F-Actin and Tubulin During Spermatogenesis in Gamasid Mites (Acari: Parasitina)

Abstract F-actin and tubulin behaviour was investigated using fluorescence probes and electron microscopy in the course of spermatogenesis in two gamasid mites, Porrhostaspis lunulata Müller (Parasitidae) and Pergamasus truatellus Athias-Henriot (Pergamasidae). In spermatogonia and primary spermatocytes of both species, the proteins were localized mainly in the intercellular bridges and, in lesser quantities, in the cytoplasm. Overall, actin was present along the plasma-lemmal contact sites of the gonial cells. At the beginning of spermatid elongation, actin could be detected in two regions: in perinuclear cytoplasm and under the plasmalemma. Subplasmalemmal actin, visible as threads running along acrosome-adhering protrusions of the nuclear envelope, is supposedly located within the electron-dense material filling the subacrosomal gap. Tubulin was found on both sides of each actin thread; its location was consistent with two sets of microtubules adhering to the inner acrosomal membrane. Their involvement in acrosome shaping is suggested. As spermatid elongation terminated, the previous pattern of proteins disappeared. In Pergamasus, however, actin emerged briefly near the centrifugal ends of spermatids (granular bodies zone). In spermatocyte-containing cysts, actin and tubulin fluorescence (more pronounced in Porrhostaspis) was associated with intercellular junctions between the cyst cells. In both species, diffuse actin fluorescence was also detected in the cytoplasm of cyst cells assembling elongated spermatids; the reaction was intensified at the end of the elongation process, when the cytoplasm of cyst cells aggregated around the centripetal ends of spermatids.     

Microstructural Reconstruction for the Testicular Cysts of Wolf Spider Using 3D Volume Rendering Technique

In insects, the alignment of neighboring spermatid in the late stages is nearly perfect, so that a transverse section of a cyst containing late spermatids transects all the spermatids at approximately the same level. However, the testicular cysts of spiders are spherical, most cysts are arranged in order of increasing maturity from the periphery to the center of the testis. For this reason, it is difficult to observe the whole spermatids within a single microscopic slide and count them. Therefore, we demonstrate microstructural reconstruction technique enabling to count exact number of sperm cells per cyst with aid of 3D volume rendering. For image processing and reconstruction, serially sectioned histologic specimens were scanned with microscopy and 3D images were reconstructed using Amira 5.3.2 software from the image stacks of the germ cells and surrounding testicular cysts subsequentially. With the information gathered by 3D reconstruction, it has finally been counted that exactly 32 (2⁵) cells of the secondary spermatocytes per cyst. This means that most cysts in P. laura contain exactly 64 (2⁶) spermatids or spermatozoa, which presumably arose from four synchronous mitotic and two meiotic divisions. In addition, the number of divisions occurring in a cyst appears to be constant for this spider because it has been known that the number of spermatids per cyst is characteristic for each species.     

F-actin-based extensions of the head cyst cell adhere to the maturing spermatids to maintain them in a tight bundle and prevent their premature release in Drosophila testis

Background  

In Drosophila, all the 64 clonally derived spermatocytes differentiate in syncytium inside two somatic-origin cyst cells. They elongate to form slender spermatids, which are individualized and then released into the seminal vesicle. During individualization, differentiating spermatids are organized in a tight bundle inside the cyst, which is expected to play an important role in sperm selection. However, actual significance of this process and its underlying mechanism are unclear.     

