Female ixodid ticks grow endocuticle during the rapid phase of engorgement

Lees (Proc Zool Soc Lond 121:759–772, 1952) concluded that the ixodid tick Ixodes ricinus grows endocuticle during the slow but not during the rapid, phase of engorgement, a conclusion supported by Andersen and Roepstorff (Insect Biochem Mol Biol 35:1181–1188, 2005) for the same species. In this study analysis of dimensional data and cuticle weight measurements from female ixodid ticks (Amblyomma hebraeum) were used to test this hypothesis. Both approaches showed that endocuticle growth continues during the rapid phase, tapering to zero at a fed/unfed weight ratio of ~60. Of the total mass of cuticle in the engorged tick 32–43% was formed during the rapid phase. We demonstrate that if cuticle growth stopped at the end of the slow phase, there would not be sufficient cuticle to account for the thickness of cuticle observed at the end of engorgement. This finding is consistent with prior studies of Rhipicephalus (Boophilus) microplus, and with a dimensional analysis of the cuticle thickness data of Lees for I. ricinus, in contradiction to his conclusion from an analysis of tick cuticle weight measurements. An examination of cuticle weight measurements for I. ricinus by Andersen and Roepstorff similarly supports the finding of cuticle growth during the rapid phase. All ixodid ticks undergo major body expansion, typically tenfold or more, during a rapid phase of engorgement and require sufficient cuticle at the end of that process to contain their body. The fact that cuticle grows during the rapid phase of engorgement in three species suggests that this is a general characteristic of the family Ixodidae.     

A comparison of the cuticular properties of the female ticks <Emphasis Type="Italic">Ixodes pacificus</Emphasis> and <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) throughout the feeding period

The mechanical properties of the cuticle of Ixodes pacificus (Ip) are compared to those of Amblyomma hebraeum (Ah) from our earlier work. The 10-fold size difference between the species is expected to lead to significant differences in mechanical properties, because cuticular stretch depends on high internal hydrostatic pressure during the rapid phase of engorgement. We demonstrate here: (1) The cuticle of partially fed Ip is less stiff and viscous than that of Ah. (2) A stretch-recoil cycle in both ticks consists of recoverable deformation (ESv) and permanent deformation (ESp); ESp is higher in Ip, and increases sharply during the slow phase of engorgement, but not in Ah. (3) Injected dopamine (DA) increases ESp and reduces all measures of stiffness and viscosity, suggesting that a catecholaminergic neurotransmitter plays a fundamental role in modulating mechanical properties of the cuticle. However, unlike Ah, DA’s effect was not different from that of the control (1.2% NaCl). Mere insertion of the needle may have punctured the gut, causing the release of perhaps a catecholamine that increases ESp, an hypothesis supported by the fact that inserting a needle without any injection also caused an increase in ESp. (4) Stretch reduces ESp, but subjecting loops to pH 6.5 in vitro restores it. (5) Despite the smaller size of Ip, later onset of the rapid phase of engorgement, a thinner cuticle and different mechanical properties all reduce the internal pressure needed for stretch.     

Vacuum level effects on knee contact force for unilateral transtibial amputees with elevated vacuum suspension

The elevated vacuum suspension system (EVSS) has demonstrated unique health benefits for amputees, but the effect of vacuum pressure values on knee contact force (KCF) is still unclear. The objective of this study was to investigate the effect of vacuum levels on KCF for unilateral transtibial amputees (UTA) using the EVSS. Three-dimensional gait was modeled for 9 UTA with five vacuum levels (0–20 inHg [67.73 kPa], 5 inHg [16.93 kPa] increments) and 9 non-amputees based on kinematic and ground reaction force data. The results showed that the vacuum level effects were significant for peak axial KCF, which had a relatively large value at 0 and 20 inHg (67.73 kPa). The intact limb exhibited a comparable peak axial KCF to the non-amputees at 15 inHg (50.79 kPa). At moderate vacuum levels (5 inHg [16.93 kPa] to 15 inHg [50.79 kPa]), co-contraction of quadriceps and hamstrings at peak axial KCF was similar for the intact limb, but was smaller for the residual limb comparing with the non-amputees. The intact limb showed a similar magnitude of quadriceps and hamstrings force at 15 inHg (50.79 kPa) to the non-amputees, but the muscle coordination patterns varied between the residual and intact limbs. These findings indicate that a proper vacuum level may partially compensate for the lack of ankle plantarflexor and reduce the knee loading. Of the tested vacuum levels, 15 inHg (50.79 kPa) appears most favorable, although additional analyses with more amputees are suggested to confirm these results prior to establishing clinical guidelines.     

