A revision of the Macrobiotus hufelandi group (Tardigrada, Macrobiotidae), with some observations on the taxonomic characters of eutardigrades

This study considers specimens of Macrobiotus hufelandi C. A. S. Schultze, 1834 collected from Italy and Germany, including the type locality. In addition to a qualitative analysis of the animals and eggs, statistics were performed on the measurements of the sclerified parts of the animals. The data illustrate low intra-species variability in the morphology of both the animals and the eggs. Differences in egg shell morphology, previously attributed to the variability of M. hufelandi, were found to fall into distinct types, related to different animal morphotypes. The data also indicate that M. hufelandi contains several new species: M. macrocalix sp.n., M. sandrae sp.n. and M. terminalis sp.n.; M. hufelandi is redescribed, and a neotype is assigned.     

Tardigrades of the Australian Antarctic Territories: the Windmill Islands, East Antarctica

Six species of tardigrades, Pseudechiniscus suillus, Macrobiotus sp., Hypsibius antarcticus, Ramajendas frigidus, Diphascon chilenense and Diphascon pingue were extracted from mosses and lichens from the ice-free regions of the Windmill Islands near Casey Base, East Antarctica. Significant positive associations were found between the three common species ( Pseudechiniscus suillus, Hypsibius antarcticus, Diphascon chilenense ) and bryophytes, whereas strong negative associations were found between these species and algae and lichens. There were additional interspecific associations between the common species of tardigrades as well as between tardigrades, nematodes and rotifers.     

Leaf structure and anatomy as related to leaf mass per area variation in seedlings of a wide range of woody plant species and types

The structural causes of variation in leaf mass per area, and of variations in leaf structure accounted for by leaf habit and life form, were explored in a set of laboratory-grown seedlings of 52 European woody species. The leaf traits analysed included density, thickness, saturated mass/dry mass, and leaf nitrogen per mass and per area. Other traits described the anatomy of leaves, most of them relating to the lamina (proportions of palisade and spongy parenchymata, epidermis, air space and sclerified tissues, expressed as volume per leaf area, and per-cell transversal areas of epidermis and parenchymata), and another referring to the mid rib (transversal section of sclerified tissues). Across the whole set of species leaf mass per area was correlated with leaf density but not with thickness, and this was confirmed by taxonomic relatedness tests. Denser leaves corresponded with greater proportion of sclerified tissues in the lamina, smaller cells and lower water and N contents, but no relation was found with the proportion of air space in the lamina. Taxonomic relatedness analysis statistically supported the negative association of leaf density with saturated to dry leaf mass ratio. Thicker leaves also exhibited greater volume per leaf area and greater individual cell area in each of the tissues, particularly parenchyma. Mean leaf mass per area and leaf thickness were lower in deciduous than in evergreen species, but no significant differences in leaf density, proportion of sclerified tissues in the lamina or cell area were found between the two groups. Leaf mass per area was higher in trees and subshrubs than in shrubs and climbers-plus-scramblers, this rank being equal for leaf density and proportion of sclerified tissues in the lamina, and reversed for cell area. Given the standardised environment and ontogenetic phase of the seedlings, we conclude that variation in leaf structure and anatomy among species and species groups has a strong genetic basis, and is already expressed early in the development of woody plants. From an ecological viewpoint, we can interpret greater leaf mass per area across this species set as greater allocation to support and defence functions, as shown predominantly by species from resource-poor environments. Received: 16 August 1999 / Accepted: 29 March 2000     

Population density and species composition of moss-living tardigrades in a boreo-nemoral forest

This study investigates for the first time the tardigrade fauna in a variety of different mosses from a coniferous forest and an adjacent clear-cut area in southern Sweden. Tardigrades were found in a majority of the samples. Sixteen species were recorded, of which the cosmopolitan species Macrobiotus hufelandi was the far most common. Some mosses, particularly species with "wefts" growth form, contained more tardigrades than other mosses, indicating that growth form may have an impact on tardigrade abundance. Mosses of the same species collected from a forest and from a clear-cut, respectively, did not show a general trend in the overall abundance of tardigrades, but the forest tended to contain more species. Five species of tardigrades ( Murrayon dianae, Isohypsibius sattleri, Platicrista angustata, Diphascon belgicae and Diphascon pingue ) never previously reported from Sweden were recorded.     

