External morphology and developmental changes of tarsal tips and mouthparts of the invasive spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae).

External structures of insects contribute to the ability of herbivores to select and feed on their host plants. The invasive spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae) is an economically important and polyphagous insect pest in the eastern US. The lanternfly causes substantial damage to many woody plants by sucking phloem sap, reducing photosynthesis, causing weeping wounds, and creating conditions for sooty mold. Lanternfly nymphs switch host plants during their development. However, little is known about relationship between the lanternfly and its plant hosts, and particularly about morphological adaptations of the lanternfly to host plant usage at each developmental stage of the pest. In this study, we focused on assessing changes in morphology of (a) the lanternfly mouthparts (stylets and labium), and (b) the lanternfly tarsal tips (arolia and tarsal claws) at each developmental stage. Our study revealed several developmental patterns among which the presence of the indentations on mandibular stylets in late instars and adults, as well as the exponential growth of the labium and stylet length, and the tarsal claw dispersal during the lanternfly development. Our findings are critical for investigating and predicting the lanternfly host range, and the lanternfly dispersal to new host trees at each developmental stage.

Obligate development of Blastocrithidia papi (Trypanosomatidae) in the Malpighian tubules of Pyrrhocoris apterus (Hemiptera) and coordination of host-parasite life cycles.

Blastocrithidia papi is a unique trypanosomatid in that its life cycle is synchronized with that of its host, and includes an obligate stage of development in Malpighian tubules (MTs). This occurs in firebugs, which exited the winter diapause. In the short period, preceding the mating of overwintered insects, the flagellates penetrate MTs of the host, multiply attached to the epithelial surface with their flagella, and start forming cyst-like amastigotes (CLAs) in large agglomerates. By the moment of oviposition, a large number of CLAs are already available in the rectum. They are discharged on the eggs' surface with feces, used for transmission of bugs' symbiotic bacteria, which are compulsorily engulfed by the newly hatched nymphs along with the CLAs. The obligate development of B. papi in MTs is definitely linked to the life cycle synchronization. The absence of peristalsis allow the trypanosomatids to accumulate and form dense CLA-forming subpopulations, whereas the lack of peritrophic structures facilitates the extensive discharge of CLAs directly into the hindgut lumen. The massive release of CLAs associated with oviposition is indispensable for maximization of the infection efficiency at the most favorable time point.

An ungrouped cuticular protein is essential for normal endocuticle formation in the brown planthopper.

Using transcriptome analysis of tissues of the brown planthopper (BPH), Nilaparvata lugens, we identified a gene tentatively designated NlCP21.92 that was expressed at high levels in the integument. Spatiotemporal expression profiling with quantitative PCR and Western blotting verified its integument-specific expression and showed periodic expression during molting. The open reading frame was GC-rich and encoded a hydrophobic polypeptide. The polypeptide contained AAPA/V repeat motifs and other sequence features similar to several reported cuticular proteins but lacked an R&R consensus and other chitin-binding domains. Double-stranded RNA-mediated RNA interference of the NlCP21.92 resulted in abnormal and lethal morphological phenotypes, and transmission electron microscopy revealed the corresponding ultrastructural defects. Immunohistochemical staining demonstrated that the NlCP21.92 protein was primarily located in the procuticle. Our results suggest that NlCP21.92 is a novel ungrouped cuticular protein essential for normal endocuticle formation.

Development of leafhopper cell culture to trace the early infection process of a nucleorhabdovirus, rice yellow stunt virus, in insect vector cells.

BACKGROUND: In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. METHODS: In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. RESULTS: Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. CONCLUSIONS: In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests.

Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests, Laodelphax striatellus and Sogatella furcifera Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the ß-pore-forming toxins. Coexpression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73- resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis, Plutella xylostella, or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from L. striatellus Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants.IMPORTANCE In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers (Laodelphax striatellus) and white-backed planthoppers (Sogatella furcifera). To our knowledge, the proteins encoded by the cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests. These two proteins should be able to be used for integrated hemipteran pest management.

Ultrastructure of spermiogenesis and spermatozoa in Marchalina hellenica (Gennadius) (Hemiptera: Sternorrhyncha, Marchalinidae).

