Rearing of two predators,Thanasimus dubius andTemnochila virescens, for the biological control ofIps grandicollis in Australia

Rearing methods for two coleopterous predators,Thanasimus dubius andTemnochila virescens, imported into Australia for the biological control ofIps grandicollis, were developed. Bionomic data obtained from laboratory rearings between 1982–1987 showed thatT. dubius eggs took about 7 days to hatch and that duration of the larval stage was about 42 days. Observations showed thatT. dubius had a prolonged prepupal stage (x=56.4 days, range 14–274 days), which was probably non-diapausal in nature. Mean adult longevity was 50 days (range 1–358 days).Temnochila virescens eggs took almost 9 days to hatch, and a lengthy larval stage (x=155.4 days, range 73–333 days) was observed. Mean duration of the pupal stage was 14 days (range 7–28 days). A long preoviposition period (x=141 days, range 47–206 days) was observed, and adults were very long-lived (x=232.7 days, range 14–667 days). Capacity for increase (r_c) calculated from rearing data suggested that numbers ofT. dubius could be increased faster thanT. virescens. Mortality between 1982–1987 averaged about 70% for both species. However, mortality ofT. dubius in 1987 increased significantly, suggesting that inbreeding or other methodological factors could be responsible. A mass-rearing method usingIps-infested pine billets was developed as a cheaper alternative to laboratory rearing, and was shown to be effective in producing large numbers of insects for release.     

Food partitioning by lake-dwelling triclads and glossiphoniid leeches: field and laboratory experiments

The triclads Polycelis tenuis and Dugesia polychroa and the glossiphoniid leeches Glossiphonia complanata and Helobdella stagnalis are abundant on the stony shores of productive British lakes. All species are food limited and there is considerable overlap in the diets of these triclads and leeches. This paper investigates interactions between the two groups using field and laboratory experiments to try to identify the mechanism of their co-existence. Triclad and leech numbers were manipulated inside experimental enclosures, mathced by controls, erected on the stony shore of an eutrophic English lake. Increasing the numbers of P. tenuis and D. polychroa prior to the reproductive season in spring resulted in a significant decrease in the numbers and body size of G. complanata and H. stagnalis compared with control populations in the summer months, and vice versa. However, increases and decreases were temporary with a readjustment of numbers and body size to control levels in the autumn after reproduction had ceased. It is suggested that increasing the numbers of either group elevated the severity of both intra- and interspecific competition for food. The condition of prey may, in part, determine the strength of competition, and this was examined in laboratory experiments in which different densities and ratios of P. tenuis and H. stagnalis were offered either live of recently crushed Asellus aquaticus. In monospecific controls, growth rates of P. tenuis were greater when fed on crushed than live Asellus, but there was no significant difference in the growth of H. stagnalis fed either live or crushed prey. In mixed cultures of predators, P. tenuis and H. stagnalis were the superior competitors when fed on crushed and live Asellus, respectively. However, when competitive pressure was low, at low densities of predators, the presence of H. stagnalis in mixed cultures fed on live prey was beneficial to the growth of P. tenuis. These results are explained in terms of the greater ability of triclads to detect damaged prey, leaking body fluids, due to their sophisticated chemosensory system, and the ability of leeches to capture live prey due to the presence of suckers. It is concluded that co-existence of the two groups in British lakes is assisted by the partitioning of food on a live or damaged basis.     

The reaction time of leech and triclad species to crushed prey and the significance of this for their coexistence in British lakes

