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A study was made of the effect of monensin on the development of immunity to E. maxima, E. brunetti or E. tenella in chickens given repeated inoculations of oocysts. Birds were inoculated three times weekly with either 1000, 100 or 10 oocysts for a total of 13 inoculations. Faecal oocyst output was recorded throughout the experiment, and after 33 days, birds were challenged with 100,000 oocysts of each species. Whereas non-medicated birds given the schedule of 10 oocysts of E. maxima had a patent period of 12 days the duration of patency for E. brunetti and E. tenella was considerably extended. Exposure of non-medicated birds to a total of only 130 oocysts over a 28-day period resulted in a strong immunity to E. maxima and E. brunetti but protection against E. tenella was incomplete. Development of immunity in birds fed 125 parts per million monensin was dependent upon the number of oocysts administered. With the exception of the group receiving the repeated dose of 10 oocysts of E. maxima, monensin did not suppress oocyst production completely. Immunity developed in medicated birds given 100 oocysts of E. maxima or 1000 oocysts of E. brunetti Partial immunity appeared in medicated birds given 100 oocysts of E. brunetti or E. tenella. Whether sufficient coccidial development would occur to stimulate immunity in practical situations would depend upon the frequency and intensity of exposure to infective oocysts in the environment.

Chicks infected with the Weybridge strains of Eimeria maxima and E. acervulina were not protected by the normal levels of amprolium, but the sporulation of the oocysts was inhibited. With higher drug concentrations, fewer oocysts were produced. E. brunetti was not so markedly affected, although oocyst sporulation was reduced by the higher dosage levels. The effects were not enhanced by the inclusion of ethopabate.With dinitolmide the phenomenon was not so marked, although oocyats of E. maxima and E. acervulina were reduced in numbers by the normal drug concentration and sporulation was reduced when this level was increased. Much higher drug levels were required to obtain these effects with E. brunetti. Monensin at 120 ppm affected neither oocyst numbers nor their sporulation in any of the species tested. The significance of the effects of anticoccidial drugs on gametogony and sporogony is discussed.

The pathogenic effects of E. acervulina, E. brunetti, and E. maxima were modified when chickens received mixed infections with these species. Six-week-old chickens were inoculated with doses of 20,000 oocysts of E. acervulina, 1250 oocysts of E. brunetti, and 5000 oocysts of E. maxima given as a single or mixed infection. Typical signs of coccidiosis were apparent in chickens infected with a single Eimeria sp. When birds were given infections composed of two species, the weight loss was greater than that due to either given alone but when three species were given, weight loss was slightly less than that due to infection will E. brunetti alone. Oocyst production due to E. acervulina tended to be similar in birds given this species alone or with E. brunetti. Output fell to less than 50% when E. acervulina was administered with E. maxima. Oocyst production due to E. brunetti and E. maxima decreased when these species were inoculated together and when they were administered with E. acervulina. Lesions of E. acervulina and E. brunetti were superimposed on those of E. maxima in birds given mixed infections. Growth retardation was not evident in chickens inoculated with E. acervulina alone, although weight loss increased when this species was administered with either E. brunetti or E. maxima.

Eimeria brunetti, an intestinal coccidial parasite of poultry, caused the most severe mucosal damage when compared to E. mivati, E. necatrix, and E. maxima. The damage caused by E. brunetti was observed in the ileum and large intestine and consisted of complete villar destruction in these areas. Oocysts of E. brunetti were observed in tissue debris. E. necatrix (jejunum) enlarged isolated villi causing the lamina propria core to extrude through the villus tip. Damage to the mucosal surface is not as severe as lesions observed on the serosal surface would indicate. E. maxima (jejunum) caused epithelial sloughing and isolated patches of exposed connective tissue. Oocysts of E. maxima were observed under the surface epithelial layer. E. mivati damaged the villus tips of the duodenum and caused sloughing of the villar epithelia exposuncated and clubbed the duodenal villi. E. mivati oocysts were observed in the infected epithelia.

