Emanations Volume 4

ABSTRACT

Two members of the family Cuculidae, the smooth-billed ani (Crotophaga ani) and the Hispaniolan lizard cuckoo (Saurothera longisrostris) forage at the same levels of the forest canopy, which in turn exposes them to the same micro and macro ectoparasites. Both continuously line their nests with the leaves of various plants as their chicks develop, suggesting that the birds may use the leaves to help protect their brood from ectoparasites. The six most predominant species of plants used, determined by percentage of bulk weight from collected nests, were Celtis trinervia (Ulmaceae), Coccoloba diverisfolia (Polygonaceace), Citharexylum fruiticosum (Verbenaceae), Sideroxylon foetidissimum (Sapotaceae), Calyptranthes pallens (Myrtaceae), and Bursera simaruba (Burseraceae). Extracts of these plants were analyzed for bioactivity against the microfauna (five bacteria and two fungi) cultivated from C. ani feathers along with those of birds with similar foraging levels: the northern mocking bird (Mimus polyglottos) and the yellow faced grassquit (Tiaris olivacea). In addition, the extracts were tested against the following microorganisms: the gram-positive bacteria Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus; the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa; and three strains of the fungus Candida albicans. The only plant that inhibited bacterial or fungal growth was C. pallens, which composed 16.78% of the bulk mass of the C. ani nest and 14.00% of the S. longirostris nest. It inhibited two bacteria that were originally isolated from M. polyglottos and C. ani, as well as all three strains of C. albicans. Although this plant is not the predominant component of the nests, these tests provide suggestive evidence that the birds are using these plants to protect the feathers of their developing brood.

The smooth-billed ani (Crotophaga ani) and the Hispaniolan lizard cuckoo (Saurothera longisrostris) exhibit a nest lining behavior that is unique among the birds of the Dominican Republic: they identify leaves to be gathered for their nest lining by crushing them in their beaks. During nest construction, egg incubation, and rearing of the young, the adult birds constantly line the nest with fresh leaf material (Rosane et al. personal observation). It has previously been shown that European starlings (Strunus vulgaris) prefer certain plants for nest building that reduce mite population (Clark and Mason 1988). Arthropod haematophagic ectoparasites have been shown to delay egg laying, decrease hatching success by nearly 20%, increase food compensation by parents, and decrease fledgling body mass (Oppliger 1994; Tripet et al. 1997; Allander 1998).

Until recently it was thought that feather damage is mainly caused by lice, but the discovery of keratinolytic microorganisms shows that arthropods are not the sole cause of feather degradation. These keratinolytic microorganisms occur most frequently in the plumage of birds that forage on the ground. Feathers of these birds are degraded most rapidly during warm temperatures and frequent rain or dew, optimal conditions for microorganismal growth, especially within the ventral feathers that have direct contact with the soil (Burtt et al. 1999). C. ani and S. longirostris, two birds that actively forage on the ground and live in a tropical climate, are vulnerable to feather damage due to microfauna. Due to the heavy concentration of keratinolytic microorganisms on the ventral feathers of adults, vertical transmission of these parasites can occur. This study was conducted to determine if tree leaves that the birds use to line their nest defend against feather-degrading bacteria and fungi.

MATERIALS AND METHODS
Feather samples and Microfauna Isolation

Feather samples were taken from birds that were captured using mist nets set up in second-growth canopy and clearings in Punta Cana in the Province of Altagracia, Dominican Republic, from June 14 to July 2, 2001. Once a bird was caught it was gently removed using sterile latex gloves; some contour feathers were removed using flame-sterilized forceps and placed in a sterile screw top vial. The bird was also measured, weighed, and banded, and a uropygial gland secretion was taken for future studies on the avian population of the area.

