DNA barcoding for identification of cryptic species in the field and existing museum collections: a case study of Aethomys and Micaelamys (Rodentia: Muridae)

DNA barcoding has been proposed as a method for species identification. However, this method has been criticised for its over-reliance on a single mitochondrial gene. In this study, four mitochondrial gene regions and one nuclear gene region were used to investigate their different abilities to identify tissue associated with museum specimens of Aethomys chrysophilus, Aethomys ineptus and Micaelamys namaquensis. Aethomys chrysophilus and the more recently elevated A. ineptus are indistinguishable on morphological grounds; however, their ranges are largely parapatric with only one syntopic locality currently known. All of the mitochondrial gene regions were able to separate M. namaquensis from A. chrysophilus and A. ineptus, but they varied in their abilities to resolve differences between A. chrysophilus and A. ineptus. The sequence results identified a specimen from KwaZulu-Natal that was misclassified and should have been identified as A. ineptus. Seven specimens that had not been reclassified following the elevation of A. ineptus to species level were identified as A. ineptus. Individuals of A. chrysophilus from Malawi could not be classified as either A. chrysophilus or A. ineptus, and may be a hybrid or a new, distinct species. This study indicates that DNA barcoding may be used to separate M. namaquensis from A. chrysophilus and A. ineptus, and although it was not able to separate A. chrysophilus and A. ineptus, it did indicate specimens from Malawi may be a new cryptic species.     

Microclimate and Animal Life in the Equatorial Mountains

The venom apparatus in both species of Dendroaspis shows great similarity to that described in other proteroglyphs. There is a posterior bulbous portion or main gland and an anterior narrower portion, the accessory gland, that surrounds the secretory duct. The gland is enclosed in a very tough connective tissue capsule. Fibres from the m. adductor externus superficialis insert on the connective tissue capsule on the dorsal aspect of the venom gland. They serve to compress the gland and expel the venom when the snake strikes. Ventrally, fibres of the m. pterygoideus are also inserted on the investing sheath. This muscle supports the gland ventrally and also appears to play some role in venom ejection. The arrangement of the jaw musculature is similar in both species of Dendroaspis. Since the m. adductor externus superficialis is closely associated with the venom gland it has become highly specialized and exhibits great variation among snakes. This muscle in Dendroaspis differs from that in other proteroglyphs in the extension of its origin in a ventral direction over the postorbital bone to the maxilla. As in other elapids, the m. levator anguli oris is absent in both species of Dendroaspis.     

Distribution of Euphausiid Crustaceans from the Agulhas Current

Twenty-nine euphausiid species were collected during the three cruises of the S.A.S. Natal in 1958. Of these only Stylocheiron microphthalmia had not previously been taken in South African waters. The commonest euphausiid represented in the collection is a larval series which is provisionally linked with Euphausia recurva or E. mutica. The vertical and horizontal distribution of each species is re-corded and seasonal and latitudinal variation discussed. Stylocheiron suhmii, S. microphthalma and Thysanopoda tricuspidata are proposed as indicators of Agulhas Current water and Nyctiphanes capensis and Euphausia lucens as indicators of water not of Agulhas Current origin. The largest number of euphausiids was found off Port Elizabeth during both summer and winter.     

Cloning, expression and in vitro evaluation of recombinant canine Tum5, an angiostatic domain of mammalian type IV collagen

The process of new blood vessel formation within and around neoplastic tissue, termed angiogenesis, is a significant factor in the development, progression and metastasis of malignant tumours in all species. A major cause of death in cancer patients is the development of treatment‐resistant metastatic disease, which may be avoided by therapies that target the genetically stable population of vascular endothelial cells within tumours. Tumstatin is a small protein formed by the cleavage of the alpha‐3 subunit of the non‐collagenous domain of mammalian type IV collagen. Recombinant human Tumstatin has been shown to have potent angiostatic properties in vitro and in vivo. Tumstatin is a potent initiator of apoptosis and inhibits the proliferation and migration of vascular endothelial cells in cell culture. Recently, a fragment of Tumstatin, termed Tum5, has been shown to have biologic activity similar to the parent compound. The systemic administration of angiostatic proteins like Tum5 may result in the remission of established tumours, while preventing or delaying the onset of clinically detectable metastasis. Recombinant canine Tum5 (cTum5) was cloned and its protein expression induced in a prokaryotic vector. The resulting cTum5 protein caused dose‐dependent inhibition of vascular endothelial cells in vitro, which appears to be mediated through apoptosis.     