Single-electron photoreduction of the PM intermediate of cytochrome c oxidase

The P_M → F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P_M state of the oxidase was generated by exposing the oxidized enzyme to CO plus O₂. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a₃ with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼ 5-fold more rapid than the rate of electron transfer from heme a to heme a₃ in the F → O transition, but is significantly slower than formation of the F state from the P_R intermediate in the reaction of the fully reduced enzyme with O₂ to form state F (70–90 μs). The ∼ 0.3 ms P_M → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P_M → F transition is generated much later, with a time constant of 1.3 ms. In addition, the P_M → F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms.     

Holocene and Late Pleistocene climate in the sub-Mediterranean continental environment: A speleothem record from Poleva Cave (Southern Carpathians,Romania)

The PP10 stalagmite from Poleva Cave provides a Late Pleistocene and Holocene isotopic record characteristic for the SW of Romania, a sub-Mediterranean climatic region. The speleothem was dated by eight TIMS and one alpha U-series dates which showed that it was precipitated between ∼ 75 ka and ∼ 2 ka with at least two hiatuses. The basal sector of the stalagmite showed a slow-growing regime of ∼ 0.26 cm/ka, while the upper one grew relatively fast with about 5 cm/ka. The temporal resolution for the isotopic sampling is thus ∼ 2 ka/sample for the lower sector, and ∼ 150 years/sample for the upper one. The relationship between δ¹⁸O and temperature was found positive. The isotopic record of the lower sector shows two marked cold intervals during ∼ 67 and 58 ka and ∼ 40–35 ka, respectively, which correlate well with the Villars and Soreq records. The upper sector record is so far the most detailed Holocene isotopic record in Romania and the only one available for the regions located at the exterior of the Carpathians Range. The signal shows a gradual warming after the GS1 event punctuated by several cold events at ∼ 8, 7.2 and 4.2 ka and also by warm oscillations centered at about 5.2 and 3.3 ka. The results seem to indicate that if the North-Atlantic first-order signals may extend well to the south-eastern Europe, their amplitude and general trend may be diminished by the interferences with the Mediterranean circulation.     

“Male factors” in ticks: their role in feeding and egg development

Summary

Female ticks of the family Ixodidae utilize their salivary glands as the major organs for fluid balance, secreting back into the host a dilute saliva. Feeding is composed of three phases: a preparatory phase (1–2 days) during which the tick establishes the feeding lesion, a slow phase (~7 days) during which body weight increases 10-fold, and a rapid phase (~1 day) in which body weight increases a further 10-fold. Following engorgement, the salivary glands are resorbed by an autolytic process triggered by an ecdysteroid hormone. If a female is removed from the host prior to repletion, her subsequent behaviour depends mostly on two factors: the degree of engorgement achieved and whether or not she has mated. If removed during the preparatory or slow phase of engorgement, the salivary glands are not resorbed, the tick will lay virtually no eggs and she will reattach to a host if given the opportunity, all of this irrespective of whether she is virgin or mated. If removed during the rapid phase of engorgement, however, mated females will not reattach to a host even if given the opportunity. Instead, they will resorb the salivary glands within 4 days post-removal and lay a batch of eggs. Virgin females removed after exceeding 10-fold the unfed weight likewise refuse to resume feeding if given the opportunity, but salivary gland reabsorption is delayed (to 8 days post-removal); if any eggs are laid, they are infertile. A number of chemical “factors” entering the female during copulation influence her feeding behaviour and egg development. Here we discuss the complexities of these interactions and suggest how they might be adaptive to ticks in nature.     

