Attracting adult New World screwworm,Cochliomyia hominivorax,to odour-baited targets in the field

Abstract. A field study in Libya investigated aspects of Cochliomyia hominivorax behaviour relevant to the operation of a target-based control system. Flies were readily caught by electrified targets, but only when baited with the attractant ‘swormlure-4’. Target size (0.25-lm²) did not affect catch, but black targets caught twice as many flies as red ones. Omitting the most volatile component of swormlure-4, dimethyl disulphide, significantly reduced catches of both sexes of C. hominivorax in wind-orientated traps, but only reduced the capture of females significantly on targets. A new polythene sachet dispenser for odours was tested; four to six such sachets filled with swormlure-4 less dimethyl disulphide gave an equivalent catch to one of the standard bottle plus wick dispensers using the same attractant, although they dispensed odour at about half the rate. From direct observations, fewer than 40% of flies contacted the target for more than 10 s on any one visit. From catches on an intermittently-active electrocuting grid, it was calculated that there is a c. 40% rate of multiple contacts of flies with the target.     

Responses of female New World screwworm flies, Cochliomyia hominivorax, to swormlure-4 in the laboratory

The behaviour of 4-day-old virgin female New World screwworm flies Cochliomyia hominivorax Coquerel was investigated in a wind tunnel using video. Addition of the screwworm attractant swormlure-4 to the airstream resulted in an increase in the number of take-offs and in the duration of flight. In the presence of swormlure-4 the flight speed decreased and the rate of turning increased. Take-off was significantly orientated upwind during the period when swormlure-4 was added to the airstream, and significantly orientated downwind in the period after the addition of swormlure-4. The possible means by which this species finds a point source of odour in the field are discussed and compared with the mechanisms used by other flies.     

Orientation of agents of wound myiasis to hosts and artificial stimuli in Hungary

Abstract. The orientation of adults of the myiasis species Wohlfahrtia magnified and Lucilia sericata to artificial visual stimuli (cloth targets) and olfactory stimuli ('swormlure-4', a potent screwworm attractant), was studied in sheep pastures near to Sarbogard in Hungary. Experiments with odour-baited cloth targets, enclosed in electrocuting grids, demonstrated that colour was an important factor influencing catches on targets: a black target caught most flies of both species, with other colours in the following order of effectiveness, blue > white > yellow. Wohlfahrtia magnified did not respond to swormlure-4 in the strong manner that L. sericata did. The sex ratios of W. magnifica caught on targets (67.2% males) and hand-netted from fence posts (68.8% males) were similar and biased towards males, whereas that of L. sericata on targets was strongly biased towards females (15.6% males), indicating a fundamental difference in the response of these two myiasis species towards the swormlure-baited targets. The orientation of these two species towards hosts was also recorded together with that of a third species, Phormia regina . Electric nets placed beside infested sheep caught significantly more flies of all three species than those placed beside uninfested sheep or in the absence of sheep. The sex ratio of W. magnified caught around infested sheep was the reverse of that on targets, with 68.5% females. Wohlfahrtia magnifica responded more strongly to healthy, uninfested sheep than did L. sericata and P. regina . The potential for use of targets both for population monitoring and control is discussed.     

The effect of colour on the numbers, age and nutritional status of Glossina tachinoides (Diptera: Glossinidae) attracted to targets

Abstract The effect of colour on the attractiveness of cloth targets for G.tachinoides Westwood was investigated, using electrified nets. The ages and nutritional states of tsetse caught by different targets were also studied. With targets of cloth and mosquito netting panels, phthalogen blue was the most attractive colour, and yellow the least, with black, red, violet and white intermediate. The strongest landing responses in females occurred on UV-reflecting white cloth. In males, landing was high with all colours except yellow. Colour combination targets were no better than all-blue targets when mosquito netting side-panels were present; in their absence, blue-and-white targets were over twice as good as all-blue targets. No consistent differences in ages or nutritional states were found between tsetse caught by differently coloured targets, but those landing directly on the cloth portion of a target had lower fat reserves than those intercepted on an adjacent netting panel.     