Putting the Biological Species Concept to the Test: Using Mating Networks to Delimit Species

Although interfertility is the key criterion upon which Mayr’s biological species concept is based, it has never been applied directly to delimit species under natural conditions. Our study fills this gap. We used the interfertility criterion to delimit two closely related oak species in a forest stand by analyzing the network of natural mating events between individuals. The results reveal two groups of interfertile individuals connected by only few mating events. These two groups were largely congruent with those determined using other criteria (morphological similarity, genotypic similarity and individual relatedness). Our study, therefore, shows that the analysis of mating networks is an effective method to delimit species based on the interfertility criterion, provided that adequate network data can be assembled. Our study also shows that although species boundaries are highly congruent across methods of species delimitation, they are not exactly the same. Most of the differences stem from assignment of individuals to an intermediate category. The discrepancies between methods may reflect a biological reality. Indeed, the interfertility criterion is an environment-dependant criterion as species abundances typically affect rates of hybridization under natural conditions. Thus, the methods of species delimitation based on the interfertility criterion are expected to give results slightly different from those based on environment-independent criteria (such as the genotypic similarity criteria). However, whatever the criterion chosen, the challenge we face when delimiting species is to summarize continuous but non-uniform variations in biological diversity. The grade of membership model that we use in this study appears as an appropriate tool.     

Sperm utilisation by Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) queens subjected to multiple mating

Summary In most Hymenoptera species the queen mates once but in a small number of species, multiple matings can occur normally. So, in this study, physogastric M. quadrifasciata queens were mated with a second male to investigate how these queens, naturally inseminated and laying eggs, use spermatozoa stored in their spermatheca, when they are mated with a second male. Results demonstrate that spermatozoa of different males mix in the spermatheca of M. quadrifasciata queens and that there is a gradual increase in the utilisation of spermatozoa of the second male, which could be explained by a competition among spermatozoa of different drones over the way in which spermatozoa are stored in the spermatheca.     

First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships

Methods for microbial classification are not always capable of distinguishing between isolates at the species level. We have previously characterised four Ferroplasma isolates that were >98.9% similar at the 16S rDNA level, the isolates showed marked phenotypic differences, and one isolate was borderline on the 70% species boundary from DNA-DNA similarity data. In this study we have used statistical comparisons of two-dimensional polyacylamide gel electrophoresis gels for classification of closely related isolates. From the protein profile similarities an un-rooted tree was constructed that was congruent with a tree derived from DNA-DNA similarities.     

Estimation of the potential fertility based upon non-return rates of bulls: using polyacrylamide gel instead of cervical mucus in the sperm penetration test

In the present study, we aimed to develop a polyacrylamide gel that could be used instead of bovine cervical mucus in the cervical mucus penetration test (CMPT) to obtain coherent and replicable results in bulls. The frozen semen samples of six Holstein bulls, which were divided into two fertility groups as low and high according to their non-return rate (NRR), were used. In this study, the modified CMPT (mCMPT) was carried out within 0.25 mL transparent plastic straws with an inner diameter 1.7 mm. The penetration ability of spermatozoa to bovine cervical mucus and to polyacrylamide gels swollen with two different solutions [NaCl (G1) and PBS (G2)] was compared. For the penetration test, the straws filled with cervical mucus and both gels were dipped into thawed semen samples and incubated at 37 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, the frozen straws were cut at 1.5-1.75 cm (penetration distance range=PDR1), 3.25-3.5 cm (PDR2) and 5.0-5.25 cm (PDR3), beginning from open-end of the straws. The separated frozen parts were then immediately transferred onto special counting slides by pushing with a mandrel and left to thaw. Thawed samples were covered with cover glass and penetrated spermatozoa in these parts were counted. The relation between the results and fertility of bulls was determined. In the tests performed using mucus, the number of spermatozoa determined in the high fertility group was found to be higher at PDR3 (p<0.0001) compared to the low fertility group, while in G1 spermatozoa number was significantly higher at PDR1 and PDR3 (p<0.0001). However, in G2 medium, no significant difference was observed between either of the fertility groups with respect to spermatozoa number determined at all distance ranges. In the study, we have determined that the gel swollen with NaCl produces better results and this gel can be used instead of bovine cervical mucus for the CMPT. Therefore, we have concluded that the penetration test performed by polyacrylamide gel swollen with NaCl can be a suitable technique for estimation of the potential fertility of bull spermatozoa.     

