Mycelial growth of strains of the genera <Emphasis Type="Italic">Suillus</Emphasis> and <Emphasis Type="Italic">Boletinus</Emphasis> in media with a wide range of concentrations of carbon and nitrogen sources

Suillus and Boletinus were studied using Ohta medium. In media with glucose or trehalose, all tested strains grew well. With mannose and cellobiose, strains generally grew well, except for one strain of Suillus. Utilization of dextrin and soluble starch differed with each strain, and that of sucrose and glycerol was low for all strains. Utilization of four amino acids, arginine, glutamic acid, aspartic acid, and alanine, was similar to that of ammonium tartrate for Suillus strains, but mycelial growth with amino acids was clearly lower than with ammonium tartrate for the Boletinus strain. The effect of glucose and ammonium tartrate concentrations for nine strains of the genera Suillus and Boletinus was studied with ranges for glucose of 1–100 and 200g/l, respectively, and for ammonium tartrate of 0.2–5 and 20g/l, respectively. Six strains showed maximal growth at a glucose concentration greater than 25g/l, and one strain showed maximal growth at 70g/l. The results indicate that these fungi are adapted to relatively high concentrations of carbon sources. In general, glucose concentration at mycelial growth maximum decreased as ammonium tartrate concentration increased, and at higher concentrations of glucose, mycelial growth decreased more rapidly in higher concentrations of ammonium tartrate.     

Larval development and food habits of the marbled parrotfish, <Emphasis Type="Italic">Leptoscarus vaigiensis</Emphasis>, associated with drifting algae

The larval development and food habits of the marbled parrotfish, Leptoscarus vaigiensis (Scaridae) associated with drifting algae were studied. In this study, 628 L. vaigiensis of various developmental stages ranging from postflexion larvae (9.4mm in standard length, SL) to adults (192.0mmSL) were sampled from drifting algae at two fishing ports in Nakagusuku Bay of Okinawa Island. In 3969 fish comprising 65 taxa in 34 families of Teleostei collected with drifting algae, L. vaigiensis occupied 15.8% of samples and occurred generally throughout the whole year. A large number of L. vaigiensis were collected from July to October accompanied by an occurrence of drifting algae composed of Sargassum spp. Larvae and early juveniles ranging from 11.1 to 14.9mmSL appeared sporadically throughout the year, and postflexion larvae 11mmSL occurred from July to November. Their food shifted from planktonic copepods in postflexion larvae and juveniles ranging from 10.0 to 14.9mmSL to seaweed in the juveniles ranging from 15.0 to 24.9mmSL. Furthermore, adults and young over 25mmSL fed almost exclusively on seaweed, with Sargassum spp. constituting the drifting algae. These facts indicate that drifting algae may have a role concerning food and habitat, and may act as a nursery for L. vaigiensis.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

Accumulation and depuration of microcystin produced by the cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> in a freshwater snail

Seasonal changes in microcystin concentrations in a resident snail (Sinotaia histrica) and an edible clam (Corbicula sandai) in Lake Biwa were surveyed. To clarify both the accumulation and depuration of microcystins, experimental studies with microcystin were also carried out on the snail. In the field investigation, microcystin was detected from the hepatopancreas and intestine of S. histrica (up to 3.2µgg^–1 dry weight and 19.5µgg^–1 dry weight, respectively); however, no microcystin was detected in the hepatopancreas of C. sandai. In the laboratory experiment, the microcystin-LR concentration in the hepatopancreas of S. histrica reached a value of 436µgg^–1 dry weight on day 10 of 15 days of uptake, and a high value persisted despite a depuration period of 15 days. The depuration rate constant of microcystin and its biological half-life were 0.0828 day^–1 and 8.4 days, respectively. These results indicate that S. histrica has a high ability to accumulate microcystin in its tissue. Because S. histrica is predated by fish and water fowl, it is likely to play an important role as a vector for microcystin in lakes with dense blooms of toxic cyanobacteria.     

Competition between <Emphasis Type="Italic">Eucalyptus victrix</Emphasis> seedlings and grass species

