N. brasiliensis larvae was found to be age-dependent, with a maximum of between 120 and 156 days. The motility of the larvae was not found to be indicative of their ability to complete migration and to become established in the small intestine of the host following subcutaneous inoculation.