The spermiogenesis, the sperm structure and the sperm motility of Marchalina hellenica (Gennadius) were examined. In the early spermiogenesis a centriolar apparatus was identified, but this structure is not involved in the production of the sperm flagellum. As in other Coccoidea, the flagellar axoneme originates by the activity of the thickened tip of the numerous microtubules surrounding the nuclear anterior region close to the periphery of the cell. This region pushes against a narrow cytoplasmic layer, giving rise to a papilla. In this region a novel structure, consisting of a regular network of thin filaments, arranged orthogonally to the bundle of microtubules, is visible. The sperm flagellum consists of a series of about 260 microtubules, regularly arranged in rings around the axial nucleus. This latter extends in the middle part of the sperm length. As usual in scale insects, sperm form a bundle, which in M. hellenica is composed of 64 sperm cells, surrounded by somatic cyst cells. The sperm bundle has an helicoidal array, with a cap of dense material at its apex, lending the anterior and the posterior region of the sperm bundle with a different structural organization. This difference is responsible of the different speed gradient observed in the helical wave propagating along the sperm bundle.

A mitochondrial membrane protein is a target for rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV; Reoviridae) is exclusively transmitted by the brown planthopper Nilaparvata lugens in a persistent-propagative manner. It is understood that RNA viral proliferation is associated with the intracellular membranes of the insect host cells. However, the molecular mechanisms of the interaction between the RRSV proliferation and the intracellular membranes remain essentially unknown. It will be of great interest to determine whether RRSV protein(s) directly interact with intracellular membrane components of its host cells. In this study, we identified a RRSV nonstructural protein Pns10 interacting with a host oligomycin-sensitivity conferral protein (OSCP) using yeast two-hybrid system. The interaction between RRSV Pns10 and N. lugens OSCP was verified by a glutathione S-transferase pull-down assay. Confocal miscopy revealed colocalization of these two proteins in the cytoplasm of the salivary gland cells during the viral infection. The virions were further detected in the mitochondria under confocal miscopy and transmission electron microscopy combined with western blotting assay. This is the first observation that RRSV protein has a direct link with mitochondria. Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects. These findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane and provide insights concerning the mitochondrial membrane protein-based virus proliferation mode in the insect vector.

Fine structure of antennal sensilla of the spittlebug Philaenus spumarius L. (Insecta: Hemiptera: Aphrophoridae). I. Chemoreceptors and thermo-/hygroreceptors.

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Cercopoidea: Aphrophoridae), is a polyphagous species that transmits Xylella fastidiosa, a bacterium associated with "Olive Quick Decline Syndrome" in Southern Italy. In this study, the morphology and the ultrastructure of the antennal sensilla of P. spumarius were investigated. The antennae consist of three segments: a basal scape, a pedicel and a flagellum composed of a basal enlargement (ampulla) and a long segment (filament). The pedicel bears a single campaniform sensillum while the ampulla houses twelve coeloconic sensilla and three large basiconic sensilla. These latter sensilla show a smooth multiporous external cuticular wall and a total number of 27 sensory neurons per sensillum. The coeloconic sensilla belong to two morphologically distinct types: double-walled and single-walled sensilla. The sensory peg of the double-walled sensilla is smooth at the base and distally has a grooved cuticular surface with pores organized in spoke channels between each ridge. Three sensory neurons enter the lumen while at the basal level, before entering the peg, a fourth sensory neuron is found. The single-walled sensilla show an aporous thick cuticular wall and two sensory neurons entering the sensillar lumen, with a third neuron ending at the sensillum base.

Structure and Sensilla of the Mouthparts of the Spotted Lanternfly Lycorma delicatula (Hemiptera: Fulgoromorpha: Fulgoridae), a Polyphagous Invasive Planthopper.