Morphological variability of potato cyst nematodes (Globodera spp.) in the Canary Islands

Thirty populations of potato cyst nematode (Globodera spp.) from the Island of Tenerife and two populations from the UK were assessed for several morphometric and non-morphometric characters thought to discriminate between the species G. rostochiensis and G. pallida. Also 200 cysts from each population were analysed by isoelectric focusing of soluble proteins. Correlation analysis, analysis of variance and principal component analysis were used to investigate relationships between the morphometric characters, how the relationships varied between species and between populations, and which characters were most useful for discriminating between species. The two species differed significantly for each of the four morphometric characters: stylet length, fenestra length, anus-fenestra distance and the number of ridges. The stylet length and fenestra length also showed differences between populations of G. rostochiensis while stylet length and number of ridges showed differences between populations of C. pallida. In general, populations of G. pallida showed greater variation than populations of C. rostochiensis. Principal component analysis of the population means indicated that over 73% of the variation in the characters could be explained by the contrast of stylet and fenestra lengths against the anus-fenestra distance and number of ridges. A plot of the first two principal components separated the two species. Stepwise discriminant analysis provided a linear combination of these four variables which discriminated between the species. Stylet length was found to be the most useful characteristic for distinguishing the species whilst anus-fenestra distance was the least useful.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

INTERACTIVE EFFECTS OF SALINITY AND TEMPERATURE ON PLANOZYGOTE AND CYST FORMATION OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) IN CULTURE1

The factors regulating dinoflagellate life‐cycle transitions are poorly understood. However, their identification is essential to unravel the causes promoting the outbreaks of harmful algal blooms (HABs) because these blooms are often associated with the formation and germination of sexual cysts. Nevertheless, there is a lack of knowledge on the factors regulating planozygote‐cyst transitions in dinoflagellates due to the difficulties of differentiating planozygotes from vegetative stages. In the present study, two different approaches were used to clarify the relevance of environmental factors on planozygote and cyst formation of the toxic dinoflagellate Alexandrium minutum Halim. First, the effects of changes in initial phosphate (P) and nitrate (N) concentrations in the medium on the percentage of planozygotes formed were examined using flow cytometry. Second, two factorial designs were used to determine how salinity (S), temperature (T), and the density of the initial cell inoculum (I) affect planozygote and resting‐cyst formation. These experiments led to the following conclusions: 1. Low P/N ratios seem to induce gamete expression because the percentage of planozygotes recorded in the absence of added phosphate (‐P) was significantly higher than that obtained in the absence of added nitrogen (‐N), or when the concentrations of both nitrogen and phosphate were 20 times lower (N/20 + P/20). 2. Salinity (S) and temperature (T) strongly affected both planozygote and cyst formation, as sexuality in the population increased significantly as salinity decreased and temperatures increased. S, T combinations that resulted in no significant cyst formation were, however, favorable for vegetative growth, ruling out the possibility of negative effects on cell physiology. 3. The initial cell density is thought to be important for sexual cyst formation by determining the chances of gamete contact. However, the inoculum concentrations tested did not explain either planozygote formation or the appearance of resting cysts.     

Fine structural reconstruction on the testicular cyst of the furrow orb weaver,Larinioides cornutus by 3D volume rendering

Morphological study on spermatids and spermatozoa have long been performed regarding various changes of cell organelles during spermiogenesis as a potential phylogenetic inference. Based on the fact that the number of germ cells per cyst increases according to a geometric series, knowing the exact number of germ cells in a certain stage may lead to the total number of sperms produced per cyst. In spiders, however, the entire process takes place in a cyst represented by a spermatogonium, producing sperms in spherical shape. It is very difficult to count the exact number of germ cells produced per cyst through a 2D image analysis. Therefore, we applied a 3D image of testicular cyst of an orb-weaving spider to visualize the exact number of germ cells produced from a cyst. In this study, 2D images obtained from serially sectioned micrographs were scanned precisely and reconstructed using a 3D-rendering technique. Finally, this research reveals that the exact number of spermatozoa produced each cyst in Larinioides cornutus appeared to be 128 (2⁷), which indicates that a single spermatogonium undergoes five mitotic divisions and two maturing divisions (meiosis) to produce final spermatozoa.     