A preliminary survey of Chlamydia psittaci genotypes from native and introduced birds in New Zealand

AIM: To describe the Chlamydia psittaci genotypes in samples from native and introduced birds from New Zealand by analysis of the sequence variation of the ompA gene.

METHODS: DNA was extracted from samples collected from a non-random sample of birds; either swabs from live asymptomatic birds or birds with clinical signs, or formalin-fixed, paraffin-embedded (FFPE) samples from historical post-mortem cases. The presence of C. psittaci in all samples had been confirmed using a quantitative PCR assay. The C. psittaci ompA gene was amplified and sequenced from samples from 26 native and introduced infected birds comprising 12 different species. These sequences were compared to published available C. psittaci genotypes.

RESULTS: Genotypes A and C of C. psittaci were identified in the samples. Genotype A was identified in samples from nine birds, including various native and introduced species. Genotype C was identified in samples from 16 different waterfowl species, and a mixed infection of both genotypes was found in a kaka (Nestor meridionalis). In native birds, C. psittaci infection was confirmed in seven new host species.

CONCLUSIONS AND CLINICAL RELEVANCE: Two genotypes (A and C) of C. psittaci were found in samples from a wider range of both native and introduced species of birds in New Zealand than previously reported. Both genotypes have been globally associated with significant disease in birds and humans. These initial results suggest the host range of C. psittaci in New Zealand birds is under-reported. However, the prevalence of C. psittaci infection in New Zealand, and the associated impact on avian and public health, remains to be determined. There are biosecurity implications associated with the importation of birds to New Zealand if there is a limited diversity of C. psittaci genotypes present.     

Phylogenetic tests of a Cercopithecus monkey hybrid reveal X-chromosomal polyphyly of C. cephus and emerging patterns in the cephus species group radiation

A captive Cercopithecus nictitans × C. cephus male was examined at loci on the X- and Y-chromosomes as a test of previously described phylogenetic methods for identifying hybrid Cercopithecus monkeys. The results confirm the reliability of such assays, indicating that they can be of immediate utility for studies of wild populations in Gabon, where the two species have hybridised. A closer examination of the resultant sex chromosomal topologies, combined with recent mitochondrial studies, reveals two emerging patterns in the evolution of the cephus species group. First, all three genetic systems (X-DNA, Y-DNA and mtDNA) agree that the earliest divergence separates West African from Central African species, consistent with a major faunal transition zone in the vicinity of the Cross River. Second, the X-DNA and mtDNA trees reveal polyphyly of C. cephus lineages. It is unclear at present whether these polyphylies are due to ancestral hybridisation or incomplete lineage sorting, or both.     

Establishment of a proteomic reference map for the gastrocnemius muscle in the rabbit (Oryctolagus cuniculus)

In several laboratory and production species, the establishment of a proteome reference map of a specific tissue has been accomplished. The rabbit is widely used as both a production and experimental animal. A lot of physiology research involving the gastrocnemius muscle of rabbit is described, although no reference proteome map is available. In this work, the first reference map of the rabbit’s gastrocnemius muscle using 2D gel electrophoresis and the identification of proteins through peptide mass fingerprinting (PMF) was established. A total of 45 proteins were localized and identified with three major roles: cell structure and contractile apparatus; metabolic and cell defense proteins. A reference map of major proteins expressed is described enabling possible comparisons with other physiological studies.     