Mouthparts are among the most important sensory and feeding structures in insects and comparative morphological study may help explain differences in feeding behavior and diet breadth among species. The spotted lanternfly Lycorma delicatula (White) (Hemiptera: Fulgoromorpha: Fulgoridae) is a polyphagous agricultural pest originating in China, recently established and becoming widespread in Korea, and more recently introduced into eastern North America. It causes severe economic damage by sucking phloem sap and the sugary excrement produced by nymphs and adults serves as a medium for sooty mold. To facilitate future study of feeding mechanisms in this insect, the fine-structural morphology of mouthparts focusing on the distribution of sensilla located on the labium in adult L. delicatula was observed using a scanning electron microscope. The mouthparts consist of a small cone-shaped labrum, a tubular labium and a stylet fascicle consisting of two inner interlocked maxillary stylets partially surrounded by two shorter mandibular stylets similar to those found in other hemipteran insects. The five-segmented labium is unusual (most other Fulgoromorpha have four segments) and is provided with several types of sensilla and cuticular processes situated on the apex of its distal labial segment. In general, nine types of sensilla were found on the mouthparts. Six types of sensilla and four types of cuticular processes are present on sensory fields of the labial apex. The proposed taxonomic and functional significance of the sensilla are discussed. Morphological similarities in the interlocking mechanism of the stylets suggest a relationship between Fulgoromorpha and Heteroptera.

Development of ovary structures in the last larval and adult stages of psyllids (Insecta, Hemiptera, Sternorrhyncha: Psylloidea).

The development and organization of the ovaries of ten species from four Psylloidea families (Psyllidae, Triozidae, Aphalaridae and Liviidae) have been investigated. The ovaries of the last larval stage (i.e. fifth instar) of all examined species are filled with numerous clusters of cystocytes which undergo synchronous incomplete mitotic division. Cystocytes of the given cluster are arranged into a rosette with polyfusome in the centre. These clusters are associated with single somatic cells. At the end of the fifth instar, the clusters begin to separate from each other, forming spherical ovarioles which are surrounded by a single layer of somatic cells. In the ovarioles of very young females all cystocytes enter the prophase of meiosis and differentiate shortly thereafter into oocytes and trophocytes (nurse cells). Meanwhile, somatic cells differentiate into cells of the inner epithelial sheath surrounding the trophocytes and into the prefollicular cells that encompass the oocytes. During this final differentiation, the trophocytes lose their cell membranes and become syncytial. Oocytes remain cellular and most of them (termed arrested oocytes) do not grow. In the ovarioles of older females, one oocyte encompassed by its follicle cells starts growing, still connected to the syncytial tropharium by a nutritive cord. After the short phase of previtellogenesis alone, the oocyte enters its vitellogenic the growth phase in the vitellarium. At that time, the second oocyte may enter the vitellarium and start its previtellogenic growth. In the light of the obtained results, the phylogeny of psyllids, as well as phylogenetic relationships between taxa of Hemiptera: Sternorrhyncha are discussed.

Phylloxera (Daktulosphaira vitifoliae Fitch) alters the carbohydrate metabolism in root galls to allowing the compatible interaction with grapevine (Vitis ssp.) roots.

Gall forming phylloxera may compete for nutrients with meristematic tissues and develop heterotrophic structures that act as carbon sinks. In this work, we studied the underlying starch metabolism, sink-source translocation of soluble sugars towards and within root galls. We demonstrated that nodosities store carbohydrates by starch accumulation and monitored the expression of genes involved in the starch metabolic. Thereby we proved that the nodosity is symplastically connected to the source tissues through its development and that the starch metabolism is significantly affected to synthesize and degrade starch within the gall. Genes required for starch biosynthesis and degradation are up-regulated. Among the carbohydrate transporters the expression of a glucose-6-phosphate translocater, one sucrose transporter and two SWEET proteins were increases, whereas hexose transporters, tonoplast monosaccharide transporter and Erd6-like sugar transporters were decreased. We found general evidence for plant response to osmotic stress in the nodosity as previously suggested for gall induction processes. We conclude that nodosities are heterogenous plant organs that accumulate starch to serve as temporary storage structure that is gradually withdrawn by phylloxera. Phylloxera transcriptionally reprograms gall tissues beyond primary metabolism and included downstream secondary processes, including response to osmotic stress.

Approach for determination of ATP:ADP molar ratio in mixed solution by surface-enhanced Raman scattering.