Spermatogenesis in the inseminating African butterflyfish Pantodon buchholzi (Teleostei: Osteoglossiformes: Pantodontidae) with the revision of residual bodies formation

The aim of this study was to analyse spermatogenesis in the African butterflyfish, Pantodon buchholzi, using transmission electron microscopy and scanning electron microscopy. P. buchholzi is the most basal teleost that exhibits insemination and produces a highly complex introsperm with the most elongate midpiece known in teleost fishes. Their early stages (spermatogonia and spermatocytes) do not differ greatly from those of other fishes, with the exception of Golgi apparatus degradation appearing as spindle-shaped bodies (SSBs). In round, early spermatids, the development of the flagellum begins after the migration of the centriolar complex towards the nucleus. Later, the elongation of the midpiece coincides with the displacement of the mitochondria and their fusion to produce nine mitochondrial derivatives (MDs). In these spermatids, the nucleus is situated laterally to the midpiece, with condensing chromatin in the centre of the nucleus. Within the midpiece, the flagellum is located within a cytoplasmic canal and is surrounded by a cytoplasmic sleeve containing fibres, MDs and a great amount of cytoplasm located on one side. During the next phase, nuclear rotation, the highly condensed chromatin is displaced to a position above the centriolar apparatus, whereas chromatin-free nucleoplasm is transferred to the cytoplasm. Later, this nucleoplasm, still surrounded by the nuclear membrane, is eliminated into the cyst lumen as the nucleoplasmic packet. Within the highly elongate spermatids, other excess organelles (SSBs, endoplasmic reticulum and mitochondria) are eliminated as residual bodies (RBs). Fully developed spermatozoa, which contain conical-shaped nuclei, eventually coalesce to form unencapsulated sperm packets (spermatozeugmata) that are surrounded by RBs at the level of the extremely elongate midpieces. Later, RBs are removed at the periphery of the cyst by means of phagocytosis by Sertoli cells.     

The ultrastructure of spermatid development during spermiogenesis within the rosebelly lizard,Sceloporus variabilis (Reptilia,Squamata, Phrynosomatidae)

Several recent studies have mapped out the characters of spermiogenesis within several species of squamates. Many of these data have shown both conserved and possibly apomorphic morphological traits that could be important in future phylogenetic analysis within Reptilia. There, however, has not been a recent study that compares spermiogenesis and its similarities or differences between two species of reptile that reside in the same genus. Thus, the present analysis details the changes to spermiogenesis in Sceloporus variabilis and then compares spermatid morphologies to that of Sceloporus bicanthalis. Many of the morphological changes that the spermatids undergo in these two species are similar or conserved, which is similar to what has been reported in other squamates. There are six main character differences that can be observed during the development of the spermatids between these two sceloporid lizards. They include the presence (S. variabilis) or absence (S. bicanthalis) of a mitochondrial/endoplasmic reticulum complex near the Golgi apparatus during acrosome development, a shallow (S. variabilis) or deep (S. bicanthalis) nuclear indentation that accommodates the acrosomal vesicle, filamentous (S. variabilis) or granular (S. bicanthalis) chromatin condensation, no spiraling (S. variabilis) or spiraling (S. bicanthalis) of chromatin during condensation, absence (S. variabilis) or presence (S. bicanthalis) of the longitudinal manchette microtubules, and the lack of (S. variabilis) or presence (S. bicanthalis) of nuclear lacunae. This is the first study that compares spermiogenic ultrastructural characters between species within the same genus. The significance of the six character differences between two distantly related species within Sceloporus is still unknown, but these data do suggest that spermiogenesis might be a good model to study the hypothesis that spermatid ontogeny is species specific. J. Morphol. 275:258–268, 2014. © 2013 Wiley Periodicals, Inc.     