Novel 6 MV X-ray photoneutron detection and dosimetry of medical accelerators

PurposeDosimetry of fast, epithermal and thermal photoneutrons in 6 MV X-ray beams of two medical accelerators were studied by novel dosimetry methods.MethodsA Siemens ONCOR and an Elekta COMPACT medical accelerators were used. Fast, epithermal and thermal photoneutron dose equivalents in 10 cm × 10 cm 6 MV X-rays fields were determined in air and on surface of a polyethylene phantom in X and Y directions. Polycarbonate dosimeters as bare or with enriched ¹⁰B convertors (with or without cadmium covers) were used applying a 50 Hz-HV electrochemical etching method.ResultsFast, epithermal and thermal photoneutron dose equivalents were efficiently determined respectively as ∼1145.8, ∼45.3 and ∼170.6 μSv in air and ∼1888.5, ∼96.1 and ∼640.6 μSv on phantom per 100 Gy X-rays at the isocenter of Siemens ONCOR accelerator in air. The dose equivalent is maximum at the isocenter which decreases as distance from it increases reaching a constant level. Tissue-to-air ratios are constants up to 15 cm from the isocenter. No photoneutrons was detected in the Elekta COMPACT accelerator.ConclusionsFast, epithermal and thermal photoneutron dosimetry of 6 MV X-rays were made by novel dosimetry methods in a Siemens ONCOR accelerator with sum dose equivalent per Gy of ∼0.0014% μSv with ∼0.21 MeV mean energy at the isocenter; i.e. ∼150 times smaller than that of 18 MV X-rays. This observation assures clinical safety of 6 MV X-rays in particular in single-mode machines like Elekta COMPACT producing no photoneutrons due to no “beryllium exit window” in the head structure.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

High cell density fed-batch fermentation for the production of a microbial lipase

Extracellular lipase of the yeast Candida rugosa was produced via high cell density fed-batch fermentations using palm oil as the sole source of carbon and energy. Feeding strategies consisted of a pH-stat operation, foaming-dependent control and specific growth rate control in different experiments. Compared to foaming-dependent feeding and the pH-stat operation, the specific growth rate control of feeding proved to be the most successful. At the specific growth rate control set at 0.05 h⁻¹, the final lipase activity in the culture broth was the highest at ∼700 U L⁻¹. This was 2.6-fold higher than the final enzyme activity obtained at a specific growth rate control set at 0.15 h⁻¹. The peak enzyme concentration achieved using the best foaming-dependent control of feeding was around 28% of the peak activity attained using the specific growth rate control of feeding at 0.05 h⁻¹. Similarly, the peak enzyme concentration attained using the pH-stat feeding operation was a mere 9% of the peak activity attained by specific growth rate control of feeding at a set-point of 0.05 h⁻¹. Fed-batch fermentations were performed in a 2 L stirred-tank bioreactor (30 °C, pH 7) with the dissolved oxygen level controlled at 30% of air saturation.     

Feeding enrichment in an opportunistic carnivore: The red fox

In captive carnivores, species-specific behaviour is often restricted by inadequate feeding regimens. Feeding live prey is not feasible in most places and food delivery is often highly predictable in space and time which is considerably different from the situation in the wild. As a result, captive carnivores are often inactive, show little behavioural diversity and are prone to behavioural problems such as stereotypic pacing. Using artificial feeding devices to substitute for natural food resources is a way to address these problems. In a group of four red fox (Vulpes vulpes), we compared a conventional feeding method to four different methods through the use of feeding enrichment that were based on natural foraging strategies of opportunistic carnivores. Feeding enrichments consisted of electronic feeders delivering food unpredictable in time which were successively combined with one of the three additional treatments: a self-service food box (allowing control over access to food), manually scattering food (unpredictable in space), and an electronic dispenser delivering food unpredictably both in space and time. The aim of administering feeding enrichment in this study was to stimulate appetitive (food searching) behaviour and to increase time spent in feeding. Compared to conventional feeding, diversity of behaviour and overall activity were significantly enhanced in the presence of electronic feeders in all four foxes (EF > CON1 = CON2, EF + SF > CON1 = CON2, EF + MS > CON1 = CON2, EF + ED > CON1 = CON2). Behavioural diversity was highest when the foxes had control over access to food (EF + SF), while the manual scattering of food (EF + MS) and the electronic dispenser (EF + ED) enhanced food searching behaviour. These results indicate that in opportunistic carnivores natural foraging and feeding behaviour can be stimulated by simple feeding enrichment strategies, and that foraging behaviour is stimulated most when food delivery is unpredictable both in space and time.     