Competition in a natural system may be interspecific or intraspecific. In semiarid ecosystems, competition for resources between established neighboring grass species and newly recruited seedlings is very high. To examine the effects of grass species density, growing space and time of establishment on Eucalyptus victrix seedlings (interspecific competition), and the effect of density and growing space within E.victrix (intraspecific competition) we conducted an experiment under controlled conditions. We tested four hypotheses (i) E.victrix seedling growth is not affected by grass density; (ii) there is no difference in E.victrix survival and growth between early and later grass establishment; (iii) interspecific competition is not more intense than intraspecific competition in E.victrix; and (iv) growth of E.victrix seedlings is not dependent on available growing space. In a monoculture of E.victrix, seedling mortality was higher (10%) in large pots. In mixed culture pots, where E.victrix seedlings and grass seedlings were planted on the same day, E.victrix seedlings survived for up to 4weeks, but started to die after week five in the smallest pots. However, mortalities occurred in pots of all sizes when grass was established before E.victrix seedlings. Results also indicated that the resources necessary for the growth of individual E.victrix seedlings were more limiting under conditions of increased density of neighboring grass species rather than intraspecific competition. In particular, photosynthetic area of E.victrix seedlings was drastically reduced in mixed cultures. Although density, pot size and time of planting had impacts on E.victrix seedlings, the patterns of these impacts were variable.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

Milk-clotting protease production by <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. in an inexpensive medium

The milk-clotting and proteolytic activities of extracellular enzyme preparations from Nocardiopsis sp. were investigated under different culture conditions. A soybean flour medium was used, with concentrations of soybean flour and of glucose varying from 0.25 to 1% w/v and from 0 to 1% w/v, respectively. Growth was monitored with 2ml samples withdrawn from the culture medium at 8-h intervals, for determination of total protein, proteolytic activity, milk-clotting activity and sugar reduction. The best milk-clotting protease production, with a specific activity of 24.49U/mg at 40h, was obtained in the glucose-free medium containing soybean flour 1% w/v.     

Efficiency of herbivore exclusion by ants attracted to aphids on the vetch <Emphasis Type="Italic">Vicia angustifolia</Emphasis> L. (Leguminosae)

The efficiency of herbivore exclusion by ants on the vetch Vicia angustifolia L. (Leguminosae) with extrafloral nectary, mediated by ant attraction to aphids was investigated in a field census and laboratory experiments. In the field, workers of Lasius japonicus Santschi and Tetramorium tsushimae Emery frequently visited plants of the vetch parasitized by aphids of Aphis craccivora Koch, but only a few workers visited plants without aphids. An increase in the number of ants visiting a plant with increasing numbers of aphids caused a decrease in the number of larvae of the weevil, Hypera postica Gyllenhal. Therefore, the efficiency of herbivore exclusion by ants was higher on plants parasitized by Ap.craccivora aphids than that on plants unparasitized by aphids. In the laboratory experiments, L.japonicus workers frequently patrolled not only shoots with Ap.craccivora aphids but also shoots without them. However, T.tsushimae workers visited mainly shoots with Ap.craccivora aphids but less frequently on shoots without aphids. Therefore, L.japonicus workers excluded herbivores more efficiently on plants of the vetch than T.tsushimae workers. Consequently, the efficiency of herbivore exclusion by ants on the vetch can be influenced directly by differences in ant species and indirectly by the presence of aphids on plants. The present study highlights the significance of indirect interactions between ants and plants with extrafloral nectary, mediated by ant attraction to aphids for herbivore exclusion of plants.     

Consequences of harvesting for genetic diversity in American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.): a simulation study

American ginseng, Panax quinquefolius L., is one of the most heavily traded medicinal plants in North America. The effect of harvest on genetic diversity in ginseng was measured with a single generation culling simulation program. Culling scenarios included random harvest at varying levels, legal limit random harvest and legal limit mature plant harvest. The legal limit was determined by the proportion of legally harvestable plants per population (% mature plants per population). Random harvest at varying levels resulted in significant loss of genetic diversity, especially allelic richness. Relative to initial levels, average within-population genetic diversity (H _e) was significantly lower when plants were culled randomly at the legal limit (Mann–Whitney U=430, p<0.001) or when only mature plants were culled (Mann–Whitney U=394, p<0.01). Within-population genetic diversity was significantly higher with legal limit mature plant harvest (H _e=0.068) than when plants were culled randomly at the legal limit (H _e=0.064; U=202, p<0.01). Based on these simulations of harvest over one generation, we recommend that harvesting fewer than the proportion of mature plants could reduce the negative genetic effects of harvest on ginseng populations.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

<Emphasis Type="Italic">Uncisudis posteropelvis</Emphasis>, a new species of barracudina (Aulopiformes: Paralepididae) from the western North Pacific Ocean

Six specimens (2 flexion larvae: 9.5–10.4mm in notochord length; 4 postflexion larvae: 12.3–18.2mm in standard length) collected from the western North Pacific are tentatively ascribed to the genus Uncisudis of the tribe Lestidiini of the subfamily Paralepidinae (Paralepididae) in sharing remarkably elongate and filamentous pelvic fin rays, their tips reaching the origin of the anal fin. They are described as Uncisudis posteropelvis sp. nov. in uniquely having the insertion of pelvic fins closer to the origin of anal fin than to the posterior end of dorsal fin base among lestidiine species. Addition to this character, the new species has remarkably elongate and filamentous dorsal fin rays, the short distance between anus and origin of anal fin (4.2–6.1% of standard length, SL), the posteriorly located pelvic fins (prepelvic length 69.4–71.5% SL), dorsal fin rays 10, anal fin rays 28–29, myomeres 41–42+38–40=80–81 (vertebrae 38+41=79), and peritoneal pigment spots 11–12. The occurrence of larvae differing in pigment pattern from the present new species suggests another undescribed species of Uncisudis in the western South Pacific.     