Progress and perspectives on improving butanol tolerance

Fermentative production of butanol for use as a biofuel or chemical feedstock is regarded as a promising renewable technology that reduces greenhouse gas emissions and has the potential to become a substitute for non-sustainable chemical production route. However, butanol toxicity to the producing microbes remains a barrier to achieving sufficiently high titers for cost-effective butanol fermentation and recovery. Investigations of the external stress of high butanol concentration on butanol-producing microbial strains will aid in developing improved microbes with increased tolerance to butanol. With currently available molecular tool boxes, researchers have aimed to address and understand how butanol affects different microbes. This review will cover the individual organism’s inherent responses to surrounding butanol levels, and the collective efforts by researchers to improve production and tolerance. The specific microorganisms discussed here include the native butanol producer Clostridium species, the fermentation industrial model Saccharomyces cerevisiae and the photosynthetic cyanobacteria, the genetic engineering workhorse Escherichia coli, and also the butanol-tolerant lactic acid bacteria that utilize diverse substrates. The discussion will help to understand the physiology of butanol resistance and to identify specific butanol tolerance genes that will lead to informed genetic engineering strategies for new strain development.     

Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.     

Characterization and utilization of microsatellite loci in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  New World screwworm populations in North and Central America have been the targets of virtually continuous eradication attempts by sterile insect technique (SIT) since the 1950s. Nevertheless, in some areas, such as Jamaica, SIT control programmes have failed. Reasons for the failure of SIT-based control programmes in some locations are unknown, but it has been hypothesized that failure may be related to mating incompatibility between sterile and wild fly populations or to the existence of sexually incompatible cryptic species. This paper outlines the development of a suite of four new microsatellite loci which can be used to study intra-specific relationships between populations of Cochliomyia hominivorax from the Caribbean and South America, which represent those populations involved in, or earmarked for, forthcoming SIT control. Cross-amplification with the secondary screwworm, Cochliomyia macellaria , was also successful with three of the new loci. We present results which suggest that populations from Trinidad and Jamaica form distinct groupings of flies and that C. hominivorax from Trinidad appears particularly distinct.     

Landing response of Aedes (Stegomyia) polynesiensis mosquitoes to coloured targets

Aedes polynesiensis Marks (Diptera: Culicidae) is the primary vector of lymphatic filariasis (LF) in the island countries and territories of the South Pacific. In the development of a novel control tool, the response of Ae. polynesiensis to six different colours (three solid fabrics, two patterned fabrics and a plastic tarp) was measured using a digital photographic system. Adult mosquitoes were placed into an environmental chamber and allowed to choose between a white target and one of six experimental targets. Mosquito landing frequency and landing duration were calculated. Adult female Ae. polynesiensis preferred all of the experimental targets to the white control target. Mosquito landing frequency was highest for the solid targets (black, navy blue and red) followed in turn by the two colour pattern targets and the polyethylene target. Mosquito landing duration was greater for experimental targets when compared with white control targets. Mosquito landing frequencies did not change over time during the course of the assay. The response of male Ae. polynesiensis was also measured when exposed to a 100% cotton black target. Male mosquitoes preferred the black target to the white control target, although at levels lower than that observed in female mosquitoes. The results suggest that future investigations evaluating the visual responses of Ae. polynesiensis mosquitoes are warranted, with a special emphasis on semi‐field and field‐based experiments.     

One shot,one kill: the forces delivered by archer fish shots to distant targets

Archer fishes are skillful hunters of terrestrial prey, firing jets of water that dislodge insects perched on overhead vegetation. In the current investigation, we sought an answer to the question: are distant targets impractical foraging choices? Targets far from the shooter might not be hit with sufficient force to cause them to fall. However, observations from other investigators show that archer fish fire streams of water that travel in a non-ballistic fashion, which is thought to keep on-target forces high, even to targets that are several body lengths distant from the fish. We presented targets at different distances and investigated three aspects of foraging behavior: (i) on-target forces, (ii) shot velocity, (iii) a two-target choice assay to determine if fish would show any preference for downing closer targets or more distant targets. In general, shots from our fish (Toxotes chatareus) showed a mild decrease (less than 15% on average) in on-target forces at our most distant target offered (5.8 body lengths) with respect to the closest target offered (2.3 body lengths). One individual in our investigation showed slightly, but significantly, greater on-target forces as target distance increased. Forces on the furthest targets offered were found to double that of attachment forces for 200 mg insects, even for individuals whose on-target forces showed mild decreases with increases in target distance. High-speed video analysis of jet impact with the target revealed that the shot was traveling in a non-ballistic manner, even to our most distant target offered, corroborating previous suppositions that on-target forces should remain high. Fish were able to accomplish this without large changes to shot velocity, but we did find evidence that the water jets appeared to differ in the timing of their acceleration as target distance increased. Our two-target choice experiment revealed that fish show preference for downing the closer target first, even though impact forces on distant targets only showed mild decreases. Our overall findings (and the findings of others) suggest that archer fish modulate many aspects of their shooting behavior: from target selection to active control over the water jet that allows the fish to deliver reliably forceful impacts to prey over a wide range of distances.     

