Seasonal abundance of three life cycle stages of Rasajeyna nannyla (coccidia) in Tipula paludosa and T. vittata

Tipula larvae contract coccidial infections in feeding with ease and regularity, yet only a minor portion of the population shows infections. The numbers of schizonts and macrogametes, two stages in the life cycle of Rasajeyna nannyla which parasitize the midguts of leatherjackets, increased throughout the life cycle of the host. A peak number of pathogens occurred in midwinter and another in summer. The pathogen was discharged from the host as oocysts, and the numbers of this stage were high at the begining and end of the leatherjacket life cycle but were low throughout the intervening winter months. Counts of macrogametes were higher than the numbers of schizonts. A difference in number of these stages is reported from Tipula paludosa and T. vittata larvae.     

Differentiation between larvae of Tipula paludosa and Tipula oleracea (Diptera: Tipulidae) using isoelectric focusing, and their occurrence in grassland in northern Britain

Thin layer agarose isoelectric focusing (IEF) was used to separate proteins from the larvae of Tipula oleracea Linnaeus and Tipula paludosa Meigen (Diptera, Nematocera). Silver staining revealed protein banding patterns which consistently distinguished these two species at any stage in their life cycles. Within the pH range 5–6 Tipula paludosa contained one major protein band and T. oleracea two bands, with the pI value of the T. paludosa protein being slightly higher than those of T. oleracea. The IEF method is particularly useful for the larval stages of these species because they are often visually inseparable. Leather jackets obtained during a survey of Northern Ireland and north-eastern and western Scotland were tested to determine the distribution and frequency of these species occurring in grassland. In each area surveyed T. paludosa predominated with only one field in Northern Ireland and north-eastern Scotland found to contain T. oleracea. In western Scotland T. oleracea occurred in five fields each year of the survey, and was locally abundant on the Isle of Bute in the 1990/91 survey making up 15% of the total larvae tested.     

Genetic and electron-microscopic characterization of Rickettsiella tipulae, an intracellular bacterial pathogen of the crane fly, Tipula paludosa

Rickettsiella tipulae is an intracellular bacterial pathogen of larvae of the crane fly, Tipula paludosa (Diptera: Tipulidae) and has previously been claimed to represent an independent species within the genus Rickettsiella. Recently, this taxon has been reorganized and transferred as a whole from the α-proteobacterial order Rickettsiales to the γ-proteobacterial order Legionellales. Here we present the electron-microscopic identification of this rickettsial pathogen together with the first DNA sequence information for R. tipulae. The results of our 16S rDNA-based phylogenetic analysis demonstrate that the transfer to the order Legionellales is justified for R. tipulae. However, there is no phylogenetic basis to consider R. tipulae an independent species, but instead conclusive evidence substantiating its species level co-assignment with Rickettsiella melolonthae. Furthermore, implications of our results for a possible reorganization of the internal structure of the genus Rickettsiella are discussed.     

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Digestion and excretion of nitrogen and carbohydrate by the cranefly larva Tipula paludosa (Diptera: Tipulidae)

The nitrogenous and carbohydrate components of ryegrass and faeces from larvae of Tipula paludosa Meigen, fed on ryegrass (Lolium perenne L.), were compared. Proteins in ryegrass were efficiently digested and uric acid was the major nitrogenous excretory product. The alkaline midgut (pH 9.1) was considered to enhance the digestibility of hemicellulose, by removing inhibitory acetyl groups, and of cellulose by altering its crystallinity. T. paludosa larvae assimilated 50% of ryegrass cellulose, and 50% of an isolated ¹⁴C-labelled cellulose, whereas 86% of hemicellulose was digested.     

