The ultrastructure of the cuticle of Nematoda. II. The cephalic cuticle of Stilbonematinae (Adenophorea,Desmodoridae)

Summary  The ultrastructure of the cephalic cuticle of 6 of 7 representative genera of Stilbonematinae (Eubostrichus, Catanema, Laxus, Robbea, Leptonemella, Stilbonema) is investigated using SEM and TEM techniques. Additionally, one species of Spirinia (Spiriniinae) and one of Desmodora (Desmodorinae) were studied for outgroup comparison. Most of the studied species show modifications of the cephalic cuticle. Furthermore, at least four different pathways have been developed to reinforce the head within Stilbonematinae. Species with a coarsely annulated somatic cuticle (Leptonemella sp., Stilbonema majum, and Desmodora ovigera) developed a rigid, non-annulated cephalic capsule by modifying the main constructing element of the median zone, the ’’ring body.’’ In faintly annulated Laxus oneistus, the annulated cephalic capsule results from a newly inserted ’’block layer’’ between the median and basal zone. The non-annulated cephalic capsule of Robbea sp. is formed by both the block layer and the ring body element. The annulated capsule of Catanema sp. stems from a doubled number of fiber layers within the basal zone. In Spirinia sp., only the amphidial region is strengthened in what could be termed an amphidial shield. All forms with cephalic capsules show mechanisms to keep the oral region pliable. Only Eubostrichus topiarius lacks a reinforcement of the cephalic cuticle. A comparison with the literature is made to elucidate corresponding structures within the cephalic capsules of representatives of Desmodorida. It is demonstrated that the presence of a cephalic cuticle is of no systematic value above the genus level. Accepted: 3 March 1996     

Phylogenetic relationships within the superfamily Desmodoroidea (Nematoda: Desmodorida), with descriptions of two new and one known species

Three nematode species of the superfamily Desmodoroidea Filipjev, 1922, were isolated from beach sediments in Wellington, New Zealand, for morphological and molecular analyses. Two of these species, D esmodorella verscheldei sp. nov. and D racograllus ngakei sp. nov. , were new to science and are described herein. Epsilonema rugatum Lorenzen, 1973, comb. nov. , which was originally described from New Zealand material as a subspecies of Epsilonema dentatum from Chile, is redescribed and elevated to the rank of species based on cuticular ornamentation. The phylogenetic relationships amongst the three Desmodoroidea families are investigated based on new and existing sequences of the D2 and D3 expansions segments of large subunit (LSU) 28S rRNA gene and small subunit (SSU) of 18S rDNA gene. Our analyses suggest that the Draconematidae is a sister taxon to the Desmodorinae and Spiriniinae, with the Draconematidae forming a monophyletic crown group and the Desmodorinae and Spiriniinae forming a paraphyletic stem group. Phylogenetic relationships between the Epsilonematidae and Stilbonematinae, however, could not be determined with certainty. The SSU and D2‐D3 of LSU consensus trees indicate that the morphological resemblance between the Draconematidae and Epsilonematidae, which are both characterized by swollen pharyngeal body regions and mid‐posterior body regions with specialized setae, reflects distinct and independently evolved adaptations to their unusual mode of locomotion, with differences in the structure and distribution of specialized setae between the two families also consistent with convergent evolution. We show that the family Desmodoridae and superfamily Desmodoroidea as currently defined are not monophyletic. It was not possible to determine whether the Prodesmodorinae are more closely related to the Desmodoroidea or Microlaimoidea, although it is clear that they do not belong to the Desmodoridae. The single Molgolaiminae sequence available formed a distinct clade together with the superfamily Microlaimoidea, and should therefore be placed with the latter. Clarifying the phylogenetic relationships within the Desmodoroidea will require greater focus on the Pseudonchinae, Molgolaiminae, and Epsilonematidae, for which no or very few sequences are available at present. © 2016 The Linnean Society of London     

Electron microscopical localization of chitin in the cuticle of Halicryptus spinulosus and Priapulus caudatus (Priapulida) using gold-labelled wheat germ agglutinin: phylogenetic implications for the evolution of the cuticle within the Nemathelminthes