Additional file 1. Title and abstract in Spanish

Additional file 2 Detailed annotation process of 33 VOCs from Silverstoneia punctiventris. Comparison between manual and automatic annotation specifying IUPAC name of the compound, CAS number for each VOCs, theoretical retention index (RI theo), experimental retention index (RI exp), the difference between theoretical and experimental retention indexes (ΔRI), reference for theoretical RI (Ref RI theo), Classyfire Chemical Superclass, Classyfire chemical Class, Classyfire chemical Subclass/Alkaloid family, reference for behavioral function (Ref BF), retention time in minutes (rtmin), retention time in seconds (rtsec), binary code (Y/N) for selecting if there was a match with GNPS automatic deconvolution process (Match GNPS). When it was a match, in addition, we provided GNPS links of the annotation suspect list for each compound, cosine scores, and balance scores of the selected annotation. Manual annotation was performed using MSD ChemStation D.02.00.275 (Agilent technologies) employing NIST 14 database, and Daly et al. (2005) database. Automatic annotation was performed on the GNPS-GC-MS pipeline employing NIST, Wiley, University of CORSICA databases. When available, links of the annotation list for each compound were provided in the Supplementary material as well as experimental and theoretical retention indexes.

Brunetti, A. E., Hermida, G. N., Luna, M. C., Barsotti, A. M. G., Jared, C., Antoniazzi, M. M., Rivera-Correa, M., Berneck, B. V. M., Faivovich, J. (2015): Diversity and evolution of sexually dimorphic mental and lateral glands in Cophomantini treefrogs (Anura: Hylidae: Hylinae). Biological Journal of the Linnean Society 114: 12-34, DOI: 10.1111/bij.12406

