Determination of LC50 of permethrin acaricide in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Currently, the most efficient and widely used method of tick control still is the treatment with acaricides, especially permethrin (active ingredient of the Advantage^® Max3, Bayer), a pyrethroid with neurotoxic action. Due to the wide use of this acaricide in the control of the tick Rhipicephalus sanguineus, this study carried out laboratorial procedures to determine the LC₅₀ (lethal concentration fifty) of permethrin in semi-engorged females of R. sanguineus. Based on the result of 14 dilutions of permethrin in distilled water and later Probit analysis, the LC₅₀ of permethrin for R. sanguineus was 2062 ppm (1549-2675 ppm). This work can be used as a protocol with other chemicals, to determinate the LC₅₀, basic procedure for studies of control, resistance and behavior of ticks treated with acaricides, especially the brown dog tick R. sanguineus. Also, the knowledge of the LC₅₀ provides information on the potency of chemicals, the sensitivity of Arthropods to them and even estimates on pest control.     

Potential of the insect growth regulator, fluazuron, in the control of Rhipicephalus sanguineus nymphs (Latreille, 1806) (Acari: Ixodidae): determination of the LD95 and LD50

Conventional pesticides have suffered two main drawbacks: (a) broad spectrum of action and (b) selection of target species resistant to the different active ingredients. Thus compounds that are less harmful to the environment and to human health, more specific and that do not induce resistance need to be developed. One alternative are insect growth regulators, such as fluazuron. The present study examined the efficacy of fluazuron (active ingredient of the acaricide Acatak®) and the sensitivity of Rhipicephalus sanguineus nymphs exposed to different doses of this chemical, and determined the lethal doses of fluazuron: 95% – LD₉₅ and 50% – LD₅₀. Different doses of fluazuron were applied in duplicates on the dorsal region of hosts (“pour on”). Distilled water was used in the control group. On the first day after the treatment with fluazuron, hosts were artificially infested with R. sanguineus nymphs. After engorgement, nymphs were removed, placed on Petri dishes, identified, and maintained in BOD incubator for 15 days. Dead R. sanguineus nymphs after the treatment with 13 different doses of fluazuron were quantified and the LD₉₅ was estimated to be 100 mg/kg and LD₅₀, 19.544 mg/kg (12.478–22.636), with a confidence interval of 95%. Nymphs of R. sanguineus were sensitive to fluazuron at various levels, indicating that this insect growth regulator (IGR) may be used to control this parasite in this stage of its biological cycle, reducing the significant damage it causes.     

Responses of lateral line receptors to water flow in the Antarctic notothenioid, Trematomus bernacchii

The response properties of anterior lateral line afferent neurones in Trematomus bernacchii were recorded extracellularly while stimulating the fish with unidirectional water flows of varying velocity. Afferent neurone responses were either flow-sensitive or flow-insensitive. Flow-sensitive neurones showed linear increases in response magnitude with increasing flow rate and tonic non-adapting response properties. These findings indicate that flow-sensitive afferent neurones originate from lateral line receptors that detect absolute flow velocity. The likely explanation is that flow-sensitive afferent neurones innervate neuromasts located superficially on the skin and flow-insensitive neurones innervate neuromasts situated in sub-epidermal fluid-filled canals.     

Warmer Weather Linked to Tick Attack and Emergence of Severe Rickettsioses

The impact of climate on the vector behaviour of the worldwide dog tick Rhipicephalus sanguineus is a cause of concern. This tick is a vector for life-threatening organisms including Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, R. conorii, the agent of Mediterranean spotted fever, and the ubiquitous emerging pathogen R. massiliae. A focus of spotted fever was investigated in France in May 2007. Blood and tissue samples from two patients were tested. An entomological survey was organised with the study of climatic conditions. An experimental model was designed to test the affinity of Rh. sanguineus for biting humans in variable temperature conditions. Serological and/or molecular tools confirmed that one patient was infected by R. conorii, whereas the other was infected by R. massiliae. Dense populations of Rh. sanguineus were found. They were infected with new genotypes of clonal populations of either R. conorii (24/133; 18%) or R. massiliae (13/133; 10%). April 2007 was the warmest since 1950, with summer-like temperatures. We show herein that the human affinity of Rh. sanguineus was increased in warmer temperatures. In addition to the originality of theses cases (ophthalmic involvements, the second reported case of R. massiliae infection), we provide evidence that this cluster of cases was related to a warming-mediated increase in the aggressiveness of Rh. sanguineus, leading to increased human attacks. From a global perspective, we predict that as a result of globalisation and warming, more pathogens transmitted by the brown dog tick may emerge in the future.     

