Francis T. Ashton

The contractile apparatus of vascular smooth muscle: Intermediate high voltage stereo electron microscopy

Intermediate high voltage stereo electron microscopy of rabbit portal-anterior mesenteric vein smooth muscle showed that thick filaments are 2·2 μm long and have tapered ends. In serial transverse sections, groups of three to five thick filaments end or begin within the same section. The intermediate (10 nm diameter) filaments were associated with dense bodies, as noted in previous studies, and were never seen within the substructure of the thick filaments. Actin filaments were found inserting on both cytoplasmic and plasma membrane-bound dense bodies. The greater length of the thick filaments in smooth than in striated muscles and their “parallel arrangement” within the fiber could contribute to the ability of smooth muscle to develop equal or greater tension than striated muscle, in spite of the much lower concentration of myosin in smooth muscle.

Chemo- and thermosensory neurons: structure and function in animal parasitic nematodes.

Nematode parasites of warm-blooded hosts use chemical and thermal signals in host-finding and in the subsequent resumption of development. The free-living nematode Caenorhabditis elegans is a useful model for investigating the chemo- and thermosensory neurons of such parasites, because the functions of its amphidial neurons are well known from laser microbeam ablation studies. The neurons found in the amphidial channel detect aqueous chemoattractants and repellants; the wing cells-flattened amphidial neurons-detect volatile odorants. The finger cells-digitiform amphidial neurons-are the primary thermoreceptors. Two neuron classes, named ADF and ASI, control entry into the environmentally resistant resting and dispersal dauer larval stage, while the paired ASJ neurons control exit from this stage. Skin-penetrating nematode parasites, i.e. the dog hookworm Ancylostoma caninum, and the threadworm, Strongyloides stercoralis, use thermal and chemical signals for host-finding, while the passively ingested sheep stomach worm, Haemonchus contortus, uses environmental signals to position itself for ingestion. Amphidial neurons presumably recognize these signals. In all species, resumption of development, on entering a host, is probably triggered by host signals also perceived by amphidial neurons. In the amphids of the A. caninum infective larva, there are wing- and finger-cell neurons, as well as neurons ending in cilia-like dendritic processes, some of which presumably recognize a sequence of signals that stimulate these larvae to attach to suitable hosts. The functions of these neurons can be postulated, based on the known functions of their homologs in C. elegans. The threadworm, S. stercoralis, has a complex life cycle. After leaving the host, soil-dwelling larvae may develop either to infective larvae (the life-stage equivalent of dauer larvae) or to free-living adults. As with the dauer larva of C. elegans, two neuron classes control this developmental switch. Amphidial neurons control chemotaxis to a skin extract, and a highly modified amphidial neuron, the lamellar cell, appears to be the primary thermoreceptor, in addition to having chemosensory function. The stomach worm, Haemonchus contortus, depends on ingestion by a grazing host. Once ingested, the infective larva is exposed to profound environmental changes in the rumen. These changes stimulate resumption of development in this species. We hypothesize that resumption of development is under the control of the ASJ neuronal pair. Identification of the neurons that control the infective process could provide the basis for entirely new approaches to parasite control involving interference with development at the time and place of initial host-contact.

Developmental switching in the parasitic nematode Strongyloides stercoralis is controlled by the ASF and ASI amphidial neurons.

Parasitic nematodes of the genus Strongyloides are remarkable for their ability to switch between alternative free-living developmental pathways in response to changing internal environmental conditions. After exiting the host, soil-dwelling larval stages may develop either to infectivity via 2 microbiverous stages (homogonic development) or to free-living adulthood via 4 microbiverous larval stages (heterogonic development). The progeny of these adults then give rise to the infective stage. In the latter case, free-living existence is extended in time and the number of infective larvae is greatly amplified. Anterior chemosensory neurons (amphidial neurons) are thought to respond to environmental cues and via signal transduction pathways control the direction of larval development. We now demonstrate by laser microbeam ablation that 2 classes of amphidial neurons (ASF and ASI), acting together, control the direction of free-living larval development. Larvae in which the neurons were killed developed to infectivity via the homogonic route rather than to adulthood via the otherwise predominant heterogonic route. These neurons are probable homologues of neurons ADF (=ASF) and ASI in Caenorhabditis elegans, suggesting the control of development at the cellular level is conserved among divergent taxa of nematodes. These observations also have important implications for the evolution of nematode parasitism and the design of new prophylactic measures against parasitic nematodes of medical and veterinary medical importance.