1. Three species of leeches, Erpobdella octoculata, Glossiphonia complanata and Helobdella stagnalis, and four species of triclads, Polycelis nigra, P. tennis, Dugesia polychroa and Dendrocoelum lacteum, commonly coexist on stony shores in productive British lakes. All species are food limited and there is much overlap in their diet. For both leech and triclad communities, coexistence of species is through the occurrence of food refuges. Leeches are more successful than triclads at capturing live prey, whereas both groups feed on damaged prey, comprising incapacitated, live or dead animals that are leaking body fluids. If triclads are better than leeches at exploiting damaged prey, this could be a mechanism for their coexistence. 2. Laboratory experiments investigated the comparative speeds at which leeches and triclads responded to crushed prey. Young and adult predators were offered a crushed specimen of the oligochaete Tubifex tubifex, the snail Lymnaea peregra, the crustacean Asellus aquaticus or the chironomid Chironomus sp., and their reaction times recorded. These four prey groups constitute the main diet of the predators in the field. Only D. polychroa and D. lacteum showed a significantly different reaction time between young and adults to crushed prey, and the reason for this is unclear. All predators, except H. stagnalis and D. polychroa, showed a difference in reaction time to the four types of prey, presumably a consequence of differences in both the ‘quality’ and ‘concentration’ of the different prey fluids, and there were some differences between predators in their speed of reaction to the same prey type. The following sequence, from fastest to slowest, in general reaction time to prey was obtained: E. octoculata, D. polychroa, P. tenuis, D. lacteum, P. nigra, H. stagnalis and G. complanata. 3. The location of the damaged food by the predators can be explained partly in terms of their foraging behaviour, with E. octoculata, D, polychroa and P. tenuis exhibiting a more seek-out strategy than other species which have a more sit-and-wait behaviour, and partly on the level of sophistication of their chemosensory system used to detect leaked prey fluids. This system is highly developed in triclad species but poorly developed in leeches. 4. In a second type of experiment in which prey, L. peregra, A. aquaticus or Chironomus sp., were offered at different time intervals after crushing to H, stagnalis and P. tenuis, few predators fed on food crushed for 24 h or longer, although a few leeches fed on Chironomus crushed for up to 72 h. 5. It is concluded that coexistence of leech and triclad species on stony shores in lakes is assisted by partitioning of food on a damaged or live basis.     

Microdistribution of sulfate-reducing bacteria in sediments of a hypertrophic lake and their response to the addition of organic matter

To clarify the ecological significance of the association of sulfate-reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers ofl-SRB anda-SRB were 10⁴–10⁵ colony forming units (CFU) per ml in the 0–3 cm layer and 10²–10³ CFU ml⁻¹ in the 10–13 cm layer while the numbers ofp-SRB were one or two orders lower than those ofl-SRB anda-SRB. A sediment suspension was fractionated into four fractions (<1, 1–10, 10–94 and >94 μm). The highest proportions ofl-SRB anda-SRB were found in the 10–94 μm fraction: 66–97% forl-SRB and 53–98% fora-SRB. The highest proportion ofp-SRB was found in the >94 μm fraction (70–74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar toDesulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples,l-SRB anda-SRB in the <10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free-living and small particle-associated (<10 μm) SRB is higher than that by SRB associated with larger particles.     

Continuing dispersal of freshwater triclads (Platyhelminthes; Turbellaria) in Britain with particular reference to lakes

1. A survey of lakes in Anglesey and Snowdonia, Wales, in the summer of 1997 recorded changes in the triclad fauna which had occurred since earlier studies in the 1950s and in 1973. Two native species, Polycelis tenuis and Dugesia polychroa, and two immigrants, Planaria torva and especially Dugesia tigrina, have increased their range. The triclad fauna has remained unchanged with time in eleven out of sixteen lakes in Snowdonia, in contrast to only one out of fourteen lakes on Anglesey. This supports the hypothesis produced in the 1950s, that the triclad fauna of the ion-poor, unproductive lakes in Snowdonia would change little with time, being maintained by ecological interactions, whereas the ion-rich, productive lakes on Anglesey, from which native Dugesia spp. and Dendrocoelum lacteum are historically absent, would increase in triclad diversity. 2. An examination of national records for the distribution of D. tigrina and Pl. torva indicated their continuing dispersal in both still and running waters in mainland Britain, with the former species having the greater frequency of occurrence. 3. The reasons for this may be that D. tigrina is (a) more easily dispersed, (b) a more opportunistic, catholic feeder, (c) a more vigorous competitor, (d) able to reproduce asexually and (e) able to prey to some extent on other triclad species, as shown in the laboratory but not yet confirmed in the field. The absence of D. tigrina and Pl. torva from unproductive lakes may be explained in terms of low temperature and feeding mode, and a low standing crop of gastropods, respectively. 4. Seven surveys (1961–1997) of the triclad fauna (six species) of Colemere, England, revealed that D. tigrina was confined to one small area of the littoral zone until the mid-1980s, after which it spread fairly rapidly to occupy the entire shore by 1997. Its expansion has been to the detriment of all the native species, particularly P. tenuis, except Dd. lacteum. The possible extinction of Pl. torva may be linked to a numerical reduction in snails, particularly Potamopyrgus jenkinsi. 5. Dugesia tigrina has an adverse effect on the native triclad fauna in lakes, particularly when only a few species are present. This has been explained in terms of the availability of a wider variety of food. The presence of a larger number of triclad species denies the immigrant food items, particularly crustaceans and snails, the food refuges of Dd. lacteum and Dugesia spp., respectively. However, the long-term study of Colemere has shown that more diverse triclad faunas may also be vulnerable to this immigrant. The effect of Pl. torva on native triclads remains uncertain.     