The temperature and humidity preferences of Haemaphysalis longicornis, Ixodes Holocyclus and Rhipicephalus sanguineus (Ixodidae): studies on eggs

The eggs of Haemaphysalis longicornis, Ixodes holocydus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to compare the temperature and humidity requirements of ticks from climatically dissimilar habitats. The response to changes in humidity was also studied with H. longicornis. Hatching in I. holocydus eggs occurred from 18 to 28°C and at a saturation deficit of 2 mm Hg or less. The eggs of R. sanguineus hatched between 18 and 38°C and tolerated saturation deficits up to 20 mm Hg. The eggs of H. longicornis hatched between 18 and 35°C and at saturation deficits up to 8 mm Hg. When eggs of H. longicornis were exposed to moderately dry conditions for different periods of time and then transferred to moist conditions, incubation periods and mortality were reduced in comparison with eggs kept continuously under dry conditions. On the other hand, mortality of H. longicornis eggs was reduced in moderately dry conditions, provided that they had been exposed to a moist environment for about 10 days previously. Weight loss of eggs in dry air and the rate of development of eggs were examined and are discussed. The eggs of I. holocylus lost weight very rapidly in dry air, whereas weight loss from R. sanguineus eggs was much slower. Weight loss from H. longicornis eggs fell between these two rates. The rate of development was fastest in R. sanguineus and slowest in I. holocydus.     

Conspecific tolerance and heterospecific competition as mechanisms for overcoming resistance to invasion by an intertidal crab

The success of the invasive Asian shore crab, Hemigrapsus sanguineus, stems partly from its ability to exclude established crab species from preferred rocky and cobble intertidal habitat. Here, we assessed preference and competition for habitat types (cobble vs. sand) for H. sanguineus and two competitor species; the previous invasive green crab, Carcinus maenas, and the native rock crab, Cancer irroratus, in New England. In simple laboratory experiments, we paired similarly sized heterospecifics and conspecifics from each species, and also grouped combinations of C. maenas and H. sanguineus in a series of four-individual sets in order to dissect the outcome of intra- and inter-specific competition at different densities. Individually, all three species preferred cobble substrate. With paired conspecifics, H. sanguineus individuals would cohabitate in cobble, whereas C. maenas and C. irroratus individuals each excluded conspecifics from cobble. In heterospecific pairs, H. sanguineus excluded both C. maenas and C. irroratus from cobble. C. maenas and C. irroratus, were equally likely to exclude the other species, but rarely excluded H. sanguineus. In larger assemblages, H. sanguineus preferentially grouped under cobble, whereas C. maenas were more evenly distributed among habitat types. These observations demonstrate that conspecific tolerance and heterospecific competition can be effective, complementary mechanisms for overcoming invasion resistance. Such mechanisms help explain the well-studied success of H. sanguineus following its introduction into New England coastal habitats, and the resulting exclusion of preexisting crab species.     

Experimental evaluation of birds as disseminators of the cosmopolitan tick Rhipicephalus sanguineus (Acari: Ixodidae)

Rhipicephalus sanguineus is believed to be the most widespread tick species of the world and its dissemination seems to rely on the diffusion of its main host, the dog. Empirical observations indicate that several bird species in urban areas regularly steal dog food. Such circumstances create a chance for R. sanguineus ticks to climb on birds and carry ticks to another site. In this work we evaluated experimentally the likelihood of birds (chicks) to either feed and/or carry R. sanguineus ticks from an infested site to another and to infest a host (rabbit) in the new location. Chicks were not suitable hosts for R. sanguineus ticks. Not a single adult tick engorged on chicks, yield as well as weight of engorged larvae and nymphs were very low and feeding period of these ticks was very long. However, a few larvae and, chiefly, nymphs were delivered to a new location either mechanically or after attachment and engorging total or partially on chicks. A few of these ticks fed successfully on rabbits. Further evidence on the capacity of birds to introduce R. sanguineus into non-infested dog settings should be provided by systematic examination of birds from urban areas, close to tick infested households.     