Evidence of a climatic niche shift following North American introductions of two crane flies (Diptera; genus Tipula)

Ecological niche models assume a species niche should be conserved over space and time. Increasingly, studies have determined that niche shifts may occur during biological invasion events. The aim of this study is to examine niche conservation for two invasive crane flies, Tipula oleracea Linnaeus and Tipula paludosa Meigen, after introductions into North America. These species have broadly sympatric invasive distributions but differ in time since introduction and dispersal abilities. As these factors may impact the area accessible to dispersal, I examined the impact of background area delineation on conclusions of niche conservation. Results indicated that alternative delineations of accessible area (i.e., background area) had no affect on measures of niche equivalence. Neither Tipula species was found to be occupying invasive niche space equivalent to that of their native ranges. Niche dissimilarity was found for both species, with results strongly impacted by the choice of background area. T. paludosa introductions displayed a niche shift across both invasive introductions when the model area drew climatic information from an area that buffered occurrences by 40 km. The eastern T. oleracea introduction displayed a niche shift when background information was drawn from within a 400 km buffered area. This study suggests that invasive populations may be displaying a niche shift when evaluated against one scale of background but conserved when evaluated against another scale. Dispersal limitations for T. oleracea in its eastern introduction and anthropogenic habitat associations for T. paludosa across both invasive introductions are indicated as causes for the observed niche shifts. The results of this study highlight the importance of carefully delineating the area accessible to invasive species in studies of niche conservation. Furthermore, it indicates that examining several spatial extents of background areas can be beneficial when examining niche conservation for species in non-equilibrium states.     

Susceptibility of the leatherjackets Tipula oleracea and T. paludosa to soil flooding

A relation was found between temperature and the survival of larvae of Tipula oleracea and T. paludosa when submerged in tap water alone or on turf growing on a sandy loam flooded with still or disturbed water (experiments with soil always starting with freshly flooded soil). Larvae under water alone usually survived until they starved, whereas larvae submerged on soil died in a relatively short time. The periods of survival at a depth of 25 mm were the same for both large and small larvae of either species at any one temperature. The regression of log.-log. survival time on temperature was a straight line, mean arithmetic values ranging from about 5 h at 20 °C to 122 h at 0 °C. Survival times at 25 mm were not increased by disturbing the water and the times at greater depths were no different, providing the water was still. If the water was regularly disturbed then the greater the depth the longer the survival time at any one temperature. Survival times in deoxygenated water indicated that lethal conditions may not be entirely due to deoxygenation of the water.     

A survey for pathogens of the black field cricket, Teleogryllus commodus, in the Western District of Victoria,Australia

A pathogen survey of the black field cricket, Teleogryllus commodus, in the Western District of Victoria, Australia, during 1979 revealed that cricket paralysis virus (CrPV) was present in 42.7% of the 232 sites sampled. The fungus Metarhizium anisopliae was detected in 5.2% of the sites and represents a new pathogen record for T. commodus. The distribution of both pathogens throughout the sites sampled appeared to be random. There was a positive correlation between sample size and the likelihood of detecting a pathogen, while analysis showed that approximately 30% of the sites were probably virus free. The results are discussed in terms of the potential of CrPV and M. anisopliae as biological control agents for the black field cricket.     

Within‐generation dynamics of leatherjackets (Tipula paludosa Meig.)

Larvae of Tipula paludosa (leatherjackets) are pests of grassland and cereals, and important prey items for a range of farmland birds. Their population dynamics from year to year have been the subject of study, but within‐generation dynamics have received much less attention. The duration and occurrence of instars and size of the population is important to both pest management and bird conservation. Leatherjackets were collected monthly from four grass fields over five generations (October to September). Count data were analysed for sources of variability and used to estimate the timing and duration of instars and population changes over a single generation. Instar durations were correlated with temperature. From the first to third instars, sample counts were strongly influenced by the location of the site and the generation. Fourth instar larvae showed a similar decline in numbers across all sites and generations. The duration and timing of first to third instars was more variable than that of the fourth instar, but there was limited evidence that this was influenced by temperature. Only the durations of the third and fourth instars were correlated. The population decline from April to September coincided with the onset of the fourth instar. Our results show that the factors influencing leatherjacket numbers change over the life cycle, but by the fourth instar, these are biotically driven. The predictability of its numerical decline indicates defined windows for crop damage and availability as prey. The stability of its timing suggests that day length may synchronize the annual cycle. Fixed timing of the fourth instar will have important consequences for the timing of crop damage and the conservation of farmland birds as climate changes.     