Life Cycle of the Tick Haemaphysalis Leporis-palustris (Acari: Ixodidae) Under Laboratory Conditions

The life cycles of two separate populations (colonies A and B) of the rabbit tick, Haemaphysalis leporis-palustris, were studied under laboratory conditions. Domestic New Zealand rabbits, Oryctolagus cuniculus, and wild rabbits, Sylvilagus brasiliensis, were used as hosts for ticks from colony B and only O. cuniculus rabbits were used as hosts for ticks from colony A. Developmental periods were observed in an incubator at 27±1°C and RH 90±5%. Larvae from colonies A and B fed for 8.0±3.7 days and 8.5±1.3 days, respectively, on O. cuniculus. On S. brasiliensis larvae from colony B fed for 7.2±1.3 days. Nymphs from colony A fed for 8.1±1.4 days on O. cuniculus and nymphs from colony B fed for 8.1±1.0 days on S. brasiliensis. Only one engorged nymph from colony B was recovered from O. cuniculus. Females from colony A fed for 20.9±5.9 days on O. cuniculus and females from colony B fed for 18.6±2.4 days on O. cuniculus and 18.7±3.7 days on S. brasiliensis. Engorged larvae from colony A required 13.7±3.7 days to molt while engorged larvae from colony B required 11.8±3.0 and 11.5±1.8 days to molt, after having fed on O. cuniculus and S. brasiliensis, respectively. Engorged nymphs from colonies A and B required 16.3±1.9 days and 14.7±1.4 days to molt, respectively. Engorged females from colonies A and B required 4–7 and 3–5 days, respectively, to start oviposition. Mean egg incubation periods lasted for 33–34 days. For ticks from colony B, host species accounted for significant differences (p<0.05) in larval and nymphal feeding periods, oviposition weights and CEIs. Significant differences (p<0.05) between the two colonies when ticks fed on O. cuniculus were observed for larval and nymphal feeding and premolt periods, engorged female and oviposition weights and conversion efficiency indexes (CEI). S. brasiliensis were always a more suitable host for H. leporis-palustris than O. cuniculus. Significantly more larvae and nymphs engorged and molted when fed on S. brasiliensis (p<0.001). Females fed S. brasiliensis were more successful to lay fertile eggs and showed the highest engorged and egg mass weights, and the highest CEIs. Data of H. leporis-palustris fed on wild rabbits (one of its natural host species) are reported for the first time.     

How does spittlebug oviposition affect shoot growth and bud production in two willow species?

We measured the effects of oviposition by the spittlebug Aphrophora pectoralis on shoot growth and bud production in two willow species, Salix miyabeana and Salix sachalinensis. In autumn, adult females of A.pectoralis insert their ovipositor into the apical region of 1-year-old shoots, resulting in the death of most shoot tips within 1week. Consequently, an increase in the number of dead buds and a decrease in the number of vegetative buds on 1-year-old shoots was recorded. In the following spring, the growth of current-year shoots was greatly increased on 1-year-old shoots damaged by spittlebug oviposition. Furthermore, spittlebug oviposition increased the production rate of vegetative buds in both S.miyabeana and S.sachalinensis. However, no impact on the production rate of reproductive buds was detected in either willow. We conclude that the compensatory growth of current-year shoots and an increase in vegetative buds in the two willow species was caused by oviposition of A.pectoralis.     

Assessment of within-population genetic structure in <Emphasis Type="Italic">Quercus crispula</Emphasis> and <Emphasis Type="Italic">Q. dentata</Emphasis> by amplified fragment length polymorphism analysis

The spatial genetic structure was assessed by amplified fragment length polymorphism (AFLP) technique for Quercus crispula Blume (37 individuals) and Q.dentata Thunberg (54 individuals) growing along a 550-m transect in Ishikari, Hokkaido, northern Japan. The simple Mantel test revealed significant negative correlation between genetic similarity and geographic distance in Q.dentata and negative but insignificant correlation in Q.crispula. Spatial autocorrelation analysis revealed that Mantels r generally decreased from positive to negative values with the increase of spatial distance in both oak species with significant deviation from zero for the 50–100-m (positive) and 250–300-m classes (negative) in Q.dentata. Thus, significant spatial genetic structure was revealed at least in Q.dentata     