 The ultrastructure of the cuticle of adult and larval Priapulus caudatus and Halicryptus spinulosus is investigated and new features of cuticle formation during moulting are described. For the localization of chitin by TEM wheat germ agglutinin coupled to colloidal gold was used as a marker. Proteinaceous layers of the cuticle are revealed by digestion with pronase. The cuticle of larval and adult specimens of both species consists of three main layers: the outer, very thin, electron-dense epicuticle, the electron-dense exocuticle and the fibrillar, electron-lucent endocuticle. Depending on the body region, the exocuticle comprises two or three sublayers. The endocuticle can be subdivided into two sublayers as well. In strengthened parts such as the teeth, the endocuticle becomes sclerotized and appears electron-dense. Only all endocuticular layers show an intense labelling with wheat germ agglutinin-gold conjugates in all investigated specimens. Additional weak labelling is observed in the exocuticle III layer of the larval lorica of P. caudatus. All other cuticular layers remain unlabelled. Chitinase dissolves the unsclerotized endocuticular layers almost completely, but also exocuticle II and partly the loricate exocuticle III. The epicuticle, the homogeneous exocuticle I and the sclerotized endocuticle are not affected by chitinase. The labelling is completely prevented in all layers after incubation with chitinase. Pronase dissolves all exocuticular layers, but not evenly. The presumably sclerotized regions of exocuticle I are not affected as well as the complete epicuticle and the endocuticle. All cuticular features of the Priapulida are compared with the cuticle of each high-ranked taxon within the Nemathelminthes with special regard to the occurrence of chitin. Based on this out-group comparison it can be concluded that: (1) a two-layered cuticle with a trilaminate epicuticle and a proteinaceous basal layer represents an autapomorphic feature of the Nemathelminthes, (2) the stem species of the Cycloneuralia have already evolved an additional basal chitinous layer, (3) such a three-layered cuticle is maintained as a plesiomophy in the ground pattern of the Scalidophora and (4) in the Nematoida, the chitinous basal layer is replaced by a collagenous one at least in the adults; the synthesis of chitin is restricted to early developmental phases or the pharyngeal cuticle. Accepted: 12 March 1998     

Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus‐growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species‐specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.     

The subepithelial gland in ants: a novel exocrine gland closely associated with the cuticle surface

Two glandular systems were discovered that secrete their products onto the cuticular surface in ants. The first, the subepithelial gland, was previously undescribed in ants, and is found throughout the body just beneath the epithelium. This gland consists of independent secretory units, each made up of a single gland cell and an associated duct cell that penetrates the cuticle. Its ultrastructural appearance is consistent with possible hydrocarbon production. Examining 84 ant species, the subepithelial gland was found in eight subfamilies (out of 13), although not necessarily in all species. In a single ant species, Harpegnathos saltator, it was the epithelium itself that was enlarged and functioned as a gland. The enlarged epithelial cells secrete their products directly onto the cuticle through distinct cuticular crevasses.     

Structure of the integument in Paranthessius anemoniae claus,a copepod associate of the snakelocks anemone Anemonia sulcata (Pennant)

The integument of Paranthessius anemoniae has been studied with light and electron microscopy. A cuticle with clearly defined epicuticular, exocuticular and endocuticular regions overlies a cellular hypodermal layer. The distribution of carbohydrate, lipid and protein components of the cuticle were demonstrated histochemically. Parabolic striations in oblique sections of cuticle suggest that its molecular architecture fits a “twisted sheet” theory proposed for other species. Arthrodial membranes at body and limb joints have a homogeneous structure, lacking exocuticle and endocuticle. Subcuticular glands appear to secrete substances thought to be responsible for the immunity which Paranthessius seems to have to the nematocysts of its host. Small hairs, situated in cuticular cups which occur over the dorsal body surface are considered to function as rheoreceptors.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

The integumental ultrastructure of Lamippe rubra Bruzelius and Enalcyonium rubicundum Olsson (Copepoda,Poecilostomatoida)

The integument of Lamippe rubra Bruzelius and of Enalcyonium rubicundum Olsson has been studied with the electron microscope.Most of the cuticle covering the body of Lamippe is represented by the epicuticle, which shows an average thickness of about 2.0 µm, but in sclerified zones it consists of a thin epicuticle (0.2 µm) and a stratified laminated procuticle (0.5–1.5 µm) without bow-shaped structure. A complex system of epithelial microvilli or a well-developed system of membranes running parallel to the cuticle is also present.The cuticle of Enalcyonium consists of a thin procuticle (0.4–0.5 µm) covered with a uniform fibrillar coat (0.5 µm), whereas in sclerotized areas it is composed of a stratified procuticle (0.7–3.5 µm) with bow-shaped structures.In both species, cuticular hairs and gland vents occur at the dorsal and ventral surfaces. Some of the hairs are considered to be sensory in nature.The cuticular ultrastructure of L. rubra and of E. rubicundum is compared with that of some other copepods.     