Protoperidinium tricingulatum sp. nov. (Dinophyceae), a new motile form of a round,brown, and spiny dinoflagellate cyst

A small, broadly ovoidal and heterotrophic dinoflagellate containing round, brownish, and spiny cyst was found in the water column of Huibertsplaat in the Wadden Sea off the coast of the Netherlands. This dinoflagellate had these conspicuous morphological characters: a five‐sided first apical plate (1′), only three cingular plates, and an extremely small first antapical plate. Based on these morphological features, Protoperidinium tricingulatum Kawami, vanWezel, Koeman et Matsuoka is described as a new species. The flagellar pore of P. tricingulatum is covered with a small fin, which rises from the left side of the right sulcal plate to the large V‐shaped posterior sulcal plate. This feature suggests that P. tricingulatum is assigned to the Abé's Monovela Group. The cyst stage of P. tricingulatum was positively linked to the vegetative stage by comparison of the ribosomal 5.8S rDNA, internal transcribed spacers (ITS1 and ITS2). Living cysts of P. tricingulatum are round, brownish, and covered with many slender spines bearing capitate or cauliforate distal ends. The cyst also possesses a theropylic archeopyle formed by a slit corresponding to parasutures between three apical and two apical intercaraly plates. These morphological characters indicate that this species is morphologically related to two dinoflagellate cyst‐genera Islandinium and Echinidinium.     

Two new Australian species of the Paraulopus nigripinnis complex (Aulopiformes: Paraulopidae)

Two new species, Paraulopus longianalis n.sp. and Paraulopus melanostomus n.sp., are described from western and southern Australian waters. Both are referable to the Paraulopus nigripinnis group of Sato and Nakabo (2002b, 2003) in having 4.5–5.5 scales above the lateral line, supraocular ridges and large adult body size, but differ from other species of the group in having two rows of cheek scales. Paraulopus longianalis is characterized by a deep anal fin in males, tiny adipose dorsal fin and supraocular ridges extending nearly to the predorsal scales, and P. melanostomus is most easily distinguished by its black buccal cavity and relatively broad, depressed head. A key to the six described species in the P. nigripinnis group is provided.     

Cytology of lepidoptera. III. Giant cysts: A morphological trait of apyrene spermatogenesis in an Ephestia kuehniella strain

A comparative investigation of testicular eupyrene cysts (in larvae) and apyrene cysts (in pupae) of Ephestia kuehniella laboratory strains was conducted using light and electron microscopy. Eupyrene cysts in the first meiotic division contained 64 spermatocytes, which showed only moderate asynchrony. In one of the strains, a wild-type strain, L, normal-sized cysts occurred together with abnormally large cysts. These are called giant cysts in this article. One of the premeiotic cysts, early giant cysts, studied in detail, contained approximately a fourfold number of cells compared with the number in a eupyrene cyst of the same stage. In cysts harboring spermatocytes and spermatids, late giant cysts, cell differentiation was highly asynchronous. Failure in one of two control mechanisms in early cyst development may have caused the appearance of the cysts. Control of cell division might have been sloppy in apyrene spermatogonia. Hence, the spermatogonia within the cyst could have passed through additional division cycles. Alternatively, the giant cysts may have originated from more than one predefinitive gonial cell enclosed in a common envelope of sheath cells. As a third possibility, giant cysts could have arisen by fusion of normal cysts at a later stage. In either case, this is evidence that separation of eupyrene and apyrene pathways is earlier than was previously expected. In two other Ephestia strains, apyrene sperm development proceeded without formation of giant cysts. One was a mutant strain, a, and the other one was a recently established wild-type strain, Sbr. Apyrene sperm development is considered an example of degenerate evolution in which enhanced variability between species and even between populations of one species is a common phenomenon.     

