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Clostridium difficile is an increasingly common cause of nosocomial infection. C. difficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage to mild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. It is not yet fully understood why this spectrum is seen, however, it is believed that the immune response mounted by an individual plays an important role in determining the outcome of infection. This thesis comprises three studies. Firstly, a comparative study of immune cell populations within the lamina propria of colonic tissue not exhibiting pathological changes and taken from individuals with symptomatic CDI (cases); asymptomatic carriers; and non-colonised controls. Effector T cells, B cells, plasma cells and macrophages were enumerated by means of immunohistochemical staining of tissue sections. Secondly, a study to establish the prevalence within these three study groups of specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8 genes by PCR genotyping and to determine whether an association existed between these genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms of moxifloxacin resistance in a collection of clinical isolates. This study also sought to determine whether the competitive advantage conferred by resistance to moxifloxacin influenced the fitness of C. difficile isolates, in particular growth and the expression of the virulence factors toxins A and B. Carriers were found to have fewer of all four immune cell types quantified than both cases and controls. However, in only one instance, that of plasma cells, was this difference statistically significant. Cases had fewer of all cell types than controls but these differences were not significant. These findings suggest that individuals who become infected, both symptomatically and asymptomatically, with C. difficile display altered mucosal immune cell populations when compared with those of uninfected individuals. The data regarding host polymorphisms are suggestive of an association between the presence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2 genotypes were carried by cases and carriers while the variant TLR5 genotype was carried by cases only. No variant genotypes were present in control subjects. All moxifloxacin resistant isolates characterised in this study, with the exception of an isolate with intermediate resistance and a third-generation mutant with reduced susceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Efflux pumps are known to play a role in multi-drug resistance in many bacterial species. Semiquantitative PCR analysis of expression of the putative efflux pumps cme and cdeA found no correlation between overexpression and moxifloxacin resistance, suggesting that these genes do not play a role. Three novel mutations in the putative promoter region of CD3197, a MerR family transcriptional regulator found immediately upstream of cme, were identified. No association between the presence of these mutations and overexpression of cme or resistance or sensitivity to moxifloxacin was found. The competitive advantage conferred by resistance to moxifloxacin does not influence the fitness of C. difficile isolates, as measured in terms of growth and toxin production.

Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.

Earthworms, Lumbricus terrestris, were challenged orally and intracoelomically with two bacterial species, Aeromonas hydrophila and Pseudomonas aeruginosa, and mortality rates were observed. Neither were found to be particularly pathogenic at injected doses of up to 108 bacteria per earthworm. The influence of Cu++ (as CuSO4) on the earthworm's response to bacterial challenge was investigated by exposing earthworms to sublethal levels of Cu++ prior to bacterial challenge. Exposure at sublethal concentrations up to 3 m g/cm2 did not have a pronounced influence on host resistance to challenge as measured by earthworm mortality. Cu++ increased the earthworm's ability to agglutinate rabbit erythrocytes, indicating that Cu++ exposure caused coelomocyte death, autolysis and release of agglutinins into the coelom, possibly explaining resistance to bacterial challenge.