The cuticle of the nematode Caenorhabditis elegans: A complex collagen structure

The cuticle of the nematode Caenorhabditis elegans forms the barrier between the animal and its environment. In addition to being a protective layer, it is an exoskeleton which is important in maintaining and defining the normal shape of the nematode. The cuticle is an extracellular matrix consisting predominantly of small collagen-like proteins that are extensively crosslinked. Although it also contains other protein and non-protein compounds that undoubtedly play a significant part in its function, the specific role of collagen in cuticle structure and morphology is considered here. The C. elegans genome contains between 50 and 150 collagen genes, most of which are believed to encode cuticular collagens. Mutations that result in cuticular defects and grossly altered body form have been identified in more than 40 genes. Six of these genes are now known to encode cuticular collagens, a finding that confirms the importance of this group of structural proteins to the formation of the cuticle and the role of the cuticle as an exoskeleton in shaping the worm. It is likely that many more of the genes identified by mutations giving altered body form, will be collagen genes. Mutations in the cuticular collagen genes provide a powerful tool for investigating the mechanisms by which this group of proteins interact to form the nematode cuticle.     

Intraspecific nestmate recognition in two parabiotic ant species: acquired recognition cues and low inter-colony discrimination

Parabiotic ants—ants that share their nest with another ant species—need to tolerate not only conspecific nestmates, but also nestmates of a foreign species. The parabiotic ants Camponotus rufifemur and Crematogaster modiglianii display high interspecific tolerance, which exceeds their respective partner colony and extends to alien colonies of the partner species. The tolerance appears to be related to unusual cuticular substances in both species. Both species possess hydrocarbons of unusually high chain lengths. In addition, Cr. modiglianii carries high quantities of hereto unknown compounds on its cuticle. These unusual features of the cuticular profiles may affect nestmate recognition within both respective species as well. In the present study, we therefore examined inter-colony discrimination within the two parabiotic species in relation to chemical differentiation. Cr. modiglianii was highly aggressive against workers from alien conspecific colonies in experimental confrontations. In spite of high inter-colony variation in the unknown compounds, however, Cr. modiglianii failed to differentiate between intracolonial and allocolonial unknown compounds. Instead, the cuticular hydrocarbons functioned as recognition cues despite low variation across colonies. Moreover, inter-colony aggression within Cr. modiglianii was significantly influenced by the presence of two methylbranched alkenes acquired from its Ca. rufifemur partner. Ca. rufifemur occurs in two varieties (‘red’ and ‘black’) with almost no overlap in their cuticular hydrocarbons. Workers of this species showed low aggression against conspecifics from foreign colonies of the same variety, but attacked workers from the respective other variety. The low inter-colony discrimination within a variety may be related to low chemical differentiation between the colonies. Ca. rufifemur majors elicited significantly more inter-colony aggression than medium-sized workers. This may be explained by the density of recognition cues: majors carried significantly higher quantities of cuticular hydrocarbons per body surface.     

Calcium translocations during the moulting cycle of the semiterrestrial isopod <Emphasis Type="Italic">Ligia hawaiiensis</Emphasis> (Oniscidea,Crustacea)

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO₃ deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. ⁴⁵Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.     