THE STRUCTURE OF INSECT VIRUS PARTICLES

Thin sections have been cut of the virus particles from four types of insect virus diseases: cytoplasmic polyhedroses of lepidopterous larvae, a nuclear polyhedrosis of Tipula paludosa (Diptera), a granulosis from Melanchra persicariae (Lepidoptera), and a new virus disease without polyhedra from T. paludosa. The cytoplasmic polyhedral viruses are thought to have composite particles in some cases. The shape and enveloping membranes of the different virus particles are compared. In the new virus disease of T. paludosa some of the virus particles appear to be empty; inclusion bodies surrounded by complicated membranes are also demonstrated.     

The epiphytic diatom flora of mosses from Strømness Bay area, South Georgia

A total of 104 taxa, belonging to 27 genera, were recorded from 36 moss samples collected in the Strømness Bay area. Cluster analysis revealed two groups of samples. Group 1 consisted of the Achnanthes incognita–Fragilaria germainii assemblage and the Eunotia paludosa var. paludosa assemblage. Group 2 included the Pinnularia borealis assemblage. The major ecological factor dividing the two groups appears to be the moisture content of the samples. Group 1 represents wetter conditions, and the second group is found on very dry mosses. The assemblages of group 1 were separated from each other by pH. The Achnanthes incognita–Fragilaria germainii assemblage occurs in wet mosses with a circumneutral pH. The Eunotia paludosa var. paludosa assemblage prefers bryophyte habitats with low pH values. The size of Pinnularia borealis drops significantly according to the dryness of the moss habitat.     

Lack of Host Specialization on Winter Annual Grasses in the Fungal Seed Bank Pathogen Pyrenophora semeniperda

Generalist plant pathogens may have wide host ranges, but many exhibit varying degrees of host specialization, with multiple pathogen races that have narrower host ranges. These races are often genetically distinct, with each race causing highest disease incidence on its host of origin. We examined host specialization in the seed pathogen Pyrenophora semeniperda by reciprocally inoculating pathogen strains from Bromus tectorum and from four other winter annual grass weeds (Bromus diandrus, Bromus rubens, Bromus arvensis and Taeniatherum caput-medusae) onto dormant seeds of B. tectorum and each alternate host. We found that host species varied in resistance and pathogen strains varied in aggressiveness, but there was no evidence for host specialization. Most variation in aggressiveness was among strains within populations and was expressed similarly on both hosts, resulting in a positive correlation between strain-level disease incidence on B. tectorum and on the alternate host. In spite of this lack of host specialization, we detected weak but significant population genetic structure as a function of host species using two neutral marker systems that yielded similar results. This genetic structure is most likely due to founder effects, as the pathogen is known to be dispersed with host seeds. All host species were highly susceptible to their own pathogen races. Tolerance to infection (i.e., the ability to germinate even when infected and thereby avoid seed mortality) increased as a function of seed germination rate, which in turn increased as dormancy was lost. Pyrenophora semeniperda apparently does not require host specialization to fully exploit these winter annual grass species, which share many life history features that make them ideal hosts for this pathogen.     

The effect of soil flooding on the survival and development of the eggs of Tipula oleracea L. and T.paludosa Meigen (Diptera: Tipulidae)

A comparison is made of the survival and development of eggs of Tipula oleracea and T.paludosa, at certain temperatures, under water only or under water standing over soil. The significance the activity of the soil microflora and mechanical disturbance of the water is discussed.     

mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP_UV) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP_UV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP_UV fluorescence.     