The temperature and humidity preferences of Haemaphysalis longicornis,ixodes holocyclus and Rhipicephalus sanguineus (ixodidae): Studies on engorged larvae

The engorged larvae of Haemaphysalis longicornis, Ixodes holocyclus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to see whether the nature of their requirements in the laboratory were similar to the climate within the geographic range of each species. The response of H. longicornis and I. holocyclus to changes in humidity was also studied. Moulting of I. holocyclus larvae occurred from 18 to 28°C and at a saturation deficit of 4 mm Hg or less. The larvae of R. sanguineus moulted between 18 and 38°C and tolerated saturation deficits up to 35 mm Hg. The larvae of H. longicornis moulted between 15 and 38°C at saturation deficits up to 8 mm Hg. When engorged larvae of H. longicornis and I. holocyclus were exposed to very dry conditions for different periods of time and then transferred to moist conditions, the minimum pre-moult period and mortality was increased in comparison with larvae kept continuously under moist conditions. On the other hand, mortality of H. longicornis larvae was reduced in very dry conditions provided that they had been exposed to a moist environment for about 5 days previously. The larvae of I. holocyclus required at least 12 days exposure to a moist environment before any survived to moult in moderately dry conditions. The engorged larvae of both H. longicornis and I. holocyclus lost weight rapidly in dry air, whereas weight loss from R. sanguineus larvae was much slower. The rate of development was fastest in R. sanguineus and slowest in I. holocyclus.     

Incongruent effects of two isolates of <Emphasis Type="Italic">Rickettsia conorii</Emphasis> on the survival of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> ticks

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35–66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.     

Morphology of the central projections of physiologically characterised neurones from the locust metathoracic femoral chordotonal organ

Summary The metathoracic femoral chordotonal organ of the locust (Locusta migratoria) is an internal proprioceptor composed of mechanosensory neurones which respond to tibial position, velocity, or acceleration, or to combinations of these parameters. Discriminant function analyses confirmed the visual observation that neurones with different responses to tibial movements had different central branching patterns. Some aspects of the projections were consistent for all neurones (e.g., the path taken by the main neurite through the metathoracic ganglion), whereas other regions of branches were consistently reduced or missing in some response classes. Some position-and-acceleration receptors had no main branches off the main neurite, and must therefore make relatively restricted contact with motor neurones and interneurones. Phasic or tonic neurones which responded in ranges of tibial extension had branches which projected further medial in Dorsal Commissures III and IV than similar neurones which responded in ranges of tibial flexion. I compare my results to previous studies of mapping in the insect CNS.Abbreviations (ms) (mt)FCO (mesothoracic) (metathoracic) femoral chordotonal organ - ANOVA Analysis of Variance     

Anti-Oxidative Defences Are Modulated Differentially in Three Freshwater Teleosts in Response to Ammonia-Induced Oxidative Stress

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH₄HCO₃) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h–48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Unlike in trout, H₂O₂ and MDA accumulation in carp and goldfish liver was restored to control levels (84 h–180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.     

Cuticular fatty acid profile analysis of three Rhipicephalus tick species (Acari: Ixodidae)

Cuticular fatty acids (CFA) are important constituents of the arthropod exoskeleton, serving as structural and defense components, and participating in intra-species communication. Here we describe for the first time a comparative analysis of the CFA profiles of three tick species of the genus Rhipicephalus: R. annulatus, R. bursa and R. sanguineus. CFA profiles were determined for R. bursa and R. sanguineus grown both on rabbit or calf, and for R. annulatus grown on calf. CFA composition was compared for each species before and after ethanol treatment, for different hosts of each species, and between the different species. Our data suggest that adsorption of the host’s fatty acids changes the apparent CFA composition. Ethanol treatment efficiently removed the unbound fatty acids from the ticks and revealed the actual composition. Comparison between ticks grown on rabbit versus calf showed significant difference in the relative abundance of fatty acids C14 and 9,12-C18:2 for R. bursa, and a difference in the relative abundance of C14 for R. sanguineus. Comparison of the CFA between the three species revealed significant differences in the abundance of fatty acids C16, 9,12-C18:2, 9-C18:1, C18 and C20. Our results show that while the host had a minor effect on CFA composition within each species, significant differences were observed in the CFA profiles of different species. We suggest that CFA profiles may be used to distinguish between related species. CFA analysis can also be used in studies of communication and defense mechanisms in ticks and other arthropods.     