Feeding responses and food preferences in the tropical, fruit-feeding butterfly, Bicyclus anynana

In the tropical butterfly Bicyclus anynana (Nymphalidae) essential components of fitness (such as fecundity and longevity) depend to a large degree on exogenous adult-derived nutrients, particularly carbohydrates. We investigated which of the nutrients/compounds found in the adult diet act as feeding stimuli, and whether butterflies show preferences for particular nutrients or combinations. Only sugars and alcohols acted as feeding stimuli, the highest responses being found for sucrose, glucose, ethanol, butanol and propanol. Various other compounds (e.g. amino acids, acetic acid, vitamins, lipids, salts, and yeast) did not elicit any probing or feeding responses. Behavioural tests revealed a clear preference hierarchy for sugars (sucrose > glucose > fructose > maltose), but not for alcohols. Butterflies did not discriminate between sucrose solutions enriched with different nutrients and plain sucrose solutions, although they showed a preference for acetic acid and an aversion to salts and ascorbic acid when offered in combination with sucrose. Throughout, both sexes showed very similar patterns. We conclude that locating carbohydrate sources seems sufficient to cover all the butterflies’ nutritional needs, while alcohols function primarily as long range signals, guiding the butterflies to food sources. Thus, fruit-feeding butterflies, in contrast to nectar-feeding butterflies, appear not to have distinctive preferences for e.g. amino acids or salts, but do share a common primary preference for sucrose.     

Visual behaviors of the migratory grasshopper,Melanoplus sanguinipes F. (Orthoptera: Acrididae)

The visual behavior of adult Melanoplus sanguinipesF. (Orthoptera: Acrididae) was investigated by placing individuals in the center of an arena and recording their orientation responses to visual targets at the perimeter of the arena. Targets that reflected more 540- to 570-nm light were approached more frequently; however, when reflectance in the 540 -to 570-nm region was combined with reflectance in the 400- to 520-nm region, orientation responses were reduced significantly. This suggests that spectral discrimination in M. sanguinipesinvolves at least two classes of photoreceptors, which respond to different regions of the wavelength spectrum. In addition, grasshoppers oriented to vertical, but not horizontal, contrasting stripes. However, when vertical stripes were added to targets reflecting 520- to 650-nm light, responses to verticals on these targets were not enhanced relative to verticals presented against a target background of 400- to 520-nm + 520- to 650-nm light. Thus, spectral discrimination and vertical stripe fixation appear to be two distinct visual behaviors, controlled by outputs from two classes of photoreceptors and a single class of photoreceptors, respectively, and may be used in different physiological or ecological contexts.     

New Targets for Drug Discovery against Malaria

A mathematical model which predicts the intraerythrocytic stages of Plasmodium falciparum infection was developed using data from malaria-infected mice. Variables selected accounted for levels of healthy red blood cells, merozoite (Plasmodium asexual phase) infected red blood cells, gametocyte (Plasmodium sexual phase) infected red blood cells and a phenomenological variable which accounts for the mean activity of the immune system of the host. The model built was able to reproduce the behavior of three different scenarios of malaria. It predicts the later dynamics of malaria-infected humans well after the first peak of parasitemia, the qualitative response of malaria-infected monkeys to vaccination and the changes observed in malaria-infected mice when they are treated with antimalarial drugs. The mathematical model was used to identify new targets to be focused on drug design. Optimization methodologies were applied to identify five targets for minimizing the parasite load; four of the targets thus identified have never before been taken into account in drug design. The potential targets include: 1) increasing the death rate of the gametocytes, 2) decreasing the invasion rate of the red blood cells by the merozoites, 3) increasing the transformation of merozoites into gametocytes, 4) decreasing the activation of the immune system by the gametocytes, and finally 5) a combination of the previous target with decreasing the recycling rate of the red blood cells. The first target is already used in current therapies, whereas the remainders are proposals for potential new targets. Furthermore, the combined target (the simultaneous decrease of the activation of IS by gRBC and the decrease of the influence of IS on the recycling of hRBC) is interesting, since this combination does not affect the parasite directly. Thus, it is not expected to generate selective pressure on the parasites, which means that it would not produce resistance in Plasmodium.     