La piel de los anfibios es un órgano dinámico con una enorme diversidad de funciones. Una característica compartida por prácticamente todas las especies de anfibios es lapresencia de dos tipos de glándulas dérmicas, mucosas y serosas. A pesar de la similitudestructural, estas glándulas pueden presentar una gran variabilidad morfológicaalcanzando un alto grado de especialización en el cumplimiento de dos funciones, lacomunicación química y la defensa contra predadores. Desde un aspecto bioquímico, elanálisis de las secreciones glandulares posee un potencial enorme dado la diversidad decompuestos. Los estudios desarrollados en algunos de ellos (i.e., péptidos, alcaloides,aminas biogénicas) permite tener una idea aproximada de la variación estructural yfuncional. Sin embargo, es limitado el conocimiento que se tiene sobre la química de loscompuestos volátiles y de los componentes secretados por las glándulas de la pielsexualmente dimórficas (SDSGs); fuente potencial de feromonas. Tradicionalmente losestudios de comunicación en los anuros se focalizaron en el análisis de las señalesacústicas, aunque existían reportes aislados de la participación de otras señales (i.e.,visual, química, táctil) en algunas especies. Los trabajos recientes muestran que estasseñales tienen una distribución taxonómica mucho mayor a la considerada inicialmente,en tanto que la co-ocurrencia de distintas señales sugieren la existencia decomunicación multimodal en algunas especies. En la presente tesis se abordaronaspectos morfológicos, químicos y comportamentales de las secreciones glandulares endos especies de Hypsiboas, considerando un contexto reproductivo en H. punctatus y uncontexto defensivo en H. pulchellus. Se analizó la estructura y la ultraestructura de la piel de las regiones mental,lateral, dorsal y ventral en machos y hembras de H. punctatus. Se identificaron dos tiposde SDSGs, en las regiones mental y lateral de los machos, representando el primer casode co-ocurrencia de diferentes SDSGs en una misma especie de anuro. A partir de estosresultados se caracterizó la variación estructural e histoquímica, y se estudió laevolución de las glándulas mentales y laterales en 28 especies de Cophomantini,incluyendo representantes de prácticamente todos los grupos de especies. La ocurrenciade estas glándulas, al igual que la variación morfológica, presenta una distribucióntaxonómica compleja con diferentes niveles de homoplasia. A partir del análisis de lassecreciones de las glándulas dorsales de machos y hembras se determinó la estructuraprimaria de 13 péptidos y se identificaron dos compuestos relaciones con alcaloides. Seidentificó un péptido presente exclusivamente en las secreciones de las glándulas mentaly lateral de los machos. Los estudios comportamentales en el campo y en condicionescontroladas de laboratorio, mostraron que los machos de H. punctatus son territoriales yposeen un repertorio vocal complejo con al menos siete cantos diferentes asociados acontextos particulares. Las hembras elegirían a los machos en base a la información delas señales acústicas y químicas, lo que sugiere la existencia de comunicaciónmultimodal en esta especie. El estudio morfológico e histoquímico de las glándulas de H. pulchellusevidenció la presencia de las dos glándulas comunes de los anfibios (mucosas yserosas). Sin embargo, los análisis de estructura, ultraestructura y de los mecanismosfisiológicos de descarga permiten inferir que las glándulas serosas cumplirían diversasfunciones, entre ellas almacenar los componentes volátiles secretados en situaciones deestrés. Estas glándulas presentan una mayor concentración en la región escapular, lo quepodría relacionarse con estrategias defensivas. A partir del análisis de las secreciones delas glándulas dorsales de machos y hembras se determinó la estructura primaria de 17péptidos y se identificaron dos compuestos relaciones con alcaloides. Las secuenciaspeptídicas de H. pulchellus e H. punctatus presentan una gran variabilidad. Estoscompuestos no fueron publicados anteriormente, aunque algunos de los péptidospresentaron una gran similitud con los descriptos en otras especies de Cophomantini. Secompararon las secuencias de todos los péptidos identificados hasta el momento en estatribu. El solapamiento de las secuencias de los péptidos de H. pulchellus e H. punctatuscon partes de péptidos mayores de otras especies sugiere la existencia deendopeptidasas específicas. Se caracterizó la fracción volátil de las secreciones de laregión dorsal y lateral en H. pulchellus e H. riojanus. Se identificaron 42 compuestos (40 en H. pulchellus y 37 en H. riojanus) pertenecientes a nueve grupos funcionales, loque constituye la mayor diversidad de compuestos volátiles identificados en una especiede anfibio. Los diferentes componentes tendrían distinto origen y función. The skin of amphibians is a dynamic organ with diverse functions. Almost all thespecies of this group of vertebrates possess two types of dermal glands, mucous andserous glands. Despite the structural similarities within each glandular type, they mayhave a great morphological variation getting a high degree of specialization related tochemical communication and defense against predators. From a biochemicalperspective, and given the high diversity of their compounds, researches on theglandular secretions have an enormous potential. In particular, the studies performed onsome of them (i.e., peptides, alkaloids, biogenic amines) allowed a generalunderstanding of their structural and functional diversity. However, knowledge of thechemical composition of the volatile secretions, as well as from the secretion ofsexually dimorphic skin glands (SDSGs; a likely source of sexual pheromones) inanurans, is very limited. Although there are isolated reports of the existence of differentsignals used for communication in some species (i.e., visual, chemical, tactile), studiesof communication in anurans have traditionally focused in the analysis of acousticsignals. Nonetheless, recent investigations show that those considered rare signals havea wide taxonomic distribution, whereas the simultaneous occurrence of some of themsuggest the existence of multimodal communication in several species. In this thesis areexamined morphological, chemical and behavioral aspects from de glandular secretionsof two species of Hypsiboas, considering a reproductive context in H. punctatus, and adefensive context in H. pulchellus. The structure and ultrastructure of skin from mental, lateral, dorsal, and ventralregions, was examined in males and females of H. punctatus. Two types of SDSGswere identified en mental and lateral regions of males. These results represent the firstcase of co-occurrence of different SDSGs in a single species. The structural andhistochemical variation, as well as the evolution, of mental and lateral glands wasanalyzed in Cophomantini from samples of 28 species including at least a representativeof almost all group of species of this clade. The occurrence of mental and lateral glandsand their morphological variation have an intricate taxonomic distribution with differentlevels of homoplasy. Thirteen peptides and two compounds related with alkaloids wereidentified from dorsal secretions of males and females. One peptide was foundexclusively from mental and lateral secretions of males. Behavioral studies performedin the field and under laboratory controlled conditions showed that males are territorial. They emit at least seven different type of calls associated to specific biological contexts. Females would choose their mates on acoustic and chemical signals, which suggest theexistence of multimodal communication in this species. Morphological and histochemical studies of the skin of H. pulchellus revealedthe presence of the two typical mucous and serous glands of amphibians. However, adetailed structural and ultrastructural analysis, as well as the physiological assessmentof the release mechanism of gland secretion, suggests that serous glands have severalfunctions. One of these functions is to accumulate, and under stress situations, secretevolatile compounds. These glands are highly concentrated in the scapular region, whichis possible related to defensive strategies. Seventeen peptides and two compoundsrelated with alkaloids were identified from dorsal secretions of males and females. Thepeptide sequences of H. pulchellus and H. punctatus are highly variable. Thesecompounds have not been published before, but some of them showed great similaritieswith peptides described in other species of Cophomantini. All peptides reported so farin species of this tribe were compared through sequence alignments. The sequences of H. pulchellus and H. punctatus overlapped longer sequences of other species suggestingthe existence of specific endopeptidase. The volatile secretions of H. pulchellus and H.riojanus were characterized from dorsal and lateral skin by a simulated stress situation. Forty-two compounds (40 in H. pulchellus and 37 in H. riojanus) from nine differentfunctional groups were identified in all samples. This represent the highest number ofvolatile compounds identified in a species of amphibian. The components would havedifferent origin and functions. Fil: Brunetti, Andrés E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.