Amphidial structure of ivermectin-resistant and susceptible laboratory and field strains of Haemonchus contortus

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians, pet owners, and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides. As a result, resistance to ivermectin, moxidectin, nemadectin, and doramectin by Haemonchus contortus has been documented throughout the world. Sensory neurons located in the cephalic end of nematodes are in close contact with the external environment. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible strains of H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveal that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. These findings raise a number of questions regarding the relationship between amphidial structure and ivermectin resistance as well as the role of amphids as a means of entry for ivermectin. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to macrocyclic lactones.

The neurons of class ALD mediate thermotaxis in the parasitic nematode, Strongyloides stercoralis

Strongyloides stercoralis, a skin-penetrating nematode parasite of homeotherms, migrates to warmth. In nematodes, the amphids, anteriorly positioned, paired sensilla, each contain a bundle of sensory neurons. In the amphids of the free-living nematode Caenorhabditis elegans, a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors, and have been named the finger cells (class AFD). A similar neuron pair in the amphids of the parasite Haemonchus contortus is also known to be thermosensory. Strongyloides stercoralis lacks finger cells but, in its amphids, it has a pair of neurons whose dendrites end in a multi-layered complex of lamellae, the so-called lamellar cells (class ALD). Consequently, it was hypothesised that these lamellar cells might mediate thermotaxis by the skin-penetrating infective larva of this species. To investigate this, first stage S. stercoralis larvae were anaesthetised and the paired ALD class neurons were ablated with a laser microbeam. The larvae were then cultured to the infective third stage (L3) and assayed for thermotaxis on a thermal gradient. L3 with ablated ALD class neuron pairs showed significantly reduced thermotaxis compared with control groups. The thermoreceptive function of the ALD class neurons (i) associates this neuron pair with the host-finding process of S. stercoralis and (ii) demonstrates a functional similarity with the neurons of class AFD in C. elegans. The structural and positional characteristics of the ALD neurons suggest that these neurons may, in fact, be homologous with one pair of flattened dendritic processes known as wing cells (AWC) in C. elegans, while their florid development and thermosensory function suggest homology with the finger cells (AFD) of that nematode.

Amphids in strongyloides stercoralis and other parasitic nematodes

In this review, Francis Ashton and Gerhard Schad examine the ultrastructure of the amphids of several animal parasitic nematodes. These structures are the main chemosensory organs of these worms and probably play an important role in host-finding behavior and the control of development. Reconstructions made from serial micrographs of the neurons in the amphids of the threadworm Strongyloides stercoralis are shown. These stereo images permit three-dimensional visualization of these complex sense organs. The association between each amphidial neuron and its cell body has not been made previously for a parasitic nematode; however, this has been done for the free-living nematode Caenorhabditis elegans, which served as a model for these studies. Recognition of the cell bodies will provide a point of departure for laser microbeam ablation studies to determine individual neuronal function.

Sensory neuroanatomy of a passively ingested nematode parasite, Haemonchus contortus: amphidial neurons of the first stage larva.

When infective larvae of Haemonchus contortus (a highly pathogenic, economically important, gastric parasite of ruminants) are ingested by grazing hosts, they are exposed to environmental changes in the rumen, which stimulate resumption of development. Presumably, resumption is controlled by sensory neurons in sensilla known as amphids. Neuronal function can be determined by ablation of specifically recognized neurons in hatchling larvae (L1) in which neuronal cell bodies are easily visualized using differential interference microscopy. Using three‐dimensional reconstructions from electron micrographs of serial transverse sections, amphidial structure of the L1 is described. Each amphid of H. contortus is innervated by 12 neurons. The ciliated dendritic processes of 10 neurons lie in the amphidial channel. Three of these end in double processes, resulting in 13 sensory cilia in the channel. One process, that of the so‐called finger cell, ends in a number of digitiform projections. Another specialized dendrite enters the amphidial channel, but leaves it to end within the sheath cell, a hollow, flask‐shaped cell that forms the base of the amphidial channel. Although not flattened, this process is otherwise similar to the wing cells in Caenorhabditis elegans; we consider it AWC of this group. Two other neurons, ASA and ADB, appear to be homologs of wing cells AWA and AWB in C. elegans, although they end as ciliated processes in the amphidial channel, rather than as flattened endings seen in C. elegans. Each of the 12 amphidial neurons was traced to its cell body in the lateral ganglion, posterior to the worms nerve ring. The positions of these bodies were similar to their counterparts in C. elegans; they were named accordingly. A map for identifying the amphidial cell bodies in the living L1 was prepared, so that laser microbeam ablation studies can be conducted. These will determine which neurons are involved in the infective process, as well as others important in establishing the host‐parasite relationship. J. Comp. Neurol. 417:299–314, 2000. Published 2000 Wiley‐Liss, Inc.