Host habitat patchiness and the distance decay of similarity among gastro-intestinal nematode communities in two species of <Emphasis Type="Italic">Mastomys</Emphasis> (southeastern Senegal)

Beta-diversity, or how species composition changes with geographical distance, has seldom been studied for different habitats. We present here quantitative estimates of the relationship between geographic distance and similarity of parasitic nematode communities in two closely related rodent host species that live in habitats with very different spatial configurations. In southeastern Senegal Mastomys natalensis lives exclusively inside human villages whereas M. erythroleucus is continuously distributed outside villages. Both host species and their gastro-intestinal nematodes were sampled on the same spatial scale. Beta-diversity was found to be higher in parasite communities of M. erythroleucus than in those of M. natalensis, and significantly related to geographic distance in this first species. Even on the local spatial scale studied, host dispersal limitation, and stochastic events, may affect species turnover in nematode communities of M. erythroleucus. In M. natalensis, no relationship was found between geographic distance and nematode community similarity, however, suggesting low host dispersal rates between habitat patches. Together with previous population genetic results, this study illustrates the need for different approaches with regard to dispersal in natural populations and its effect on biodiversity.     

Sperm ultrastructure of an oviparous and an ovoviviparous onychophoran species (Peripatopsidae) with some phylogenetic considerations

The spermatozoa of the Australian oviparous Ooperipatellus insignis and the South African ovoviviparous Opisthopatus cinctipes (both: Onychophora, Peripatopsidae) were studied and compared with the spermatozoal patterns already described in the taxon. The spermatozoa of both species conform with the general plan described for the Onychophora: they are filiform cells formed, in sequence, by an elongated, fully condensed nucleus capped by an acrosome and surrounded by several spiral ridges; by a mitochondrial midpiece characteristically interpolated between the nucleus and a characteristic flagellum. Major differences between the spermatozoa of both species concern their acrosome organization. The correlation between the acrosomal pattern and the size and structure of the ovarial eggs (oocytes) in onychophorans has been investigated. A parsimony analysis was performed on 21 spermatozoal characters of the species considered. Its results are congruent with those of the traditional systematics. A new set of autapomorphies characterising onychophoran sperm is suggested and some of the spermatological homologies proposed between Onychophora and Euclitellata spermatozoa are critically discussed. Our analysis suggests that spermatozoal characters are good phylogenetic markers among onychophorans, also at low taxonomic level.     

Foliar sclereids in the Magnoliaceae

The foliar sclereids in 136 species representing 11 of the 12 genera of Magnoliaceae were studied and compared. Sclereids occur in four different cell assemblages within the leaf: diffuse idioblasts, mesophyll, dermal system, and the vein sheath including terminal elements. Tropical members tend to have the most highly sclerified leaves. In species of Manglietia the leaves have sclerified spongy mesophyll and either sclerified epidermis or hypodermis. In Talauma , sclerification affects the vein sheath and terminal cells of veinlets, with a specialized thick marginal vein in the Asian taxa but not in the American ones. Liriodendron and the magnolias native to the north temperate zone have only minimal sclerification, which usually is confined to the vein sheath of the midrib and the main lateral veins, and as idioblastic sclereids in the petiole and the midrib near the base of the blade. The two largest genera Magnolia and Michelia are heterogeneous and include species which vary as to their combinations of sclerified elements among the four possible types.     

Regional genetic variation in the major sperm protein genes of Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea)

Onchocerca volvulus and Mansonella ozzardi are two human filarial parasites present in South and Central America. In the Brazilian Amazonia they are found in sympatry, and the lack of clear morphological diagnostic characters in the microfilariae hinders their identification. The major sperm protein (MSP) gene of both species has been sequenced and characterised to determine its potential as a molecular diagnostic character. The length of the MSP gene is different in each species, and this could be used to detect and differentiate them by running the polymerase chain reaction (PCR) product in an agarose gel. Two major gene groups were identified in O. volvulus with a genetic distance of 6% between them. In M. ozzardi only one major group of genes was observed. The high similarity between the protein amino acid sequence of both filarial species confirms that the MSP has been highly conserved through nematode evolution.     

Early puberty in local Naga boar of India: assessment through epididymal spermiogram and in vivo pregnancy

Male Naga pig of India, a miniature breed is known for its meat quality and early puberty. No scientific efforts were made to verify the farmers' view that this breed reaches puberty at around 2 months of age. A preliminary study was, therefore, conducted with the objectives: (a) to find out the age at puberty based on mature spermiogram and in vivo pregnancy and (b) to record the sperm morphology in different parts of the epididymis. Animals were selected from two different age groups: group I aged 53 days and 2.4 kg and group II of 85 days and 3.0 kg. Semen samples collected from different sections of epididymis were analyzed for sperm motility, live spermatozoa, and morphological abnormalities. Motility increased (P<0.01) and live spermatozoa and total morphological abnormalities decreased (P<0.001) from caput through cauda epididymis in both the groups. Sperm motility, live spermatozoa and morphologically normal spermatozoa in each section of the epididymis were higher (P<0.01) in group II than I. Boars with >60% progressive motility, >70% live spermatozoa, <15% total morphological abnormalities and <10% abnormal acrosomes in cauda epididymal spermatozoa were considered mature spermiogram. As per this definition, pigs of group II had only mature spermiogram. In vivo pregnancy confirmation indicated that Naga boar could impregnate female as early as 90 days of age. In conclusion, Naga boar attained puberty by not later than 3 months with 3.0 kg, which is the lowest body weight at puberty in this species reported so far, as reflected by mature epididymal spermiogram and in vivo pregnancy confirmation.     