Sex chromosomes behavior and G‐banding treatment of male meiosis in nuptial gift‐giving spiders of the family Trechaleidae

Preliminary genetic studies in Trechaleidae spider family show high variation in sex chromosomes and high heterocigocity, suggesting high chromatin plasticity. The trechaleids Paratrechalea ornata, Trechalea bucculenta and Trechaleoides biocellata are present in Uruguay. Males offering nuptial gifts during courtship have been reported in P. ornata and T. bucculenta but not in T. biocellata. Nuptial gifts are an inherited trait probably highly affected by environmental factors, which play an important role in gene expression. We hypothesize that this trait could be associated with tissue‐specific genes existing in G‐bands. We investigate the male meiosis in these 3 species, their sex chromosome system and the effects of G‐banding on their chromosomes, and elucidate genetic differences among them. Meiotic stages of the 3 species were submitted to Giemsa‐staining and G‐banding treatments. We observed a haploid number of n= 11 in P. ornata and n= 13 in both T. bucculenta and T. biocellata. Males from the 3 species presented an X₁X₂0 sex chromosome system, which is suggested as ancestral in Araneae. In P. ornata and T. bucculenta, both sex chromosomes were together and aligned in parallel until the segregation during anaphase I. In contrast to these species, sex chromosomes of T. biocellata usually remained distant from each other until diakinesis when they were observed associated in parallel disposition. Interstitial G‐bands were similar in P. ornata and T. bucculenta, and they both differed from those in T. biocellata. The special behavior of sex chromosomes in T. biocellata as well as the different G‐banding pattern of this species suggests the existence of novel modifications in this species.     

Uptake of milk with and without solid feed during the monogastric phase: Effect on fibrolytic and methanogenic microorganisms in the gastrointestinal tract of calves

Microbial communities are affected by diet and play a role in the successful transition from milk to a solid diet. The response of microorganisms in the gastrointestinal tract of Holstein bull calves to the uptake of milk with solid feed (control treatment; CT), or milk without solid feed (milk‐only treatment; MT) during the first 3 weeks of life was investigated. Samples were collected from the rumen (fluid and tissue), abomasum (fluid), cecum (fluid and tissue) and feces at 7, 14 and 20 days of age. Calf weight was higher on days 14 and 20 in the MT than the CT. In the rumen at 14 days, the fibrolytic bacteria Fibrobacter succinogenes and Prevotella ruminicola increased in the CT and Ruminococcus flavefaciens increased in the MT. This suggests that R. flavefaciens is not strictly fibrolytic and that it might use milk as a substrate or other microbial species might supply a substrate. Diet affected methanogens, but this may have been due to an indirect effect via an association with Geobacter spp. or other syntrophic partners. The treatments also affected microorganisms in the abomasum, cecum and feces. Our results contribute to an understanding of diet, microbes in the gastrointestinal tract and weaning.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

Goblet Cell Types in Intestine of Tiger Barb and Black Tetra (Cyprinidae,Characidae: Teleostei)

Histochemical properties of goblet cells in intestine of a stomach‐less teleost, tiger barb (Puntius tetrazona), and a stomach‐containing teleost, black tetra (Gymnocorymbus ternetzi), are described and compared. The intestine goblet cells were mostly wide in both species, but in tiger barb, some of them were markedly thinner. In black tetra, all the intestine goblet cells displayed magenta colour after PAS, whereas in the tiger barb, only the thinner goblet cells displayed such affinity. The latter cell type was coloured strongly magenta when the tissue was treated with alcian blue (pH 2.5) followed by PAS, whereas the wide goblet cells in tiger barb and all goblet cells in black tetra displayed mainly a blue colour after such treatment. Further, the goblet cells in both species were coloured cleanly blue after high iron diamine followed by alcian blue (pH 2.5). The intestine goblet cells in both species displayed a moderate affinity to WGA and concanavalin A lectins and no affinity to DBA. Most of the goblet cells displayed no affinity to PNA, but some of them in the tiger barb displayed a moderate or strong affinity to this lectin. The affinity to WGA was somewhat strengthened after pre‐treatment with neuraminidase. These results suggest that tiger barb contains two types or variants of intestinal goblet cells: high numbers of wide cells filled by acidic, non‐sulphated mucin and some thinner cells filled by neutral mucin. The intestine goblet cells in black tetra were filled by variable amounts of neutral and acidic mucin, but the total number of such cells is much less than in tiger barb. The present lectin and neuraminidase results suggest that the intestinal mucins in both species contain significant amounts of N‐acetylglucosamine, sialic acid and glucose/mannose, but seem to lack N‐acetylgalactosamine. However, some of these cells in tiger barb contain moderate to large amounts of galactose. Together, these results suggest significant species‐specific features of the intestine goblet cells and mucin types in tiger barb and black tetra. In conclusion, the present results suggest that the diet and feeding habits in stomach‐less teleosts compared with stomach‐containing teleosts, greatly influence the number of intestine goblet cells and type of mucin in these cells.     