Induction of defensive morphological changes in ciliates

Freshwater ciliates of the genusEuplotes change their morphology in response to the presence of predators. The morphological transformations limit the ability of the predators to ingest the ciliates. Induction of defensive morphology by the predatorsStenostomum sphagnetorum (Turbellaria),Lembadion bullinum (Ciliata),L. magnum, andAmoeba proteus (Rhizopoda) was studied inEuplotes aediculatus andE. octocarinatus. The results suggest the possibility of natural occurrence of predator-induced defensive morphology inEuplotes. A density of 1 predator ml⁻¹ was sufficient to induce significant changes in the morphology ofEuplotes. L. magnum was found at natural population densities of 5 individuals ml⁻¹. Transformations can take place within 2–4 h. Morphological changes are induced by signal substances released from the predators; direct contact between prey and predators is not necessary. The extent of transformation depends on the concentration of the signal substance. The size frequency distribution of the populations only had one peak that was related to predator density. All individuals of a population ofEuplotes changed their morphology according to the predator abundance. This may reduce energy costs for the prey by prevention of unnecessary morphological changes.Amoeba proteus induces morphological changes inE. octocarinatus, but not inE. aediculatus.     

Effects of recent acidification on Cladocera in small clear-water lakes studied by means of sedimentary remains

Cladoceran populations, as revealed by sedimentary remains, were studied in six lakes which by diatom evidence have become strongly acidified during the past 30 years. The accumulation rates of cladoceran remains and in two of the most strongly acidified lakes both relative and absolute numbers ofBosmina greatly increased during this period. Species numbers were not reduced, but there were shifts in relative abundances of cladoceran species.Bosmina longispina dominated all the lakes. Low pH (4.7) seemed not to be straight-forwardly harmful toDaphnia longispina, but the relative abundances of this taxon were reduced because ofBosmina.     

Leech predation on juvenile freshwater snails: effects of size,species and substrate

Summary The leechGlossiphonia complanata does not appear to have substantial impact on snail populations, but this may be due to most studies focusing on adult snails rather than juvenile snails. In this study I investigated how predation rates ofG. complanata feeding on newly-hatched and juvenile snails was affected by snail species, snail size, snail density and substrate, in a laboratory experiment. Number of snails eaten increased with increasing density resulting in a type II functional response curve. Predation rates were higher when leeches were feeding onLymnaea emarginata than onPhysa gyrina, whereas there was no significant difference in predation rates when they were feeding onL. emarginata andHelisoma anceps. Sandy substrates and greater snail size resulted in decreased predation rates. Sand reduced movement speed ofG. complanata, which probably reduced encounter rates. Thus, there was a comparatively large effect of leech predation on newly-hatched snails, due to a high probability of encounter and high predation rates, but spatial and temporal refuges probably reduce the importance of leech predation as a structuring force in freshwater snail assemblages.     

Effects of a top invertebrate predator (<Emphasis Type="Italic">Dytiscus alaskanus</Emphasis>; Coleoptera: Dytiscidae) on fishless pond ecosystems

We investigated the predatory effects of Dytiscus alaskanus, a large predaceous diving beetle, on the biomass, species composition and diversity of fishless pond communities. The effects were tested using presence and absence treatments of D. alaskanus in 24 mesocosms distributed among six ponds. We sampled phytoplankton, zooplankton and macroinvertebrates every two weeks for a six week period. Periphyton was sampled from the mesocosm walls on the final day. Total macroinvertebrate biomass decreased in the presence of dytiscids while species richness was not affected. Macroinvertebrate predators, snails and Gammarus lacustris decreased in the dytiscid treatments. Laboratory feeding experiments confirmed feeding preferences consistent with the mesocosm results. Periphyton biomass was six times greater in the dytiscid enclosures, concomitant with the decreased grazing by gastropods and other invertebrate primary consumers indicating a benthic trophic cascade. Top–down effects of dytiscids on other predatory invertebrates led to increased total zooplankton biomass, largely due to increased abundances of large and small cladocerans. Zooplankton species richness increased in the dytiscid enclosures. Inconsistent with trophic cascade theory, phytoplankton did not respond to top–down effects of D. alaskanus within the study period. Overall, the results show D. alaskanus predation caused trophic effects via two distinct food chains, a dytiscid–snail–periphyton trophic cascade, and a dytiscid–predatory macroinvertebrates–zooplankton partial trophic cascade.     