Cytoplasmic bridges,intercellular junctions,and individualization of germ cells during spermatogenesis in Dermatobia hominis (Diptera: Cuterebridae)

During mitotic and meiotic divisions in Dermatobia hominis spermatogenesis, the germ cells stay interlinked by cytoplasmic bridges as a result of incomplete cytokinesis. By the end of each division, cytoplasmic bridges flow to the center of the cyst, forming a complex, called the fusoma. During meiotic prophase I, spermatocytes I present desmosome-like junctions and meiotic cytoplasmic bridges. At the beginning of spermiogenesis, the fusoma moves to the future caudal end of the cyst, and at this time the early spermatids are linked by desmosome-like junctions. Throughout spermiogenesis, new and sometimes broad cytoplasmic bridges are formed among spermatids at times making them share cytoplasm. In this case the individualization of cells is assured by the presence of smooth cisternae that outline their structures. The more differentiated spermatids have in addition to narrow cytoplasmic bridges, plasmic membranes junctions. By the end of spermiogenesis, the excess cytoplasmic mass is eliminated leading to spermatid individualization. Desmosome-like junctions of spermatocytes I and early spermatids appear during the fusoma readjustment and segregations; on the other hand, plasmic membrane junctions appear in differentiating spermatids and are eliminated along with the cytoplasmic excess. These circumstances suggest that belt desmosome-like and plasmic membrane junctions are involved in the maintenance of the relative positions of male germ cells in D. hominis while they are inside the cysts. © 1996 Wiley-Liss, Inc.     

The amount of heterochromatic proteins in the egg is correlated with sex determination in <Emphasis Type="Italic">Planococcus citri</Emphasis> (Homoptera,Coccoidea)

In the mealybug Planococcus citri, there are no identifiable sex chromosomes. Early in the development of embryos destined to become males, the genome contributed by the sperm undergoes heterochromatization and, following an inverted type of meiosis, will be eliminated. Only two vital sperms are therefore produced, both carrying the same maternally derived genome. A differential distribution observed on the two spermatids during male germline cyst formation of chromatin remodeling factors such as HP1 and methylated K9 histone H3 prompted us to propose an imprinting/sex determination model in which the imprinted sperm is the one to undergo heterochromatization at syngamy. The sex ratio is normally 1:1, but aged females are known to produce almost exclusively male progeny, suggesting that the imprinting pattern of the male gamete in P. citri, though necessary, is apparently not sufficient for sex determination. We report here that egg cells of aged females show larger amounts of HP1 and Su(Var)3–9 than egg cells of young females. These data suggest that a determinant of sex may be the amount of maternally derived heterochromatic proteins.     

Structure and development of sperm bundles in the dragonfly Aeshna juncea L. (Odonata)

A re-examination of the origin and development of sperm bundles in aeshnid dragonflies (Odonata, Anisoptera) was carried out using light and electron microscopy. During their elongation, intracyst spermatids of the testis of the dragonfly Aeshna juncea L. form a slender cytoplasmic protrusion, the acrosomal conicoid, beyond the nucleus and acrosome rodlet. Gathering and parallel alignment of the transforming spermatids into a tight bundle take place inside the cyst. The original, rigid spermatid foreparts eventually associate, initially by becoming adhesive and swelling progressively to intertwine, and thus come to constitute a cap that binds together all sperm heads within a cyst in a spermatodesma. The development of the spermatodesma seems to occur disjunct from somatic cyst cells. Bundled in this form, the sperms are transferred to the intratestis canal and moved down the spermiduct to the seminal vesicle. They are then forwarded to the male copulatory apparatus, from which they are transmitted to the female. Individual, fully formed sperms are seen to be liberated from the bundle when in the female receptaculum seminis. The remnant of the cytoplasmic acrosomal conicoid, which is considered an envelope of the acrosome rodlet, is then dissolved. The spermatodesmata are large sperm aggregates that constitute efficient vehicles for transmission of amounts of filamentous sperm to the female. J. Morphol. 235:239–247, 1998. © 1998 Wiley-Liss, Inc.     