Potent humanin analogue (HNG) protects human sperm from freeze-thaw-induced damage

This study was aimed to investigate the protective effect of potent humanin analogue (HNG) supplementation to freezing media on freezing-thawing induced human sperm damage. We collected semen samples with normal sperm parameters from 15 healthy men. After the swim-up processing, the motile spermatozoa from each of the men were allocated to four equal groups: In the control group, the spermatozoa were frozen in media without HNG supplementation. In the other three groups, the spermatozoa were frozen in media supplemented with different concentrations of HNG (2 μM, 10 μM and 20 μM, respectively). We analyzed the sperm motility, viability, sperm mitochondrial membrane potential, apoptosis, sperm DNA fragmentation index (DFI), reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and caspase-3 activity for the sperm in each group. As a result, supplementation of HNG with 2 μM, 10 μM and 20 μM to the freezing media all significantly improved sperm motility and viability (all p < 0.05) when compared with the control group. Similarly, we found that supplementation of HNG reduced the damage to the mitochondrial membrane and DNA integrity, and inhibited the reaction of oxidative stress and the activity of caspase-3 in sperm. Although these protective effects increased with the elevated concentration of HNG in the freezing media, a final HNG concentration of 20 μM failed to exert significant improvements when compared with the concentration of 10 μM (all p > 0.05). In conclusion, our results suggested that HNG supplementation to the freezing media could protect sperm cells from freezing-thawing induced sperm damage.     

Disruption of interfascicular fiber differentiation in an Arabidopsis mutant.

Arabidopsis develops interfascicular fibers in stems for needed support of shoots. To study the molecular mechanisms controlling fiber differentiation, we isolated an interfascicular fiber mutant (ifl1) by screening ethyl methanesulfonate-mutagenized Arabidopsis populations. This mutant lacks normal interfascicular fibers in stems. Interestingly, some interfascicular cells were sclerified in the upper parts but not in the basal parts of the ifl1 stems. These sclerified cells were differentiated at a position different from that of interfascicular fibers in the wild type. Lack of interfascicular fibers correlated with a dramatic change of stem strength. Stems of the mutant could not stand erect and were easily broken by bending. Quantitative measurement showed that it took approximately six times less force to break basal stems of the mutant than of the wild type. In addition, noticeable morphological changes were associated with the mutant, including long stems, dark green leaves with delayed senescence, and reduced numbers of cauline leaves and branches. Genetic analysis showed that the ifl1 mutation was monogenic and recessive. The ifl1 locus was mapped to a region between the 17C2 and 7H9L markers on chromosome 5. Isolation of the ifl1 mutant provides a novel means to study the genetic control of fiber differentiation.     

Relationship between total superoxide dismutase activity with lipid peroxidation, dynamics and morphological parameters in canine semen

There is a lack of information regarding biochemical studies on canine semen. The knowledge of canine sperm metabolism is important because this species has differences in fertility among males, but biochemical studies are not well developed in this species. Reactive oxygen species (ROS) are active molecules produced during the oxygen reduction that have been implicated in several deleterious effects on the function and viability of spermatozoa when they are produced at high concentrations. Peroxidation of the plasma membrane phospholipids leads to a loss of motility, viability, and cytosolic elements, metabolic changes and structural alterations in spermatozoa. The most important antioxidant enzyme involved in ROS detoxification in mammalian spermatozoa is superoxide dismutase (SOD). The aims of the present study were to determine the presence of SOD-like activity in different fractions of canine semen, and to determine the relationship between SOD-like activity and different morphological, dynamical and biochemical parameters in canine spermatozoa. We demonstrated the presence of SOD-like activity in canine seminal plasma and spermatozoa; the negative correlation between SOD-like activity and lipid peroxidation concentrations in spermatozoa suggests a protective effect of this enzymatic activity against the potential oxidative stress in canine spermatozoa. A significant negative correlation between percentage of abnormal spermatozoa, and the percentage of progressive motility or vigor was found. Abnormal spermatozoa not only showed a deformed shape, moreover there was an energetic motility system failure. The presence of SOD-like activity in seminal plasma, spermatic, and postspermatic fractions of canine semen was demonstrated for the first time.     