Due to the difficulty of catching S. longirostris, feathers were taken from C. ani and other species that forage on the ground: the northern mocking bird (Mimus polyglottos) and the yellow-faced grassquit (Tiaris olivacea) (Table 1). Inside a laminar flow hood, two feathers from each individual were used to inoculate a fungal nutrient agar plate and a bacterial nutrient agar plate. One set of control plates was used to assess contamination inside the hood. The plates were incubated for 24 hours at 35°C, after which the colonies were separated on the basis of color, morphology and comparison to organisms that grew on the control plates. Five bacteria and two fungi were isolated.

Leaf Collection and Extraction

Extracts were made from the leaves of the following trees: Celtis trinervia (Ulmaceae), Coccoloba diverisfolia (Polygonaceace), Citharexylum fruiticosum (Verbenaceae), Sideroxylon foetidissimum (Sapotaceae), Calyptranthes pallens (Myrtaceae), and Bursera simaruba (Burseraceae). These species compromised the largest percentage by mass of the C. ani and S. longirostris nest lining, as calculated from three C. ani nests and one S. longirostris nest that were collected the previous year: C. trinervia 36.5% of S. longirostris nest, C. diverisfolia 9.7% of C. ani nest, C. fruiticosum 18.53% of C. ani nest, S. foetidissimum 9.2% of S. longirostris nest, C. pallens 16.78% of C. ani & 14% of S. longirostris nests, and B. simaruba 31.07% of S. longirostris nest (Rosane et al. unpubl. data). The samples were taken from locations where there was no risk of pesticide use. Voucher specimens were made and deposited in the Cornell University L.H. Bailey Hortorium and the Jardín Botánico Nacional Dr. Rafael M. Moscoso, Santo Domingo. The extracts consisted of 1 gram dried, ground leaf material per 10 ml ethanol.

Bioassays

Three types of bioassays were performed: disc diffusion assay, phototoxicity disc diffusion assay, and a brine shrimp assay. In the disk diffusion assay, the six plant extracts were tested for anti-bacterial and anti-fungal activity against the gram-positive bacteria Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus; the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa; three strains of the fungus Candida albicans, and five bacteria and two fungi isolated from bird feathers. Sterile discs impregnated with 40uL of an extract and allowed to air dry were placed on agar plates each inoculated with one of the above microorganisms. The plates were incubated for 24 hours at 35°C, and each assay was checked for zones of growth inhibition (defined as a clear ring of at least 2mm around the impregnated disc). For the phototoxicity assay, a second group of plates was placed under a long wave UV light for one hour before being incubated in the same manner. This assay determines if the plant extracts become active with exposure to UV radiation. The brine shrimp assay was used to determine the cytotoxicity of the plant extracts as described in Meyer et al. 1982.

RESULTS

The disc diffusion assay was the only test to yield positive results. The brine shrimp assay showed no cytotoxicity and the phototoxicity assay showed no change in activity from the disc diffusion assay. The only bioactive extract was C. pallens, which inhibited the growth of two of the bacteria isolated from the bird feathers as well as B. subitilis and all three strains of C. albicans.

DISCUSSION

This study has demonstrated that the leaves used by C. ani and S. longirostris inhibit the growth of some microorganisms that colonize the birds’ feathers. Tree species other than C. pallens may deter arthropod parasites or other microfauna not tested here. In addition, due to field/lab conditions, there was a continual problem with extracts becoming contaminated with fungi. This contamination may have masked the activity of many of the samples.

Other members of the Cuculidae family should also be studied to determine if green plant material use in C. ani and S. longirostris is a convergently evolved adaptation in response to parasite pressures or if it is synapomorphy of nest architecture in the Cuculidae family. Crotophaga and Saurothera have been shown to be sister taxon in phylogenetic trees based on two different bodies of work; one that was constructed using phylogentic analyses of behavior and ecological characters, while the other used mitochondrial DNA (Hughes 1996; Hedges 1995).
Many questions remained unanswered, such as the identity of the microorganisms isolated and whether or not they are keratinolytic. Further research is necessary to isolate, identify, and test a wider variety of microorganisms from the birds in this study.

WORKS CITED

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