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS–mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from ∼ 3–50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of ∼ 1.6 × 10⁷ CFU ml^− 1 (∼ 1.6 × 10⁴ cells spot^− 1), and bound to proteins of ∼ 50 and ∼ 39 kDa. Other MAbs, binding to proteins ranging from ∼ 3–14 and ∼ 40 kDa, detected VVB (but not VVC) with high sensitivity at ∼ 1.6 × 10⁵ and 4 × 10⁶ CFU ml^− 1 (∼ 1.6 × 10² and 4 × 10³ cells spot^− 1), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.     

Novel instrumented probe for measuring 3D pressure distribution along the vaginal canal

We developed an intravaginal instrumented probe (covered with a 10 × 10 matrix of capacitive sensors) for assessing the three-dimensional (3D) spatiotemporal pressure profile of the vaginal canal. The pressure profile was compared to the pelvic floor (PF) digital assessment, and the reliability of the instrument and repeatability of the protocol was tested. We also tested its ability to characterize and differentiate two tasks: PF maximum contraction and Valsalva maneuver (maximum intra-abdominal effort with downward movement of the PF). Peak pressures were calculated for the total matrix, for three major sub-regions, and for 5 planes and 10 rings throughout the vaginal canal. Intraclass correlation coefficients indicated excellent inter- and intra-rater reliability and intra-trial repeatability for the total and medial areas, with moderate reliability for the cranial and caudal areas. There was a moderate correlation between peak pressure and PF digital palpation [Spearman’s coefficient r = 0.55 (p < 0.001)]. Spatiotemporal profiles were completely different between tasks (2-way ANOVAs for repeated measures) with notably higher pressures (above 30 kPa) for the maximum contraction task compared to Valsalva (below 15 kPa). At maximum contraction, higher pressures occurred in the mid-antero-posterior zone, with earlier peak pressure onsets and more variable along the vaginal depth (from rings 3 to 10-caudal). During Valsalva, the highest pressures were observed in rings 4–6, with peak pressure onsets more synchronized between rings. With this protocol and novel instrument, we obtained a high-resolution and highly reliable innovative 3D pressure distribution map of the PF capable of distinguishing vaginal sub-regions, planes, rings and tasks.     

Marine snow formation by the toxin-producing diatom,Pseudo-nitzschia australis

The formation of marine snow (MS) by the toxic diatom Pseudo-nitschia australis was simulated using a roller table experiment. Concentrations of particulate and dissolved domoic acid (pDA and dDA) differed significantly among exponential phase and MS formation under simulated near surface conditions (16 °C/12:12-dark:light cycle) and also differed compared to subsequent particle decomposition at 4 °C in the dark, mimicking conditions in deeper waters. Particulate DA was first detected at the onset of exponential growth, reached maximum levels associated with MS aggregates (1.21 ± 0.24 ng mL⁻¹) and declined at an average loss rate of ∼1.2% pDA day⁻¹ during particle decomposition. Dissolved DA concentrations increased throughout the experiment and reached a maximum of ∼20 ng mL⁻¹ at final sampling on day 88. The succession by P. australis from active growth to aggregation resulted in increasing MS toxicity and based on DA loading of particles and known in situ sinking speeds, a significant amount of toxin could have easily reached the deeper ocean or seafloor. MS formation was further associated with significant dDA accumulation at a ratio of pDA: dDA: cumulative dDA of approximately 1:10:100. Overall, this study confirms that MS functions as a major vector for toxin flux to depth, that Pseudo-nitzschia-derived aggregates should be considered ‘toxic snow’ for MS-associated organisms, and that effects of MS toxicity on interactions with aggregate-associated microbes and zooplankton consumers warrant further consideration.     

Forecasting the effect of feast and famine conditions on biological sulphate reduction in an anaerobic inverse fluidized bed reactor using artificial neural networks

