Secondary Seed Ingestion in Snakes: Germination Frequency and Rate, Seedling Viability, and Implications for Dispersal in Nature

The importance of vertebrate animals as seed dispersers (zoochory) has received increasing attention from researchers over the past 20 years, yet one category in particular, diploendozoochory, remains understudied. As the term implies, this is a two-phase seed dispersal system whereby a secondary seed predator (carnivorous vertebrate) consumes a primary seed predator or granivore (rodent and bird) with undamaged seeds in their digestive tract (mouth, cheek pouch, crop, stomach, or other organ), which are subsequently eliminated with feces. Surprisingly, although snakes are among the most abundant predators of granivorous vertebrates, they are the least studied group insofar as our knowledge of seed rescue and secondary dispersal in a diploendozoochorous system. Here, using live snake subjects of the Sonoran Desert (one viperid and two colubrid species) and seeds of the Foothill Palo Verde (Parkinsonia microphylla), a dominant tree of the same region, we experimentally tested germination frequency and rate, and seedling viability. Specifically, to mimic rodents with seed-laden cheek pouches, we tested whether wild-collected P. microphylla seeds placed in the abdomen of thawed laboratory mice and ingested by the snakes would retain their germination viability. Second, we examined whether seeds exposed to gut transit germinated at a greater frequency and rate than the controls. While we found strong statistical support for our first hypothesis, both aspects of the second one were not significant. Accordingly, we provide an explanation for these results based on specific life-history traits (dormant and non-dormant seeds) of P. microphylla. Our study provides support for the role of snakes as important agents of seed rescue and dispersal in nature, their potential as ecosystem engineers, and crucial evidence for the investment of field-based studies on diploendozoochorous systems in deserts and other ecosystems.

First reported case of a histiocytic sarcoma in an Armenian hamster (Cricetulus migratorius)

A 14-month-old male Armenian hamster ( Cricetulus migratorius) presented with a spontaneous, subcutaneous, firm mass (4.0 × 2.0 × 1.5 cm) on the ventral neck extending towards the cheek pouch causing multifocal small oral ulcerations. This animal was immunized subcutaneously on the dorsal neck for the development of monoclonal antibodies seven months before presentation. The animal was euthanized and necropsy was performed. Histopathology of the mass showed a well demarcated, multilobulated, unencapsulated, highly cellular, neoplastic mass composed of spindle cells arranged in interlacing streams and bundles, with a moderate amount of fibrovascular stroma. The neoplastic cells exhibited indistinct cell borders and a moderate to large amount of eosinophilic, fibrillar cytoplasm, marked anisocytosis and anisokaryosis, binucleated and multinucleated cells, and high mitotic rate. Based on the histomorphologic features of the mass, and the presence of renal tubular hyaline globules and myeloid hyperplasia in the bone marrow, a diagnosis of histiocytic sarcoma was made. The presumptive diagnosis was confirmed by immunohistochemistry, upon which the neoplastic cells showed strong immunoreactivity for the histiocytic cell markers Iba1 and CD11b. Histiocytic sarcomas have been reported in Syrian ( Mesocricetus auratus) and Siberian dwarf ( Phodopus sungorus) hamsters but, to our knowledge, the current report represents the first case of histiocytic sarcoma described in an Armenian hamster. It is plausible to consider the animal’s experimental immunization history and the development of the histiocytic sarcoma to be related. An association between adjuvanted vaccines and soft-tissue sarcomas has been described in cats and referred to as feline injection-site sarcomas.

Effects of Ruscus extract on muscarinic receptors: Is there a role for endothelium derived relaxing factors on macromolecular permeability protection and microvascular diameter changes?

BACKGROUND: Protective effects of Ruscus extract on macromolecular permeability depend on its capacity to stimulate muscarinic receptors on endothelial cells and induce the release of endothelium derived relaxing factors (EDRFs). OBJECTIVE: To investigate if these effects depend only on activation of muscarinic receptors or EDRFs release are also necessary. We have also investigated the participation of Ruscus extract on muscarinic-induced release of EDRFs on microvascular diameters. METHODS: Hamsters were treated daily during two weeks with Ruscus extract (50, 150 and 450 mg/kg/day) and then macromolecular permeability induced by histamine and arteriolar and venular diameters after cyclooxygenase (COX) and nitric oxide synthase (NOS) inhibitors: indomethacin and Nω-Nitro-L-arginine (LNA), respectively applied topically at 10–8M, 10–6M and 10–4M were observed on the cheek pouch preparation. RESULTS: Ruscus extract decreased macromolecular permeability in a dose-dependent fashion and did not affect microvascular diameters. NOS and COX inhibitors enhanced this effect. NOS inhibition reduced arteriolar diameter and COX blocking decreased arteriolar and venular diameters at the lowest dose and increased them at higher doses of Ruscus extract. CONCLUSION: The protective effect of Ruscus extract on macromolecular permeability seems to be mediated only via muscarinic receptors. Muscarinic activation attenuated vasoconstrictive tone through cyclooxygenase-independent endothelium derived relaxing factors.