The ATP:ADP molar ratio is an important physiological factor. However, in previous literatures, ATP and ADP could not be distinguished by Raman spectroscopy due to the high similarity of molecular structure. To challenge this problem, also considering that the γ phosphate group may interact with adenine group and cause a different variation of the Raman spectrum than that of ADP, a highly sensitive, low-cost, environment protecting, flexible and super-hydrophobic Au nanoparticles/cicada wing (Au/CW) substrate with three-dimension structure was fabricated and employed as an active surface-enhanced Raman scattering (SERS) substrate to detect the ATP:ADP molar ratios. The concentration as low as 10(-8)M for ATP and ADP was analyzed to determine the limit of detection. This SERS study on various ATP:ADP molar ratios demonstrates that ATP:ADP could be distinguished and the quantitative determination of ATP content was achieved. Moreover, a principle was speculated based on the molecular structures of ATP and ADP of the Raman peaks centered at ~685 and ~731cm(-1) to explain the linear relationship between the area ratio and the molar ratio. A new method has been developed for quantitative determination of ATP:ADP molar ratio based on Au/CW substrate by the SERS method.

The sperm of Matsucoccus feytaudi (Insecta, Coccoidea): Can the microtubular bundle be considered as a true flagellum?

In the present work the spermiogenesis and sperm structure of Matsucoccus feytaudi, a primary pest of the maritime pine in southern eastern Europe, is studied. In addition to the already known characteristics of coccid sperm, such as the absence of the acrosome and mitochondria, and the presence of a bundle of microtubules responsible for sperm motility, a peculiar structure from which the microtubule bundle takes origin is described. Such a structure--a short cylinder provided with a central hub surrounded by several microtubules with a dense wall--is regarded as a Microtubule Organizing Centre (MTOC). During spermiogenesis, quartets of fused spermatids are formed; from each spermatid, a bundle of microtubules, generated by the MTOC, projects from the cell surface. Each cell has two centrioles, suggesting the lack of a meiotic process and the occurrence of parthenogenesis. At the end of the spermiogenesis, when the cysts containing bundles of sperm are formed, part of the nuclear material together with the MTOC structure is eliminated. Based on the origin of the microtubular bundle from the MTOC, the nature of the bundle as a flagellum is discussed.

Ultrastructure of sensory equipments on the heads of Kallitaxila granulata (Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae).

The polyphagous planthopper, Kallitaxila granulata (Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae), has been recently introduced into southeastern China, the Philippine islands, and Hawaii, where it has done significant damage to agricultural and forest ecosystems. The external morphology of the heads of adult male and female K. granulata was examined using scanning electron microscopy. Seven types of sensilla were reported: trichoid sensilla and campaniform sensilla on the antennal pedicel, antennal scape and maxilla; plate organs on the antennal pedicel; coeloconic sensilla in Bourgoin's organ on the expanded flagellar base; ampullaceal sensilla on the antennal pedicel; wavy-pit sensilla on the antennal pedicel and antennal scape; and coin-shaped sensilla on each lateral side of the labium. Evans' organ was described as placoid sensilla sunk into shallow cuticular cavities below the antennae. The external morphology, distribution, and abundance of sensilla located on antennae, maxillae, and labium in K. granulata were illustrated, with a brief discussion of their taxonomic, phylogenetic, and putative functional significance.

Ultrastructural and functional aspects of the spermatheca in the american harlequin bug, Murgantia histrionica (Hemiptera: Pentatomidae)

The spermatheca of Murgantia histrionica (Hahn) was investigated using fluorescence, scanning and transmission electron microscopy. The aim of the study was to elucidate the structure of this organ, pointing out differences between mated and unmated females. Results have shown an elaborated cuticular structure associated with muscular and glandular tissues. The spermatheca is joined with the common oviduct by the spermathecal duct, forming a thin saccular dilation through two consecutive invaginations. The distal part of the organ is formed by a series of two communicating cuticular chambers. The first cylindrical-shaped chamber, corresponding to the coiled region, is wrapped by longitudinal muscular fibers suspended between two cuticular flanges. The contractions of these fibers compress a deformable zone of the cylinder, pumping the sperm toward the spermathecal duct. Without contractions the cylinder results to be isolated from the proximal part of the spermatheca by means of a valve. The second chamber, corresponding to the spermatheca, is made of two parts: a truncated-conical sub chamber, with a constant cuticular thickness, bearing on itself the distal flange, where muscular fibers are attached. The second part is a bulb-like structure wrapped in a glandular epithelium. The secretory units are composed by two cells: a secretory cell and an associated duct cell. Every evacuating duct shows a little reservoir just after the terminal apparatus, and converge inside the distal bulb after a tortuous path. The functional implications of this structure in the reproductive biology of M. histrionica are discussed.