The longevity and robustness of bioreactors used for wastewater treatment is determined by the activity of the microorganisms under steady and transient loading conditions. Two identical continuously operated inverse fluidized bed bioreactors (IFB), IFB R1 and IFB R2, were tested for sulphate removal under the same operating conditions for 140 d (Periods I–IV). Later, IFB R1 was used as the control reactor (Period V), while IFB R2 was operated under feast (Period V-A) and famine (Period V-B) feeding conditions for 66 d. The sulphate removal efficiency was comparable in both IFB, <20% in Period I and ∼70% during Periods II, III and IV. The robustness of the IFB was evident when the sulphate removal efficiency remained comparable during the feast Period (67 ± 15%) applied to IFB R2 compared to continuous feeding Periods (Period IV (71 ± 4%) for IFB R2 and Period V (61 ± 15%) for IFB R1). The IFB performance was modelled using a three-layered artificial neural networks (ANN) model (5-11-3) and a sensitivity analysis, the sulphate removal was found to be dependent on the COD:sulphate ratio. Besides, the robustness, resilience and adaptation time of the IFB were affected by the degree of mixing and the hydraulic retention time.     

Plasma gossypol dynamics in white-tailed deer: Implications for whole cottonseed as a supplemental feed

Whole cottonseed (WCS) is a potential supplemental feed for white-tailed deer (Odocoileus virginianus) in rangeland conditions because of its high digestible energy and protein content, moderate fiber content, and resistance to degradation in moist conditions. WCS also contains the polyphenolic secondary metabolite gossypol, which reduces palatability to non-target monogastric species but may be of concern for deer nutrition. Plasma gossypol stabilization when fed a constant dry matter intake, plasma gossypol depletion after WCS was removed from the diet, and the relationship between WCS consumption and plasma gossypol concentration was studied in 10 mature male (N = 5) and female (N = 5) captive white-tailed deer. Consumption of WCS by 73 free-ranging white-tailed deer (59 males, 14 females) was estimated using results of the captive study. Plasma gossypol concentrations declined exponentially and averaged 0.74 μg/mL 35 days after WCS was removed from the diet. Plasma gossypol concentration was linearly related to WCS consumption (P < 0.001), with females having 0.35 μg/mL greater (P = 0.04) plasma gossypol than males for a given rate of dry matter consumption. All female and 93% of male white-tailed deer captured in WCS supplemented pastures had detectable plasma gossypol. Female averaged 1.88 μg/mL of plasma gossypol and males averaged 4.84 μg/mL of plasma gossypol. Based on the captive deer data, these plasma values suggest an average WCS consumption of ∼2.6 g/kg BW/day for female free-ranging deer and ∼5.6 g/kg BW/day for male free-ranging deer. Inferentially, a large proportion of free-ranging white-tailed deer in rangeland conditions will consume WCS, with females consuming 125 g WCS/day and males consuming 428 g WCS/day. That plasma gossypol levels decrease rapidly after cottonseed is removed from the diet suggests that the long withdrawal periods often used prior to breeding season may not be needed. However, although 93% of gossypol was eliminated from the animals after a five-week withdrawal period, a small amount of gossypol can still be detected. While our preliminary data on these animals suggests that these levels are not detriment to animal health or reproduction, ranch managers may want to take a conservative approach to the feeding of WCS until these questions are answered.     

The CCKB antagonist CI988 reduces food intake in fasted rats via a dopamine mediated pathway

Studies have shown a reduction of food intake following peripheral and brain injection of CCK. However, it remains to be established whether endogenous central CCK is involved in the regulation of food intake. We investigated the role of central CCK in the regulation of food intake by pharmacological manipulation of the CCK_B (CCK₂) receptor system. Intracerebroventricularly (ICV) cannulated male Sprague Dawley rats were fasted for 24 h and received an ICV injection of the CCK_B receptor antagonist CI988 at a dose of 10 nmol or 49 nmol or vehicle. Another group received two consecutive ICV injections consisting of the corticotropin-releasing factor (CRF) receptor-1 (CRF₁) antagonist, CP376395 (3 nmol) or the CRF₂ receptor antagonist, K41498 (2 nmol) alone, or followed by CI988 (49 nmol). Lastly, another group of rats received an intraperitoneal (IP) injection of the dopamine antagonist, flupentixol (∼197 and ∼493 nmol/kg) alone, or followed by CI988 (49 nmol, ICV). Cumulative food intake was assessed for 11 h. Vehicle injected rats showed a robust feeding response. CI988 at 49 nmol reduced food intake by 30% starting at 2 h post injection. CP376395 and K41498 had no effect on food intake. Flupentixol injected IP at a dose of 197 and 493 nmol/kg alone did not modulate food intake whereas the higher dose blocked the CI988-induced reduction of feeding. During the dark phase, CI988 had no effect on food intake in unfasted rats. In summary, CCK_B signaling is involved in the regulation of food intake after a fast likely by downstream dopamine signaling.     