The myosin filament: IX. Determination of subfilament positions by computer processing of electron micrographs

Abstract Computer image processing of electron micrographs has been employed to delineate the position of thick filament subunits in transverse sections of extensively crosslinked vertebrate skeletal muscle. Both back projection and rotational averaging methods indicate the presence of 12 subunits arranged on an approximately hexagonal lattice similar to that proposed by Pepe (1967). The spacing between subunits and the myosin content of the thick filament indicate that these subunits probably contain more than one myosin molecule and are most likely dimers.

The amphidial neuron pair ALD controls the temperature-sensitive choice of alternative developmental pathways in the parasitic nematode, Strongyloides stercoralis

The parasitic nematode Strongyloides stercoralis, has several alternative developmental pathways. Upon exiting the host (humans, other primates and dogs) in faeces, 1st-stage larvae (L1) can enter the direct pathway, in which they moult twice to reach the infective 3rd-stage. Alternatively, if they enter the indirect pathway, they moult 4 times and become free-living adults. The choice of route depends, in part, on environmental cues. In this investigation it was shown that at temperatures below 34 degrees C the larvae enter the indirect pathway and develop to free-living adulthood. Conversely, at temperatures approaching body temperature (34 degrees C and above), that are unfavorable for the survival of free-living stages, larvae develop directly to infectivity. The time-period within the L1s development during which temperature influenced the choice of the pathway depended on the temperature, but, at any given temperature, occurred approximately in the middle of the time-span spent in the L1 stage, which varied inversely with temperature. This critical period was associated with the time-interval in which the number of cells in the genital primordium began to increase, thus providing a morphological marker for the pathway decision in individual worms. Sensing the environment is the function of the amphidial neurons, and therefore we examined the role of individual amphidial neurons in controlling entry into the direct pathway to infectivity. The temperature-sensitive developmental switch is controlled by the neuron pair ALD (which also controls thermotaxis), as seen by the loss of control when these neurons are ablated. Thus, in S. stercoralis a single amphidial neuron pair controls both developmental and behavioural functions.

Thermotaxis and thermosensory neurons in infective larvae of Haemonchus contortus, a passively ingested nematode parasite

As a basis for studies of thermal behavior of infective larvae (L3) of Haemonchus contortus resulting from ablation of amphidial neurons, the locations of the amphidial cell bodies in the hatchling larva (L1) were compared with their locations in the L3. We sought to verify that killing each targeted cell body in L1 destroys the putative corresponding dendrite of the L3. These comparisons confirmed the predicted cell body‐to‐dendrite connections, as well as similarities in the general amphidial structure of the two stages. We then conducted a series of studies using laser microbeam ablation of amphidial cell bodies in the L1 to determine the role of specific neurons in the thermal behavior of the L3. In a thermal gradient, normal L3 of H. contortus migrate to the temperature at which they were cultured and/or maintained. Larvae grown at 16° or 26°C migrate appropriately to either of these temperatures. Larvae grown to the L3 stage at 16°C and then moved to 26°C become acclimated to this temperature and thereafter migrate to it. However, when the putative thermosensory neurons, the finger cell neurons (AFD), were ablated in hatchling larvae with a laser microbeam, and these were grown to the L3 stage and tested on a radial thermal gradient, they failed to migrate to their culture temperature. Instead, they moved actively and continuously over much of the assay plate surface, with no obviously oriented cryo‐ or thermotactic movement. Ablation‐control larvae, those in which putatively chemosensory neuron classes ASE or AWC were killed, migrated normally to their culture temperature. When the RIA interneurons (identified by positional homology with those of Caenorhabditis elegans) were ablated, the operated larvae moved actively, but circled near the initial placement point; control larvae, in which other nonamphidial neurons were killed, migrated normally. These results indicate that the finger cell neurons (AFD) are the primary thermosensory class in H. contortus. The RIA‐class neurons integrate thermal responses in H. contortus, as do their putative structural homologs in C. elegans, but the behavior of H. contortus subsequent to RIA ablation is strikingly different. J. Comp. Neurol. 424:58–73, 2000.