The tardigrades of Emilia (Italy). II. Monte Rondinaio. A multihabitat study on a high altitude valley of the northern Apennines

We investigated a small valley of glacial origin in the northern Apennines (Italy). A multihabitat study was performed on the mosses and lichens on rocks, temporarily submersed mosses, beech litter, grass turf and sediments from pools, ponds and streams. A few species colonized more than one habitat. Several species were typical of high altitudes or latitudes, some were rare, and two, Hypsibius pradellii sp. nov. and Diphascon (Diphascon) secchii sp. nov. , were new to science.     

Morphology of the male reproductive system of freshwater prawn Macrobrachium carcinus (Decapoda,Caridea): Functional and comparative aspects

Testes and vasa deferentia are parts of the male reproductive system of decapod crustaceans. Both organs show morphological differences among decapod species in terms of anatomical and histological patterns reflecting the diversity of this group. Describing these features may assist in systematics, phylogenetics, and studies of reproductive behavior, especially for species of commercial interest, such as Macrobrachium carcinus, a native American species that, unusually for this genus, has no precopulation courting behavior. This study aims to describe the reproductive morphology and spermatogenesis of the male freshwater prawn M. carcinus. The male reproductive system of this species consisted of lobed testes connected to the vasa deferentia. The testis of M. carcinus was divided into several lobules. Each lobule was formed by a cluster of germ cells surrounded by connective tissue and nurse cells. This microscopic anatomy and histology of the testicular histoarchitecture has been described for many species of Decapoda and may represent a derived design of the testes. Unlike that in other decapod species, spermatogenesis proceeds in short transitory phases that produce spermatozoa at high concentrations and frequencies, corroborating the uncommon male reproductive behavior of this species. In the spermatic pathway, the lobules develop and fuse before releasing spermatozoa from the testes; however, this process has not been observed in decapods, yet. The neutral compounds secreted by the vas deferens are important for sperm nutrition as females secrete a substance for spermatophore adhesion during reproduction. This study presents different features and dynamics of the spermatogenic process in the male reproductive system of M. carcinus that have not yet been presented in the literature for decapods.     

Effects of cryoprotectant agents and freezing protocol on motility of black-lip pearl oyster (Pinctada margaritifera L.) spermatozoa

Gamete cryopreservation techniques have been applied to several bivalve mollusc species. However, research activity in this area has primarily focused on cryopreserving gametes from edible oysters (Ostreiidae). Few studies have examined the effect of cryoprotectants and freezing protocols in the preservation of spermatozoa from cultured pearl oysters (Pteriidae). Pearl oyster producers are increasingly looking towards the development of improved family lines and, as a consequence, the ability to cryopreserve gametes would bring about significant benefits to the cultured pearl industry. In response to this need, we evaluated the effect of three cryoprotectant additives (CPA) on motility of spermatozoa from the black-lip pearl oyster, Pinctada margaritifera. These additives have previously been used to cryopreserve gametes of other bivalve species. The following CPA mixtures were evaluated: (1) 0.45M trehalose and 0, 0.64, 1.02 and 1.53 M dimethyl sulfoxide (Me(2)SO); (2) 0.2M glucose and 2M Me(2)SO and (3) 1.31 M propylene glycol (PG). The effects of four different freezing protocols on motility of P. margaritifera spermatozoa were also evaluated (slow, medium, medium-rapid and rapid cooling). This study showed that total motility was best retained when spermatozoa were cryopreserved in 0.45 M trehalose and 0, 0.64, 1.02 or 1.53 M Me(2)SO and frozen using slow to medium-rapid cooling rates (2.1-5.2 degrees Cmin(-1)). Rapid freezing through direct plunging of spermatozoa into liquid nitrogen resulted in the lowest overall retention of motility regardless of the CPA additive; however, CPA mixture also influenced retention of motility, with 0.2M glucose in 2M Me(2)SO and 1.31 M PG retaining the lowest levels of motility for the CPAs evaluated.     

Steroid metabolism by monkey and human spermatozoa

Freshly ejaculated spermatozoa from monkey and human were washed and incubated with tritium labelled androgens or estradiol to study the pattern of spermatozoa steroid metabolism. When equal concentrations of steroid substrates were used for incubation, monkey and human spermatozoa showed very similar pattern of steroid conversion. Spermatozoa from both species converted testosterone mainly to androstenedione, but reverse conversion of androstenedione to testosterone was negligible. Estradiol-17 beta was converted mainly to estrone. The close similarity between the spermatozoa of monkey and men in their steroid metabolic pattern indicates that the rhesus monkey could be an useful animal model to study the effect of drugs on the metabolic pattern of human spermatozoa.