Relative population sizes and trends,and hybridization of fur seals Arctocephalus tropicalis and A. gazella at the Prince Edward Islands,Southern Ocean

Fur seals were counted at the Prince Edward Islands during the 1981/1982 austral summer. Classified counts, adjusted for pup undercounting and mortality, pregnancy rate and seasonal haul-out patterns, of Arctocephalus tropicalis and A. gazella are presented. These populations have entered a phase of rapid growth, as indicated by higher rates of population growth than previously found. The possible role of A. gazella immigration is unknown. There has been an increase in the number of breeding localities used by both species. At present the extent of hybridization between these two species appears limited, possibly by behavioural, ecological and genetic processes.     

Observations on the Fine Structure of the Liver in the Camel (Camelus dromedarius)

The structure of macroscopically inconspicuous livers in 23 adult camels (Camelus dromedarius) was studied by light and transmission electron microscopy. A well-developed connective tissue characterizes the camel liver. Thick trabeculae divide the liver parenchyma into lobules. Portal tracts and central veins are surrounded by a variable amount of fibrous tissue. In the perisinusoidal space (DISSE), collagen fibres form a dense three-dimensional network around the sinusoids. A mild to moderate fatty infiltration is present in hepatocytes of all animals. In the epithelial cells of the bile ducts, small to medium sized lipid inclusions are a common feature. The ultrastructure of hepatocytes in the camel liver corresponds to that of other domestic mammalian species. The endothelial cells lining the sinusoids show a multiple fenestration and are surrounded by a discontinuous basal lamina. Fat-storing cells are numerous and contain lipid droplets varying in size, number and electron density from one cell to another.     

Evaluation of reproductive parameters in male Neotropical bats during dry and rainy months in a specific area of the Cerrado biome

The aim of this study was to analyse the reproductive aspects of male bats of three common species of the Phyllostomidae family: Artibeus lituratus, Platyrrhinus lineatus and Sturnira lilium, during dry and rainy months in a specific area of the Cerrado biome. Body weight was significantly higher during the dry months for S. lilium. The gonadosomatic index (GSI) and testicular weight were not significantly different between dry and rainy periods. The tubular parameters were significantly bigger in A. lituratus than in the other two species during both periods. No difference in the tubular/interstitial ratio was observed in any of the species during both periods. In both periods, all sperm cells and germ cell developmental stages were visible on seminiferous tubules whereas sperm cells were observed in epididymides of all sampled animals. The percentage of morphologically normal sperm was low (35%–60%), with no difference between periods. Spermatozoa from A. lituratus presented a leaf-shaped head, while the head was round-shaped in the other two species. In conclusion, our data suggest that males from the three studied species did not present reproductive latency during the most critical weather periods (dry and rainy months) in the metropolitan region of Brasilia, Brazil.     

Screening for the Fish Disease Agent Aeromonas salmonicida with an Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

Serological detection of bacterial pathogens in fish tissue is an important tool for surveying epidemiological situations. Whenever antibacterial treatment of fish is recommended, it becomes necessary, however, to culture the pathogen for sensitivity testing. An enzyme-linked immunosorbent assay (ELISA) was developed to identify Aeromonas salmonicida in culture. This serological identification can partly substitute for biochemical characterization of the organism and thus decrease the time between isolation and sensitivity testing by at least 3 d. The ELISA works with only one bacterial colony and yields results within 4 h. During this time, a bacterial suspension can be prepared for the resistance test. The specificity of an antiserum, raised in rabbits, against whole cells of A. salmonicida can be increased by adsorption with strains of cross-reacting species. However, difficulties arise when serologically heterogeneous species (e.g., A. hydrophila) are used as the cross-reacting bacterium. In the present study, severalfold adsorption with four isolates did not totally rule out cross-reactivity against additional strains. Therefore, the strain in question should also be checked for colony morphology, production of pigment, or presence of cytochrome oxidase to validate the serologically obtained result.     