A novel milky disease organism from Australian scarabaeids: Ultrastructure

The unusual ultrastructure of a milky disease bacterium isolated from Aphodius tasmaniae is described and compared with a similar bacterium from Tipula paludosa. Both organisms possess an elongate sporangium with a small central spore, a multilayered exosporium, and longitudinally arranged extrasporal fibrils which arise from plates attached to the ends of the spore. The morphogenesis of the sporangium is described and illustrated for the organism from A. tasmaniae. It is suggested this organism and that from T. paludosa are closely related. Their taxonomic position remains obscure.     

Why flowers close at noon? A case study of an alpine species Gentianopsis paludosa (Gentianaceae)

Repeatable floral closure with diurnal rhythms, that is, flower opening in the morning and closing in the evening, was widely reported. However, the rhythm of flower opening in the morning but closing in the midday received much less attention. Gentianopsis paludosa, Gentianaceae, has an obvious petal movement rhythm opening in the morning and closing at noon at northeast of the Qinghai‐Tibetan Plateau. In this study, we examined the effects of temperature (T), relative humidity (RH), and illumination intensity (II) on G. paludosa''s flower closure. Furthermore, we monitored the environmental changes inside and outside of the flowers, aiming to test the effect of floral closure on the stability of microenvironment inside the flower. Finally, we artificially interrupted temporal petal closure and investigated its effects on reproductive fitness. The results showed that high/low temperature contributed more to the flower closure than low RH, while illumination intensity had no significant effect on it. The medium temperature, relative humidity and illumination intensity (environmental conditions at 10:00) did not delay flower closure when flowers at pre‐closing period or stimulate reopen when flowers full closed. Floral closure provided a stable temperature condition and a higher RH condition inside the flower. Meanwhile, compulsive opening and delayed closure of flowers decreased the seed‐set ratio while no effect was found when flowers were forced to close. We conclude that endogenous rhythm regulates floral closure. The rhythm of petal movement providing a stable microenvironment for G. paludosa, increasing the seed production and saving energy from flower opening maintenance, which might be an adaptive strategy to against unfavorable environmental conditions.     

Isozymes of coreopsis section Calliopsis (Compositae): Genetic variation within and divergence among the species

Enzyme electrophoresis was employed to examine genetic variation within and among the three diploid (n=12) species comprisingCoreopsis sectionCalliopsis: C. leavenworthii, C. paludosa andC. tinctoria. Twelve enzymes specified by 20 genes were included, and those isozymes localized in the plastids were identified for several of the enzymes. Mean genetic identities calculated for pair-wise comparisons of 22 populations (four ofC. leavenworthii, one forC. paludosa, and 17 ofC. tinctoria) reveal no genetic differentiation betweenC. leavenworthii andC. tinctoria, whereasC. paludosa exhibits lowered values with the other two taxa. These results are in general agreement with concepts of relationships inferred from morphological and biosystematic data. That is,C. paludosa has been viewed as more distinct fromC. leavenworthii andC. tinctoria than the latter two are from each other. The high similarity betweenC. leavenworthii andC. tinctoria is somewhat unexpected, however, given the several morphological features that serve consistently to distinguish them.     

Hemocytes and granular cell fragments of Tipula paludosa larvae

Five morphological categories of hemocyte (prohemocyte, ameboid plasmatocyte, spindle-shaped plasmatocyte, oenocytoid, and granular cell) were observed by light and electron microscopy of hemolymph from Tipula paludosa larvae. In addition, vast numbers of membrane-bounded granule-containing fragments were present, and were also found in the larval hemolymph of four other Tipula spp. The fragments appear to be derived from the granular cells, which readily fragment. Granules in the granular cells and the fragments appear to have similar substructure. Melanization and coagulation of the hemolymph occur rapidly on exposure to air; the granular cell fragments may be concerned with one or both of these processes.