Musical calling as a behavior ancestral to all modern primates

Music is a singular behavior humans exhibit via the creation of patterned sounds organized, for example, along rhythmic and melodic dimensions. Music-like behavior, however, also appears in many other animals including non-human primates. Calls with western musical attributes are primarily ascribed to species of gibbon, tarsier, indri, and potentially also marmoset, tamarin, and titi monkeys. A recent survey of primates, using a continuous measure of acoustic musicality, suggests that many other species—including galagos, several lemurs, and some old-world monkeys—also exhibit calls with music-like qualities. Based on this ubiquity and a recent arboreal-origins theory for musical behavior, I hypothesized that the ancestor of all living primates was likewise also somewhat musical. Ancestral character estimation was used to reconstruct ancestral phylogenetic states using scored spectrographic depictions of vocalizations of extant primates (n=58 species) to determine if musical calling is the initial primate condition. The results suggest that nearly all primate families have an ancestor with at least one moderately music-like call—two or more reappearing syllables—with the clear exception of Cercopithecine. These cheek-pouch monkeys likely experienced a multi-million year diminution in musical behavior, perhaps in conjunction with their recent adaptations for quadrupedal terrestriality. These findings largely indicate that exhibition of at least one music-like call is a normal, and likely quite ancient, constituent of primate vocalization behavior.

Seed Shadows of Northern Pigtailed Macaques within a Degraded Forest Fragment, Thailand

Research Highlights: Frugivores able to disperse large seeds over large distances are indispensable for seedling recruitment, colonization and regeneration of tropical forests. Understanding their effectiveness as seed dispersal agents in degraded habitat is becoming a pressing issue because of escalating anthropogenic disturbance. Although of paramount importance in the matter, animal behaviour’s influence on seed shadows (i.e., seed deposition pattern of a plant population) is difficult to evaluate by direct observations. Background and Objectives: We illustrated a modeling approach of seed shadows incorporating field-collected data on a troop of northern pigtailed macaques (Macaca leonina) inhabiting a degraded forest fragment in Thailand, by implementing a mechanistic model of seed deposition with random components. Materials and Methods: We parameterized the mechanistic model of seed deposition with macaque feeding behavior (i.e., consumed fruit species, seed treatments), gut and cheek pouch retention time, location of feeding and sleeping sites, monthly photoperiod and movement patterns based on monthly native fruit availability using Hidden Markov models (HMM). Results: We found that northern pigtailed macaques dispersed at least 5.5% of the seeds into plantation forests, with a majority of medium- to large-seeded species across large distances (mean > 500 m, maximum range of 2300 m), promoting genetic mixing and colonization of plantation forests. Additionally, the macaques produced complementary seed shadows, with a sparse distribution of seeds spat out locally (mean >50 m, maximum range of 870 m) that probably ensures seedling recruitment of the immediate plant populations. Conclusions: Macaques’ large dispersal distance reliability is often underestimated and overlooked; however, their behavioral flexibility places them among the last remaining dispersers of large seeds in disturbed habitats. Our study shows that this taxon is likely to maintain significant seed dispersal services and promote forest regeneration in degraded forest fragments.

Babassu (Attalea glassmanii Zona) Nut Oil Is More Effective than Olive Oil for Treating Ischemia-Reperfusion Injury

Background. Cardiovascular disease (CVD) is the leading cause of death in Western civilizations. The type of fatty acid which makes up the diet is related to the cardiovascular morbimortality and the formation of atheromas. Populations with high consumption of oils and fats have a higher number of deaths from CVD. Purpose. In the present study, the objective was to comparatively analyze the microcirculatory effects of unrefined babassu oil with olive oil in microcirculation and liver of male hamsters of the species Mesocricetus auratus, checking the permeability to macromolecules after ischemia-reperfusion (I/R) without and with topical application of histamine 5 × 10−6 M. This is an experimental study, using as model the hamster’s cheek pouch, which was prepared for intravital microscopy. The hamsters were divided into seven groups and orally treated for 14 days, twice a day (at 8 AM and 4 PM), orally received treatments in the following doses: unrefined babassu oil (BO) 0.02 mL/dose (group BO-2), 0.06 mL/dose (group BO-6), and 0.18 mL/dose (BO-18 group); extra virgin olive oil (OI) 0.02 mL/dose (group OI-2), 0.06 mL/dose (group OI-6), and 0.18 mL/dose (OI-18 group); and mineral oil (MO) 0.18 mL/dose (MO-18 group). The observations were made on the 15th day on the hamsters’ cheek pouch; the increase of vascular permeability induced by I/R with and without histamine application was evaluated, and in the liver the biological material was collected aseptically then fixed in 10% buffered formalin. Results. Microcirculatory analyses showed a significant reduction in the number of leaks after I/R with and without the topical use of histamine in animals treated with unrefined BO 0.06 mL/dose (BO-6) and 0.18 mL/dose (BO-18) compared to animals treated with OI. The BO group (p<0.001) presented a dose-response relationship for decreasing leaks after I/R with and without topical use of histamine. Histological liver analyses showed no fat deposition changes in any of the treatment groups. Phytochemical analyses evidenced a chemical compound (C31H60NO8) in unrefined BO but not in OI. Conclusions. This experiment demonstrates the protective effect of unrefined BO on the microcirculatory system and its greater dose effect than that of OI. Finding a chemical compound (C31H60NO8) that is present in BO but not in OI opens the possibility of investigating whether this chemical compound was responsible for the protective effect on membrane permeability.

