Interactions between different media and follicle‐stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758)

The aim was to verify the effect of follicle‐stimulating hormone (FSH) supplementation to α‐MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non‐cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag‐NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7‐day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag‐NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7‐day culturing conditions.     

Deep‐water cirripedes colonizing dead shells of the cephalopod Nautilus macromphalus from New Caledonian waters

Fossil cephalopods are frequently encrusted by epibionts; however, determining whether encrustation occurred prior to or post‐mortem to the host, and whether the final environment of deposition corresponds to the habitat of encrustation is complex. The present paper describes cirripede epibionts, their calcareous bases and their attachment scars on 6 post‐mortem shells of Nautilus macromphalus, collected from deep water off New Caledonia. The cirripedes have left both cemented calcareous bases of Hexelasma and scars associated with bioerosion and discoloration produced by verrucomorph barnacles. Live cirripedes included a Metaverruca recta, with articulated opercular plates and organic tissue (on a shell that had been exposed on the sea floor for at least 150 years), and specimens of Hexelasma velutinum, one of which was partly attached to an internal surface of a shell. The disposition of verrucomorphs indicates that most Nautilus shells were colonized post‐mortem rather than during a floating stage. However, as cirripedes are known to have colonized living Nautilus, some Hexelasma, preserved only as calcareous eroded bases, may represent specimens that settled on a living Nautilus. The degree of bioerosion and discoloration induced by verrucomorph barnacles varies according to the surface preservation of Nautilus shells, with deeper and discolored traces preserved on old and degraded shells. Traces made by verrucomorphs described here are ellipsoidal and a new ichnotaxon, Anellusichnus ellipticus, is proposed to accommodate them. Importantly, verrucomorphs and other cirripede taxa with membranous bases that were attached to pristine shells may not leave any substantial scars, and, thus, will be difficult to detect in the fossil record.     

Biochemical responses to ultraviolet‐C radiation and methyl jasmonate in Pinus radiata seedlings that accompany induced resistance to Diplodia pinea

Irradiation of Pinus radiata seedlings with ultraviolet‐C (UV‐C) radiation or spray application of methyl jasmonate (MeJA) each resulted in induced resistance to subsequent wound inoculation with Diplodia pinea. Induced resistance was expressed by lower incidence of disease and by reduced size of stem lesions than in untreated seedlings. UV‐C was more effective than MeJA and the induced resistance was greatest in seedlings that were irradiated with UV‐C for 60 min, 1 week before pathogen inoculation. Induced resistance in the UV‐C treated stems was concomitant with increases in peroxidase (POX) and polyphenol oxidase (PPO) activity but showed no correlation with concentrations of α‐pinene and β‐pinene or total phenolics. Furthermore, POX induction in stem tissue was cumulative and was greatest after three repeat treatments with UV‐C at 6, 3 and 1 week before inoculation. In contrast to UV‐C, MeJA induced a significant increase in β‐pinene concentration in stem tissue but did not affect PPO activity. POX activity was induced by MeJA in stems, although to a lesser extent than by UV‐C, but was not affected in needles. This appears to be the first report demonstrating the use of UV‐C radiation to induce resistance to fungal infection in a coniferous species. The implications of the underlying differences between UV‐C‐ and MeJA‐mediated resistance to D. pinea are discussed.     

Trichoderma atroviride promotes growth and enhances systemic resistance to Diplodia pinea in radiata pine (Pinus radiata) seedlings

Root drench application of Trichoderma atroviride isolates R32, R33, R40 and R84 promoted the growth of potted radiata pine seedlings. After 6 weeks, seedlings treated with R33 and R84 had thicker stems and greater stem and root biomass (p < 0.05) than untreated controls. Treatment with R32 increased seedling root biomass whilst R40 increased stem diameter. None of the isolates affected seedling height. One isolate, R33, induced systemic resistance to stem inoculation with Diplodia pinea and reduced dieback incidence by 20% compared with untreated controls. To our knowledge, this is the first report of systemic induced resistance by Trichoderma in a pine species. Furthermore, seedlings that were treated with R33 (root drench) plus foliar application of methyl jasmonate (MeJA) expressed elevated peroxidase activity in their stems 2 weeks later, compared with seedlings treated only with MeJA. Because R33 itself did not affect peroxidase activity, this may be indicative of treatment synergy or defence potentiation by R33. Curiously, R33 + MeJA induced terpenoids but suppressed phenylalanine ammonia‐lyase activity suggesting possible trade‐offs between phenolic and terpenoid defence pathways in the treated seedlings.     

Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris‐based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.     

Pathology of experimentally-induced, acute toxoplasmosis in macropods

SUMMARY Thirteen Tammar wallabies (Macropus eugenii) were dosed orally with 500, 1000 or 10 000 oocysts of Toxoplasma gondii, as part of a vaccination trial. Eleven animals died of acute toxoplasmosis 9 to 15 days after challenge. The lesions were similar in all animals, consisting of foci of necrosis and inflammation in the intestines, lymphoid tissue, adrenal cortex, heart, skeletal muscle and brain, and severe generalised pulmonary congestion and oedema. Free and intracellular tachyzoites of Toxoplasma were associated with the lesions. The remaining 2 animals had shown no signs of disease when euthanased four months after challenge. Small, focal, non-suppurative inflammatory lesions were seen in brain, heart and skeletal muscle of these animals and chronic Toxoplasma infection was confirmed by mouse inoculation.