Complete Mitochondrial Genome Sequence Data Provides Genetic Evidence That the Brown Dog Tick Rhipicephalus sanguineus (Acari: Ixodidae) Represents a Species Complex

Ticks are blood-sucking ectoparasites of great medical and veterinary significance that can transmit bacteria, protozoa, fungi and viruses, and cause a variety of human and animal diseases worldwide. In the present study, we sequenced the complete mitochondrial (mt) genome of Rhipicephalus sanguineus from China (RSC) and compared with that of R. sanguineus from USA (RSU). Nucleotide sequence difference in the full mt genome was 11.23% between RSC and RSU. For the 13 protein-coding genes, comparison revealed sequence divergences at both the nucleotide (9.34-15.65%) and amino acid (2.54-19.23%) levels between RSC and RSU. In addition, sequence comparison of the conserved mt cox1 and cytb genes among multiple individual R. sanguineus revealed substantial nucleotide differences between RSC and RSU but limited sequence variation within RSC. Phylogenetic analysis of ticks based on the amino acid sequence data of 13 protein-coding genes revealed that R. sanguineus from China and R. sanguineus from USA represent sister taxa (likely separate species). Taken together, the findings support the recently proposal that R. sanguineus tick may represents a species complex of at least two closely related species.     

The Product of glnL Is Not Essential for Regulation of Bacterial Nitrogen Assimilation

The glnL product is not necessary for the control of expression of glnA and other nitrogen-regulated genes, but it presumably communicates reduntant information on the availability of ammonia from an ammonia-sensitive system consisting of the products of glnB and glnD to the regulatory products of glnF and glnG.     

Potential impact of the Asian shore crab <Emphasis Type="Italic">Hemigrapsus sanguineus</Emphasis> on native northeast Pacific crabs

Asian shore crabs (Hemigrapsus sanguineus De Haan) are a dominant invasive species in the northwest Atlantic, where recent evidence suggests that they have been introduced multiple times from their native range of Japan and Korea. Despite favorable environmental conditions and a high level of shipping traffic across the Pacific, this crab has not become established in the northeast Pacific. This may be due to interactions with the native shore crab assemblage, which previous studies have found to outcompete juvenile H. sanguineus for shelter. The present study used a combination of feeding and behavioral experiments to quantify the feeding behavior of native crabs (Pachygrapsus crassipes, Hemigrapsus nudus, Hemigrapsus oregonensis) with and without the presence of H. sanguineus. H. sanguineus ate more than the native crabs combined at both 7 and 13 °C. Crabs in the mixed treatment which included all four species ate less than expected, indicating that at least one species reduced its feeding rate. However, time-lapse photography revealed similar amounts of time feeding in both treatments for all 4 species, suggesting that there were changes in feeding intensity when all crabs were together. The high feeding rates but lack of a competitive advantage for H. sanguineus make it an unlikely but potentially impactful invader in the northeast Pacific.     

Physiology of spontaneous [Ca2+]i oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca²⁺ signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca²⁺]_i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca²⁺]_i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca²⁺]_i dynamics. In rats dehydrated for 3 or 5 days almost 90% of neurones displayed spontaneous [Ca²⁺]_i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca²⁺ signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.     

A taste receptor neurone dedicated to the perception of pyrrolizidine alkaloids in the medial galeal sensillum of two polyphagous arctiid caterpillars

It is shown that pyrrolizidine alkaloids are phagostimulants for the caterpillars of two polyphagous arctiid caterpillars, Estigmene acrea and Grammia geneura. The caterpillars will also eat dry glass‐fibre discs containing only pyrrolizidine alkaloid ? an example of pharmacophagy. The tip‐recording technique is used to demonstrate that each species has a neurone in the medial galeal styloconic taste sensillum responding to pyrrolizidine alkaloids, although the species differ in their sensitivities. This neurone responds to at least four different pyrrolizidine alkaloids and their N‐oxides, and experiments indicate that it is dedicated to perception of these compounds. The sensory response is phasic–tonic and during the tonic phase remains unchanged for at least 500 ms, resembling the type of response often seen in neurones that are sensitive to plant secondary compounds producing deterrent effects.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.