Species-specific differences of the avian oesophagus: Histological and Ultrastructural study

The oesophagus is a muscular tube comprised of cervical and thoracic regions. Several studies have clarified the histological structure of the oesophagus. However, its histoarchitecture in relation to variable dietary habits of each species is still unclear. In the current study, 21 pigeons, cattle egrets and ducks, n = 7, each was used. Macroscopically, the oesophagus of cattle egrets either the cervical or thoracic parts was the longest among the pigeons and ducks. Histologically, the oesophagus comprised of four distinct tunicae: mucosa, propria submucosa, musculosa and adventitia or serosa. A great structural variation in these layers among the three investigated species was recorded. In the cervical oesophagus of pigeons, the superficial squamous cells showed perinuclear halo zone, the propria submucosa was characteristically lacked any gland. Moreover, its musculosa was very thick. On the other hand, the intraepithelial glands were characteristically distributed along the whole length of the cattle egret’s oesophagus. Interestingly, the cervical esophagus of the ducks showed submucosal associated lymphatic tissue; diffuse and nodular Ultrastructurally, the oesophageal glands showed secretory granules of variable electron densities, electron - lucent in the pigeons and ducks and electron - dense in the cattle egrets.     

Changes in Lymphocyte Subsets in the Bronchus‐associated Lymphoid Tissue of Goats Naturally Infected with Different Mycoplasma Species

The distribution of cells containing lysozyme, S‐100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus‐associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3–5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1–6), M. mycoides ssp. capri (goats no. 7–12) and M. capricolum ssp. capricolum (goats no. 13–18). Microscopically, infected animals showed a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3⁺ T lymphocytes, and the ratio of CD4⁺:CD8⁺ cells was >2. The BALT showed large germinal centres mainly composed of IgG⁺ B lymphocytes, with numerous S‐100⁺ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG⁺ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Reproductive strategies in males of the world's southernmost lizards

Reproductive and life history patterns in reptiles are tightly related to the environmental conditions, so male reproductive cycles have been historically characterized as continuous, for tropical lizards, or seasonal, for temperate lizards. However, males of Liolaemus and Phymaturus lizards (Liolaemidae), from cold temperate climates of high altitudes or latitudes in Argentina and Chile, have developed a variety of reproductive cycles to coordinate with the short female reproductive season and to deal with the low frequency of reproductive females in the population. Using gonadal histology and morphological analysis, we describe the male reproductive biology, fat storage and sexual dimorphism of the viviparous lizards Liolaemus sarmientoi and Liolaemus magellanicus that inhabit an austral grass steppe at 51°S, in the southern limit of the American continent. Males of L. sarmientoi and L. magellanicus are reproductively available during the entire activity season of approximately 5 months. In addition, males of both species exhibit greater body sizes than females in morphological variables relevant in sexual selection. Meanwhile, females of both species exhibit larger inter‐limb length than conspecific males, which suggests fecundity selection to increase space for a larger litter size. The continuous sperm production throughout the activity season allows these liolaemids to mate at any time when females ovulate, representing a selective advantage to deal with the short activity season and the adversities of the cold environment they inhabit.     

Influence of the Cumulus and Gonadotropins on the Metabolic Profile of Porcine Cumulus–Oocyte Complexes During In Vitro Maturation

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A₁ (with a dense cumulus), A₂ (with a translucent cumulus), B₁ (with the corona radiata), B₂ (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A₁ COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.     

Histology of the venom gland of the puff-adder (Bitis arietans)

The histology of the venom gland of the puff-adder (Bitis arietans) has been investigated in the resting and stimulated state. No accessory venom gland was found to be associated with the main venom gland or duct in the same position as has been reported for other snakes. In the resting state the parenchyma of the venom gland was found to consist of tubules lined by a single layer of tall columnar secretory cells. After being stimulated to secrete by repeated milkings, the histological appearance of the gland changed and the epithelium was found to be more foliaceous and the component cells to be taller and more slender. A pronounced increase of pigment was also observed in the connective tissue septae. Micro-organisms were observed in the secretion pools of resting glands and by culturing the venom it could be shown that these were Staphylococcus aureus, Staphylococcus epidermidis and Klebsiella ozaenae. The presence of these organisms would suggest that the venom is not cytotoxic to all cells at this level of production.