Density-dependent effects of multiple predators sharing a common prey in an endophytic habitat

Multiple predator species feeding on a common prey can lead to higher or lower predation than would be expected by simply combining their individual effects. Such emergent multiple predator effects may be especially prevalent if predators share feeding habitat. Despite the prevalence of endophagous insects, no studies have examined how multiple predators sharing an endophytic habitat affect prey or predator reproduction. We investigated density-dependent predation of Thanasimus dubius (Coleoptera: Cleridae) and Platysoma cylindrica (Coleoptera: Histeridae) on a bark beetle prey, Ips pini (Coleoptera: Scolytidae), in a laboratory assay. I. pini utilize aggregation pheromones to group-colonize and reproduce within the stems of conifers. T. dubius and P. cylindrica exploit these aggregation pheromones to arrive simultaneously with the herbivore. Adult T. dubius prey exophytically, while P. cylindrica adults enter and prey within the bark beetle galleries. Larvae of both predators prey endophytically. We used a multiple regression analysis, which avoids confounding predator composition with density, to examine the effects of varying predator densities alone and in combination on herbivore establishment, herbivore reproduction, and predator reproduction. Predators reduced colonization success by both sexes, and decreased I. pini reproduction on a per male and per female basis. The combined effects of these predators did not enhance or reduce prey establishment or reproduction in unexpected manners, and these predators were entirely substitutable. The herbivores net replacement rate was never reduced significantly below one at prey and predator densities emulating field conditions. Similar numbers of each predator species emerged from the logs, but predator reproduction suffered from high intraspecific interference. The net replacement rate of P. cylindrica was not affected by conspecifics or T. dubius. In contrast, the net replacement rate of T. dubius decreased with the presence of conspecifics or P. cylindrica. Combinations of both predators led to an emergent effect, a slightly increased net replacement rate of T. dubius. This may have been due to predation by larval T. dubius on pupal P. cylindrica, as P. cylindrica develops more rapidly than T. dubius within this shared habitat.     

Morphological and ecological shifts in a land snail caused by the impact of an introduced predator

Introduced predators have become major threats to native animal species in oceanic islands. A number of studies have shown that alien predators have caused serious extinctions of island endemics. However, little attention has been paid to the evolutionary impacts of alien predators on native species. The present study shows that predation by black rats, Rattus rattus, has resulted in ecological and morphological changes in the land snail Mandarina anijimana from the island of Anijima in the Ogasawara archipelago. The frequency of empty predated shells has increased over the past 17–19 years in southern areas of the island. The shells of these snails were found to be significantly higher, smaller and darker in the survey in 2006 than in the survey in 1987–1989 performed in central and southern parts of Anijima, where predation by Rattus was serious. M. anijimana were formerly restricted to shallow broad-leaved litter, whereas they are currently found in deep palm litter, where predation pressure from Rattus may be lower. This suggests that increased predation pressure by Rattus has changed the habitat use of M. anijimana. The close association between shell morphology and habitat use of Mandarina species suggests that the habitat shift induced by the predation of Rattus has caused these changes in the shell morphology of M. anijimana over a period of 17–19 years.     

The role of predators and parasites in controlling populations ofEarias insulana,heliothis armigera andBemisia tabaci on cotton in the Syrian Arab Republic

Studies on the relationship between insect pests and their predators and parasites were conducted in the Syrian cotton agro-ecosystem from 1980 up to 1983.Earias insulana (Boisduval) was found to be the most damaging pest, whileHeliothis armigera (Hübner) was not an annual returning problem. Among the entomophagous insects found, coccinellids were most numerous during the months June and July and the hemipterous predators were more abundant during August and September. When predator numbers were reduced with insecticide applications, a significant increase in the bollworm populations occurred, resulting in significant reductions in seed cotton yields. Parasites were relatively not important for the control of lepidopterous pests on cotton. Egg and larva populations ofE. insulana andH. armigera were parasitized byTrichogramma spp. andHabrobracon brevicornis Wesmael respectively. Populations ofBemisia tabaci (Gennadius) were heavily parasitized byEretmocerus mundus Mercet.      