Gap junctions between germ and somatic cells in the testis of the moth,Anagasta küehniella (Insecta: Lepidoptera)

Summary Heterocellular gap junctions were demonstrated in germ cysts of the moth Anagasta küehniella (Lepidoptera). They conjoin peripheral germ cells of a cyst and cells of their envelope. Their morphology differs according to the developmental stage of the germ cell involved. While gap junctional profiles are flat in cysts of gonia, in cysts of early spermatocytes they appear as button-like structures, the germ cell indenting the corresponding cyst cell. In cysts of late spermatocytes and of young spermatids, they are very numerous and often located at the extremity of conical protrusions of the germ cell. On the germ cell side, cytoplasmic microfilaments are associated with the junctional differentiation. Gap junctions are observed as being pinched off from the surface of the spermatids and, correspondingly, gap vesicles are found in the cyst cells. This, together with the fact that gap junctions are not found at later stages of development, suggests that internalization of the gap junctions might take place before elongation of the spermatids. The potential importance of these germsomatic cell gap junctions is evaluated in light of recent physiological findings obtained by other authors on the oocyte-cumulus system and also in relation with some particularities in the development of the male germ cells in Lepidoptera.     

Spermiogenesis in the horseshoe crab,Limulus polyphemus

Groups of spermatids of Limulus polyphemus undergo differentiation in thin-walled cysts within the seminiferous tubules. The nucleus compacts to a spherical shape, but retains a much less condensed nuclear appendage, whose unique pores are each surrounded by a microtubule. The appendage, unmodified mitochondria, glycogen, and coated vesicles, all present in the mature spermatozoon, suggest an unusual degree of metabolic self-sufficiency of the cell. The acrosome is associated with a 50 μ-long acrosomal filament that penetrates the nucleus during spermiogenesis and coils up in the cytoplasm, enveloped by two outer nuclear membranes. The filament, which eventually comes to lie in the circumnuclear cisterna, retains a covering of one membrane during its discharge at the time of the acrosome reaction. The posterior region of the head forms a thin-walled collar with peculiar internal supports around the base of the flagellum. Serverance of intercellular bridges between spermatids, cytoplasm elimination, and rupture of the cyst precede liberation of the immature spermatozoa into the lumen of the seminiferous tubules. Notwithstanding its peculiarities, the Limulus spermatozoon, with its simple shape closely resembling that of annelids and molluscs, represents the most primitive arthropod spermatozoon congruent with the evolutionary stability of the xiphosurans.     

Patterns of vertical cyst distribution and survival in 100-year-old sediment archives of three spring dinoflagellate species from the Northern Baltic Sea

The history of expansion of bloom-forming cold water dinoflagellates in the Northern Baltic Sea was studied using 100-year-old sediment archives of their resting cysts. Vertical cyst distributions of Biecheleria baltica and Apocalathium malmogiense, two dinoflagellates indistinguishable by light microscopy and not recognized as distinct species in monitoring, and chain-forming Peridiniella catenata were analysed in Pb²¹⁰ and Cs¹³⁷ dated layers of a sediment core from deep, hypoxic accumulation bottoms of the Gulf of Finland. Cyst profiles showed that B. baltica and A. malmogiense were already present in the Baltic spring phytoplankton community at the beginning of the 20th century. This confirms that B. baltica, which was only recognized in the late 1980s, is a native species in the area. A drastic increase in B. baltica cyst concentrations in the 1930s to 1960s coincided with the acceleration of anthropogenic eutrophication. Large cyst deposits accumulated over several decades in the sediment which, by the 1980s, amounted to the seed stock necessary to inoculate dominant blooms. In the cyst records A. malmogiense always contributed a minor fraction of the two species. P. catenata had a relatively short cyst record in Gulf of Finland sediments despite demonstrated long-term presence in the plankton, which emphasizes that cyst-based historic surveys are not suitable for all cyst-forming dinoflagellates. This was corroborated by correspondence analyses of long-term plankton and cyst records which validated the trends from the sediment archive for B. baltica and A. malmogiense, but failed to do so for P. catenata. Germination experiments with 100-year-old cysts revealed a remarkable long-term survival capacity of A. malmogiense, making this species a suitable model for resurrection studies testing adaptation in heavily impacted systems such as the Baltic Sea.     

Functional morphology of the gastric mills of carnivorous,omnivorous, and herbivorous land crabs

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.