Ultrastructure and redescription of Notozulia entreriana (Berg) (Hemiptera: Cercopidae) / Ultraestrutura e redescrição de Notozulia entreriana (Berg) (Hemiptera: Cercopidae)

A morfologia ultra-estrutural da cigarrinha Notozulia entreriana (Berg) é apresentada. Resultaram 32 ilustrações, utilizando-se microscópio eletrônico de varredura, bem como uma detalhada descrição da espécie.

The ultrastructure of the spittlebug Notozulia entreriana (Berg) is presented. The study resulted in 32 scanning electron microscope photos, with detailed species description.

Mealybug beta-proteobacterial endosymbionts contain gamma-proteobacterial symbionts.

Some insects have cultivated intimate relationships with mutualistic bacteria since their early evolutionary history. Most ancient 'primary' endosymbionts live within the cytoplasm of large, polyploid host cells of a specialized organ (bacteriome). Within their large, ovoid bacteriomes, mealybugs (Pseudococcidae) package the intracellular endosymbionts into 'mucus-filled' spheres, which surround the host cell nucleus and occupy most of the cytoplasm. The genesis of symbiotic spheres has not been determined, and they are structurally unlike eukaryotic cell vesicles. Recent molecular phylogenetic and fluorescent in situ hybridization (FISH) studies suggested that two unrelated bacterial species may share individual host cells, and that bacteria within spheres comprise these two species. Here we show that mealybug host cells do indeed harbour both beta- and gamma-subdivision Proteobacteria, but they are not co-inhabitants of the spheres. Rather, we show that the symbiotic spheres themselves are beta-proteobacterial cells. Thus, gamma-Proteobacteria live symbiotically inside beta-Proteobacteria. This is the first report, to our knowledge, of an intracellular symbiosis involving two species of bacteria.

Electron tomography of insect flight muscle in rigor and AMPPNP at 23 degrees C.

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23 degrees C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23 degrees C reveal many double-headed and some single-headed "lead" bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situ crossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar comparison using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.

Role of MAPs and motors in the bundling and shimmering of native microtubules from insect ovarioles.

Bundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark-field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent-extracted and reactivated. We have studied the nucleotide-specificity and the drug-sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated.

X-ray diffraction and electron microscopy from Lethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol.

The low-angle X-ray diffraction pattern from Lethocerus flight muscle fibres was recorded in rigor or under two conditions that modify crossbridge structure and behaviour, aqueous adenylylimidodiphosphate (AMPPNP) and AMPPNP + calcium in an ethylene glycol-water mixture. The effects on the 38.7 nm layer-line peaks (hk.6) of the diffraction patterns were studied in detail. In aqueous AMPPNP at room temperature, a condition in which rigor tension drops to half without loss of stiffness, the peaks remained nearly as intense as in rigor except for the 10.6, which dropped to half. In 20% (v/v) ethylene glycol-AMPPNP + 100 microM-Ca2+ at 23 degrees C (gly + pnp + Ca), a condition which removed muscle tension but left stiffness close to the rigor value, the 10.6 and 11.6 peaks greatly decreased but the 31.6 remained relatively high. The 14.5 nm meridional peak (00.16) became stronger on addition of AMPPNP and again on adding glycol + calcium. Considered in terms of constructively interfering filaments and crossbridges, the X-ray data indicated a transfer of diffracting crossbridge mass towards the thick filament as relaxation proceeds. We compared the X-ray diffraction patterns and crossbridge structure seen with electron microscopy (EM) under the same chemical conditions. EM and X-ray observations were mutually quite consistent overall. However, X-ray data indicated that more crossbridge mass was stereospecifically related to actin before fixation in the partially relaxed state (gly + pnp + Ca) than was suggested by the disordered crossbridge profiles seen by EM. We conclude that myosin heads at the start of the power stroke may both be closely related to their thick filament origins and form actin-determined attachments to the thin filament.