Effect of corticosteroid (triamcinolone acetonide) and chlorhexidin on chemotherapy- induced oxidative stress in buccal mucosa of rats

Oral mucositis (OM) refers to erythematous and ulcerative lesions of the oral mucosa. This pathology can occur by various causes. Cancer therapy is one of the well-known causes of OM such as chemotherapy and/or with radiation therapy. It has been widely mentioned that oxidative stress parameters such as lipid peroxidation (LP) levels increase during cancer process. Glutathione (GSH) is one of the major intracellular enzymes to detoxify oxidant molecules. The aim of this study was to investigate and compare the effects of Triamcinolone Acetonide (TA), a synthetic steroid chlorhexidine (CHX), a chemical antiseptic, on 5- fluorouracil (5-FU), a chemotherapeutic agent and soft abrasion induced OM in buccal mucosa of rats. OM was induced in rats through a combination of 5-FU treatment and mild abrasion of the cheek pouch with a wire brush. Buccal mucosa lipid peroxidation (LP) levels were higher (p< 0.05) in 5-FU group than in control although LP levels were lower (p<0.05) in TA group than in control group. The reduced glutathione levels were lower (p<0.05) in 5-FU group than in the control group although its level was higher (p<0.05) in TA and CHX groups than in the 5-FU group. Glutathione peroxidase activity was also higher (p<0.05) in TA group than the 5- FU group. In histopathological analyses, treatment with TA reduced 5-FU induced inflammatory cell infiltration and ulceration (p<0.001) but not with CHX. In conclusion, we observed that TA and CHX treatment modulated chemotherapy induced oxidative injury in the rat OM. However, only TA histopathologically ameliorated the 5-FU induced OM of rats. These findings suggest that TA is a useful agent for management of experimental oxidative injury and OM caused by the chemotherapy.

Hydrogel Formulations Incorporating Drug Nanocrystals Enhance the Therapeutic Effect of Rebamipide in a Hamster Model for Oral Mucositis

A mouthwash formulation of rebamipide (REB) is commonly used to treat oral mucositis; however, this formulation does not provide sufficient treatment or prevention in cases of serious oral mucositis. To improve treatment, we attempted to design a hydrogel incorporating REB nanocrystals (R-NPs gel). The R-NPs gel was prepared by a bead mill method using carbopol hydrogel, methylcellulose and 2-hydroxypropyl-β-cyclodextrin, and another hydrogel incorporating REB microcrystals (R-MPs gel) was prepared following the same protocol but without the bead mill treatment. The REB particle size in the R-MPs gel was 0.15–25 μm, and while the REB particle size was 50–180 nm in the R-NPs gel. Next, we investigated the therapeutic effect of REB nanocrystals on oral mucositis using a hamster model. Almost all of the REB was released as drug nanocrystals from the R-NPs gel, and the REB content in the cheek pouch of hamsters treated with R-NPs gel was significantly higher than that of hamsters treated with R-MPs gel. Further, treatment with REB hydrogels enhanced the healing of oral wounds in the hamsters. REB accumulation in the cheek pouch of hamsters treated with the R-NPs gel was prevented by an inhibitor of clathrin-dependent endocytosis (CME) (40 μM dynasore). In conclusion, we designed an R-NPs gel and found that REB nanocrystals are taken up by tissues through CME, where they provide a persistent effect resulting in an enhancement of oral wound healing.

Effect of Topical 2% Eucalyptus Extract on 5-FU-Induced Oral Mucositis in Male Golden Hamsters

Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p<0.05). Also, a lower malondialdehyde level and higher myeloperoxidase and glutathione activities were detected in the eucalyptus group in comparison to the calendula and control groups (p<0.001). The thickness of the mucosa and submucosa increased in the eucalyptus group. The numerical density of the fibroblast and the volume density of the collagen significantly increased in the eucalyptus group. In conclusion, the use of eucalyptus hydroalcoholic extract may be associated with reduced intensity of oral mucositis, diminished concentration of malondialdehyde, increased activity of myeloperoxidase and glutathione, increased volume of mucosa and submucosa, increased fibroblast and collagen in the induced oral mucositis in golden hamsters undergoing 5-FU consumption.