Phase state and surface topography of palmitoyl-ceramide monolayers

In cell biology (and in many biophysical) studies there is a natural tendency to consider ceramide as a highly condensed, solid-type lipid conferring rigidity and close packing to biomembranes. In the present work we advanced the understanding of the phase behavior of palmitoyl-ceramide restricted to a planar interface using Langmuir monolayers under strictly controlled and known surface packing conditions. Surface pressure–molecular area isotherms were complemented with molecular area–temperature isobars and with observations of the surface topography by Brewster Angle Microscopy. The results described herein indicate that palmitoyl-ceramide can exhibit expanded, as well as condensed phase states. Formation of three phases was found, depending on the surface pressure and temperature: a solid (1.80 nm thick), a liquid-condensed (1.73 nm thick, likely tilted) and a liquid-expanded (1.54 nm thick) phase over the temperature range 5–62 °C. A large hysteretic behavior is observed for the S phase monolayer that may indicate high resistance to domain boundary deformation. A second (or higher) order S → LC phase transition is observed at about room temperature while a first order LC → LE transition occurs in a range of temperature encompassing the physiological one (observed above 30 °C at low surface pressure). This phase behavior broadens the view of ceramide as a type of lipid not-always-rigid but able to exhibit polymorphic properties.     

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus microplus associated with resistance to flumethrin but not to cypermethrin

A mutation in the domain II S4–5 linker region of the para-sodium channel gene has been associated previously with synthetic pyrethroid (SP) resistance in the cattle tick (Rhipicephalus microplus) in Australia. This is a C → A mutation at nucleotide position 190, which results in a leucine to isoleucine amino acid substitution (L64I). In a survey of 15 cattle tick populations with known SP resistance status, sourced from Queensland and New South Wales in Australia, there was a strong relationship (r = 0.98) between the proportion of ticks carrying the L64I homozygous resistant genotype and the survival percentage after exposure to a discriminating concentration of cypermethrin in the bioassay, as expected. However, among populations resistant only to flumethrin, the L64I homozygous genotype was not found. The sequence obtained for a 167 bp region including domain II S4–5 linker in flumethrin-resistant ticks identified a G → T non-synonymous mutation at nucleotide position 214 that results in a glycine to valine substitution (G72V). The frequency of the G72V homozygous genotype in each population was found to be moderately related to the survival percentage at the discriminating concentration of flumethrin in the larval packet test (r = 0.74). However, a much stronger relationship between genotype and resistance to flumethrin was observed when the heterozygotes of L64I and G72V were added to the G72V homozygotes (r = 0.93). These results suggest that there is an interaction between the two mutations in the same gene, such that flumethrin resistance might be conferred by either two copies of the G72V mutation or by being a L64I and G72V heterozygote.     

Biomechanics of invasive growth by Armillaria rhizomorphs

Rhizomorphs of wood-decay basidiomycetes are root-like structures produced by the coordinated growth of thousands of hyphae. Very little is known about their development nor the way that they penetrate soils and rotting wood. In this study, we applied techniques used in previous studies on hyphae to explore the mechanics of the invasive growth process in Armillaria gallica. Growth rate measurements were made in media with different gel strengths. The osmolyte composition of rhizomorph sap was determined spectroscopically and the forces exerted by growing tips were measured using a force transducer. Cultured rhizomorphs extended at much faster rates than unbundled hyphae (3.5 mm d⁻¹ versus 1.5 mm d⁻¹) and their growth accelerated in response to increased medium gel strength (to 7.4 mm d⁻¹). Measurements of rhizomorph osmolality provided a turgor pressure estimate of 760 kPa (7.5 atm.), and spectroscopic analysis showed that this pressure was generated by the accumulation of erythritol, mannitol, and KCl. Forces exerted by growing tips ranged from 1 to 6 mN, corresponding to pressures of 40–300 kPa (0.4–3.0 atm.). Pressures exerted by extending rhizomorphs are comparable to those produced by individual vegetative hyphae. This suggests that the mechanical behavior of hyphae is similar whether they grow as unbundled cells or aggregate to form macroscopic rhizomorphs.