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.     

Regeneration studies on a crayfish neuromuscular system. II. Effect of changing the nerve entry point into the muscle field on the gradient of innervation

The superficial flexor muscle of the crayfish is a neuromuscular system in which the neurons form position-dependent connectivity patterns with the muscle fibers. This system could be formed with the help of a single medial-to-lateral gradient during development that embodies positional information. To test this gradient hypothesis we changed the nerve's normal medial entry point into the muscle by transplanting it to the middle of the muscle sheet. When all the muscle fibers were present in the target area, most of the neurons studied passed through a stage during regeneration in which they showed preference for either medial or lateral synapse formation. Those neurons that in normal animals innervated preferentially the medial fibers showed a medial preference for new contacts; the neuron that normally innervated the lateral fibers showed a lateral preference for new contacts; the neuron that normally innervated everywhere regenerated equally well into both medial and lateral fibers. Therefore, these neurons are able to detect information regarding their position within the muscle mass and respond to it by preferential synapse formation. The effect of a positional gradient could not be detected when half of the target field was removed prior to regeneration. In this instance, the neuron that innervated the missing target area now regenerated to almost all the available fibers. It is suggested that the interplay of positional cues with other factors at different points in time could determine the final connectivity patterns formed by these cells.     

Climatological correlates of screwworm (Cochliomyia hominivorax) abundance in Texas, U.S.A.

The causes of screwworm (Cochliomyia hominivorax) outbreaks in North America are not well understood, but the literature suggests that climate was historically important. Screwworm case incidence in each of seven climatological divisions of Texas was examined for the years 1962-83, the period when sterile-male releases were made. Weak but statistically significant correlations were found between winter and summer cases and mean winter and summer rainfall and temperature when the independent variables were examined one at a time. Multiple regression of log case incidence on previous quarterly cases and current rainfall and temperatures showed a significant, negative effect of temperature on summer cases. Lagged screwworm cases accounted for most of the variation in quarterly cases. No climatic effects were detected in the other seasons. Rainfall was statistically unrelated to screwworm abundance in any season even in an arid region. The analysis does not support a climatic genesis of screwworm outbreaks or eradication. The sterile-male method is a credible explanation for screwworm disappearance.     

Syntheses of Four Methyl-branched Secondary Acetates and a Methyl-branched Ketone as Possible Candidates for the Female Pheromone of the Screwworm Fly,Cochliomyia hominivorax

6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.     

The use of different eye regions in the mantis shrimp Hemisquilla californiensis Stephenson, 1967 (Crustacea: Stomatopoda) for detecting objects

A behavioral assay was used to assess the ability of the stomatopod Hemisquilla californiensis to perceive and respond to a moving target under different wavelengths and intensities of light illumination. Subjects responded to targets rotating horizontally across their visual field by a brief startle response of their eyes or antennules but did not track the targets. Under white light responses were elicited down to a light intensity of 0.9 μW cm^− 2. Responses were seen in blue light at intensities as low as 0.5 μW cm^− 2, and in green light down to 1.0 μW cm^− 2. The animals were less sensitive to red light, with no responses seen at intensities below 3.0 μW cm^− 2. Subjects did not respond to the targets at all under infrared light. This response pattern mirrors the computed sensitivity spectrum of ommatidia in the species' peripheral hemispheres but not that in most of the central bands. We conclude that this species uses the monochromatic vision in the peripheral hemispheres of its eyes to recognize objects and that the sharply tuned color receptors of the central band serve to add supplemental information if light conditions allow.     

rMCP-2, the Major Rat Mucosal Mast Cell Protease,an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins

Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.     

Design,construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Designed ankyrin repeat proteins (DARPins) are well‐established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin‐target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase‐7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.