Effects of domestication and agronomic selection on phytoalexin antifungal defense in <Emphasis Type="Italic">Phaseolus</Emphasis> beans

Systems of wild and cultivated relatives are good experimental systems to test chemical defense theory because they provide closely related varieties that differ in discrete traits. To determine the relationship between resistance and chemical defense diversity among wild, landrace and cultivar accessions of Phaseolus vulgaris, we measured resistance to fungal infection in laboratory and field experiments, quantified phytoalexin diversity, and assessed the effectiveness of phytoalexin mixtures extracted from live tissue. Results show a gradient of resistance to fungal infections between wild, landrace and cultivar varieties. In the laboratory, wild seedlings were more resistant (93% non-infected) than landrace seedlings (80% non-infected) and modern cultivar seedlings (68% non-infected). Under field conditions wild seedlings were more resistant (97% non-infected) than cultivar seedlings (71% non-infected). Wild seedlings presented the highest phytoalexin diversity (H=1.11), while those of the landrace presented an intermediate level (H=0.97) and cultivar seedlings presented the lowest diversity (H=0.93). No differences were found in total concentrations. The in vitro inhibitory properties on hyphal growth of the isoflavonoid mixtures produced by individual seedlings showed a similar trend. Our results are consistent with similar gradients in other species of Phaseolus beans and resistance to Colletotrichum sublineolum in sorghum.     

Seed dispersal curve of a Mediterranean myrmecochore: Influence of ant size and the distance to nests

Euphorbia characias is a Mediterranean spurge with a diplochorous dispersal system: after a ballistic dispersal that scatters the seeds, some ant species find and retrieve the seeds to their nest (myrmecochorous dispersal). The seed dispersal curve generated by ants in an abandoned field was described and partitioned according to ant size and to the distance to nest entrance from where seeds fell after ballistic dispersal. Both variables (ant size, distance to nest) affected dispersal distance. The seed dispersal curve showed a peak at short distance (median=1m) and a tail extending to 9.4m. The peak and the tail are explained differently. Short distances are usually generated by small ants (Pheidole pallidula and Tapinoma nigerrimum; 0.56±0.41m [n=48]) both from the nearest or farther nest entrances. The tail of the curve is generated disproportionately by big ants (Aphaenogaster senilis and Messor barbarus; 2.09±1.71m [n=61]) from farther nests. Seeds have a much greater probability (P=0.734) of being transported to nests which are not the nearest. This effect is largely due to transportation by big ants.     

Development and distribution of the early life stages of the longfin pearleye <Emphasis Type="Italic">Benthalbella linguidens</Emphasis> (Aulopiformes: Scopelarchidae) in the western North Pacific

The early life history and development of the scopelarchid Benthalbella linguidens was studied, based on 203 specimens (from 5.3 to 89.7mm in body length: BL) collected from Kuroshio, Oyashio waters and transition waters of the western North Pacific. The early life stages of B. linguidens are distinguished from those of other species of Benthalbella that inhabit the North Pacific by the characters of 62–64 myomeres in the larval stage and 26–28 anal fin rays in juvenile and transforming specimen. Larvae are elongate; notochord flexion begins at ca. 12mm BL and is completed at ca. 15mm BL. The fin ray complements are established at ca. 40mm BL. The single transforming specimen (89.7mm BL) that has peritoneal pigment was collected from transition waters. All larvae were collected from Kuroshio and transition waters from winter to early summer; however, the size of larvae in Kuroshio waters was apparently smaller than that in transition waters, with ranges of 5.3–32.4mm BL (mean 17.1) and 15.3–35.3mm BL (mean 27.5), respectively. Juveniles were distributed in transition and Oyashio waters and were absent in Kuroshio waters, where adults are commonly distributed. These occurrences of larvae and juveniles in the western North Pacific indicate that B. linguidens spawns in Kuroshio waters in winter and uses transition waters as nursery grounds.     

Improvement of Kefir yeast by mutation with N-methyl-N- nitrosoguanidine

A commercial culture of Kefir grew well at 30°C and 37°C with NaCl up to 4% (w/v) but at 42°C and 4% (w/v) NaCl it grew very slowly. These conditions were selected for the mutation with N-methyl-N-nitrosoguanidine in order to improve cell yield at high temperatures and salt concentration. The mutated cells had higher biomass and growth rate compared with the original culture. The improvement of Kefir at high temperatures and salt concentrations offers advantages for uses in single cell protein production and alcohol production from whey containing high salt.