Fine structural observations on the epidermis

Summary In species of Apium, Eryngium and Humulus, the cuticular membrane of the petiole could be resolved into two parts, of which the inner one appeared amorphous and after staining appeared to be penetrated by an electron-dense reticulum, whereas the outer layer showed a lamellate structure consisting of electron-dense and electron-transparent plates, 50–80 Å in thickness. These layers are considered to correspond with the cuticular layer and the cuticle proper, respectively. In species of Abutilon and Rumex the cuticle proper did not exhibit the lamellate structure. In the leaves of Eryngium the outer lamellated structure was present in the cuticle of both young and mature leaves. Both the lamellate and non-lamellate types of the cuticle proper increased in thickness with age of the specimen. The results are discussed in relation to earlier investigations.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

Coordinated development in the fly foot: Sequential cuticle secretion

Development of the adult fly foot falls into clearly defined phases of cell division, growth, cuticle secretion and cell death. The pulvillus is composed dorsally of two giant cells and ventrally of thousands of minute tenent cells; the former produce the dorsal footpad cuticle and the latter the thousands of tenent hairs. Cell divisions are still occurring in future tenent cells when increase in size of the cells and in polyteny of the chromosomes is already occurring in the two dorsal cells. Also cell death occurs considerably earlier in the tenent cells, yet the sequential secretion of some six cuticular layers takes place at comparable times in dorsal and ventral cuticles. The cuticular layers formed are, in their order of secretion: ecdysial membrane, cuticulin of the epicuticle, dense exocuticle, homogeneous exocuticle, an intermediate layer, wax of the epicuticle, and an extensive mass of endocuticle. The ecdysial membrane seems to perform an important mechanical role in maintaining the shape of the delicate cytoplasmic projections of the tenent cells, before and during cuticle secretion, and in establishing the cuticular pattern of ridges in the dorsal cuticle. Comparisons are made with trichogen cell cuticle development and with tracheal cuticle. Tracheal, trichogen and dorsal footpad cuticle patterns are compared. Details of giant cell activity provide a working basis for studies of nuclear-cytoplasmic interactions, and the whole system raises many unsolved problems in the general field of cell differentiation and pattern formation.     

Water composition and loss by body color and form mutants of the German cockroach (Dictyoptera: Blattellidae)

Body water composition and rates of water loss were determined for adult males of five body color or form mutants of the German cockroach, Blattella germanica (L.). Body water content ranged from 68.4% for a yellow body strain to 74.7% for the balloon wing strain. Dark body strains (black and black×orange) contained less water than lighter color strains (orange and yellow). There were no differences in whole-body lipid content among the strains. Cuticular permeability at 30°C ranged from 18.44 to 24.8 μg cm^-2 h^-1 mmHg^-1 for black body and balloon wing strains, respectively; dark body strains had lower cuticular permeabilities than light body strains. Brief, whole-body extraction with hexane increased cuticular permeability from 5.6-fold for the balloon strain to 8.3-fold for the orange body strain. Cuticular permeabilities of hexane-extracted dark body strains again had lower cuticular permeabilities than those of light body strains, indicating that differences in permeability were due to the cuticle itself and not differences in the amount or composition of epicuticular lipids. Measurements of cuticle water content indicated that light body strains contained significantly more water than dark body strains. Rate of percent original mass and percentage of total body water lost increased linearly with time. Implications of cuticular water content to cuticular permeability are discussed.     

Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: A case study with ant cuticular bacteria

High‐throughput sequencing of the 16S rRNA gene has considerably helped revealing the essential role of bacteria living on insect cuticles in the ecophysiology and behaviour of their hosts. However, our understanding of host‐cuticular microbiota feedbacks remains hampered by the difficulties of working with low bacterial DNA quantities as with individual insect cuticle samples, which are more prone to molecular biases and contaminations. Herein, we conducted a methodological benchmark on the cuticular bacterial loads retrieved from two Neotropical ant species of different body size and ecology: Atta cephalotes (~15 mm) and Pseudomyrmex penetrator (~5 mm). We evaluated the richness and composition of the cuticular microbiota, as well as the amount of biases and contamination produced by four DNA extraction protocols. We also addressed how bacterial community characteristics would be affected by the number of individuals or individual body size used for DNA extraction. Most extraction methods yielded similar results in terms of bacterial diversity and composition for A. cephalotes (~15 mm). In contrast, greater amounts of artefactual sequences and contaminations, as well as noticeable differences in bacterial community characteristics were observed between extraction methods for P. penetrator (~5 mm). We also found that large (~15 mm) and small (~5 mm) A. cephalotes individuals harbour different bacterial communities. Our benchmark suggests that cuticular microbiota of single individual insects can be reliably retrieved provided that blank controls, appropriate data cleaning, and individual body size and functional role within insect society are considered in the experiment.     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.