Predation and the distribution and abundance of a pulmonate pond snail

Summary The abundances of a freshwater pulmonate snail, Lymnaea elodes were studied in a temporary pond and a permanent, more productive pond in northeastern Indiana, USA. When snails from both populations were reared in each of the ponds in containers excluding predators, snails grew to be 1.3 to 2 times as large in the more productive pond, and laid 9 times as many eggs. However, field sampling data showed adults to be more abundant in the temporary pond. The only obvious difference between the two ponds was the presence of the molluscivorous central mudminnow (Umbra limi) in the permanent pond. These fish fed upon L. elodes when eggs and juvenile snails were abundant. In an experiment in the temporary pond, addition of mudminnows lowered egg and juvenile snail survival in pens where snail abundances had been increased. We suggest that vertebrate predators like the mudminnow can be significant sources of mortality for thin shelled species like L. elodes, possibly excluding them from habitats like lakes and rivers.Address for offprint requests     

Application of a rapid thin section method for observations on decomposing litter in mor humus form in a subalpine coniferous forest

Morphological changes in the decomposing litter ofAbies spp. andBetula spp. in a mor humus form were studied by a rapid thin section method. According to the morphological characteristics, the epidermis, mesophyll and vascular bundleof Abies needle litter were classified into four types: (i) newly fallen; (ii) slightly decomposed; (iii) moderately decomposed; and (iv) greatly decomposed. The distribution of these tissue types along the profile of the forest floor was then investigated. The morphological changes in other litter types, such as branches, scales andBetula leaves during decomposition were observed directly with microscope and electron microscope. Five vertical thin sections and 80 horizontal thin sections were used for these observations and investigations. the decomposition ofAbies litter was slower than that ofBetula litter. The relative decomposition rate of the tissues was in the order of: mesophyll>vascular bundle >epidermis inAbies needles; mesophyll≥epidermis>vascular bundles inBetula leaves; and inner bark >xylem>outer bark in bothAbies andBetula branches. The last remains of the litter were usually stomata, segments of seminiferous scale and outer bark ofAbies. The decomposition of plant litter occurred mainly within the L and F layers of the soil (0–5 cm in depth).Abies needles andBetula leaves completely disappeared at depths of 0–6 cm and 0–4 cm, respectively. Branches disappeared within the top of 5 cm and 6–8 cm forBetula and forAbies, respectively. The scales ofAbies were most slowly decomposed in the soil layers.     

Determining the frequency of heteropteran predation on sweetpotato whitefly and pink bollworm using multiple ELISAs

The gut contents of field-collected, predaceous Heteroptera were assayed for the presence of eggs of the sweetpotato whitefly,Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) and the pink bollworm,Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) using multiple enzyme-linked immunosorbent assays (ELISAs). Of seven species examined,Geocoris species andOrius tristicolor (Say) were the most frequent predators of sweetpotato whitefly with 32–39% of the individuals tested over the whole season scoring positive for whitefly antigens. With the exception ofLygus hesperus Knight, a major insect pest as well as a predator, the frequency of predation on pink bollworm eggs was much lower (0.7–14.3% positive over the season). Relatively few predators tested positive for both antigens (0.3–12.5%).     

Sulphur requirements of Curvularia pallescens Boed., Alternaria citri Ell. and Pierce and A. tenuis auct

Sulphur requirements ofCurvularia pallescens, Alternaria citri, andA. tenuis have been studied. The fungi exhibited selectivity towards different sulphur compounds. Magnesium sulphate gave best growth and sporulation of the three fungi and it could not be replaced by any other sulphur compound.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

RFLP analyses of severalBrassica representatives

Molecular variability was determined between, 17Brassica napus cultivars, 8 new selections 4 F₁ hybrids, 5 cultivars ofB. oleracea, B. campestris andB. peruvianum with 4 rDNA probes fromLycopersicon esculentum, 3 probes from a PstI library ofB. oleracea, 3 probes from EcoRI libraries ofB. napus andB. oleracea and the acetolactatsynthase gene fromNicotiana tabacum. Sporadic interspecific and rare intraspecific variability was detected. Contribution presented to the 5^th Czechoslovak Seminar “Plant Gene Engineering”, organized by the Institute of Plant Molecular Biology in České Budějovice, 2–13 September 1991.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Regulation of nebramycin biosynthesis by inorganic phosphate

The effect of inorganic phosphate on the biosynthesis of nebramycin factors2, 4 and5′ was studied inStreptomyces tenebrarius strain A (forming2, 4 and5′ in natural ratios) and its mutants B (forming predominantly2), C (forming2 as the only major product) and D (forming predominantly5′). In phosphate-supplemented complex media, the production of2 in A, B and C was reduced by 20–70%, while the yields of5′ remained unchanged in A and decreased by 30–60% in B. The production of4 increased by 50–90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.