Interleukin-l0 (IL-10) helps to limit the duration of potentially harmful inflammatory responses but has also been implicated in the persistence of a number of chronic viral infections. This thesis aimed to investigate the phenotype and function of mv -specific IL-l0-producing cells in chronic HIV-I infection, and the effect of IL-10 blockade on responses to candidate HIV -I vaccines. A cytokine capture assay was used to determine the HIV -specific cellular sources of IL- 10 in PBMC from 55 chronically infected individuals. A rare subset of CD8+ T cells was found to be the major HIV -I Gag-specific IL-10-producing population; these cells were restricted to ART-naive individuals and did not express the regulatory T cell markers CD25 or FoxP3 but could co-express IFN-y. A proportion of the population (median 48% and 9% respectively) expressed the P7 chain of the gut-homing integrin a4p7 and the chemokine receptor CXCR3, which mediates lymphocyte migration to sites of inflammation. Experimental depletion of Gag-specific IL-10+ CD8+ T cells did not affect T cell activation, or the production of cytokines such as IL-2 or IFN-y during short-term culture. However, depletion was associated with a significant increase in CD38 expression on CDI4+ monocytes, a trend towards increased HLA-DR expression on the same cells, and a significant increase in the concentration of the pro-inflammatory cytokine IL-6 in culture supernatants. There was also a significant increase in the number of HIV-infected (p24 antigen+) CD4+ T cells in cultures depleted of Gag- specific IL-10+ CD8+ T cells after 3 days, indicating that this population may contribute to control of viral replication. In order to determine the effect of IL-10 blockade on vaccine immunogenicity, IL-10R blocking antibody was administered to BALB/c mice prior to immunisation with two mV-I candidate vaccines, HIVA and HIVconsv. IL-10R blockade resulted in a trend towards increased IFN-y production by CD8+ T cells in response to the dominant H (Env) and P (Pol) epitopes of HIV A, and a significant increase in IFN-y ELISPOT responses to the subdominant Gl (Gag) epitope of HIV consv in vitro. Collectively, these data suggest that IL-10 producing cell populations may play critical but different roles in chronic infection and vaccination. Further research into how the timing of IL-10 responses affects disease outcome may allow IL-IO blockade to be explored as a therapeutic strategy in humans

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Insects are confronted by a large variety of potentially harmful microorganisms to which they are resistant as they are able to build up an efficient innate defense system that relies on three tightly interconnected reactions. One of the reactions is the transient and rapid synthesis of a battery of antimicrobial peptides (AMPs). Development of antimicrobial therapeutic drugs and vaccines is very crucial due to factors such as the emergence of multiple-drug resistance. AMPs have been termed natural antibiotics because of their large spectrum of activity. The current study focused on the isolation and identification of cationic antimicrobial peptides and the analysis of immune response in the South African Euoniticellus intermedius embryonic (SAEIE08) cell line upon exposure to pathogens. E. intermedius is of the Coleopteran order in the Scarabaeoidea superfamily. Liquid growth inhibition assay showed higher antimicrobial activity in SAEIE08 that was treated with heat-killed E. coli compared to untreated. Further evidence for antimicrobial activity was seen as a clear zone of inhibition in solid growth inhibition assay when a gel run with protein extracts was plated and overlayed with live E. coli. Changes in protein expression patterns that were analysed in SDS-PAGE and 2-D PAGE indicated the most intense bands and spots at low molecular weight sizes around 10 kDa and/or 16 kDa which implicated increased induction of AMP expression upon exposure to pathogen. Homologues of Saccharomyces cerevisiae proteins were found in some of the 5′/3′ RACE sequences. Possible explanation for matches to these homologues could be that short sequences were used for database searches. The proteins were identified as flavin-containing monooxygenase, long-chain fatty acyl-CoA synthetase, severe depolymerization of actin protein and serine/threonine protein kinase. Interestingly, these proteins play roles in metabolism, cell proliferation and/or molecular pathways which do occur when cells are exposed to stress. There was also an insect peptide allatotropin from Spodoptera frugiperda. The results show that there is inducible antimicrobial activity in embryonic E. intermedius cell line.

A desnutrição protéico-energética (DPE) afeta mais de 1 bilhão de pessoas no mundo, principalmente crianças, idosos e pacientes hospitalizados. Ela provoca alterações metabólicas e hormonais, além de afetar o tecido hemopoético. O comprometimento da hemopoese provoca anemia e leucopenia, modificando a resposta imune inata e adquirida do organismo. Dessa forma, é comum a associação entre desnutrição e infecção, levando ao comprometimento do tratamento e aumento da morbidade e mortalidade de indivíduos hospitalizados. Após a recuperação nutricional, é relatada a reversão das alterações bioquímicas e hormonais, bem como das alterações na hemopoese e na resposta imune. Porém, muitos resultados são controversos, existindo dúvidas quanto à reversibilidade das alterações. Assim, nos propusemos a avaliar os efeitos da recuperação nutricional nos parâmetros bioquímicos, hormonais, hematológicos e imunológicos em modelo murino de desnutrição. Os animais desnutridos apresentaram perda de peso significativa, redução de proteínas totais, albumina, glicose, insulina e IGF-1, bem como aumento de glutamina plasmática, glutamina sintetase muscular e corticosterona. Houve redução dos parâmetros hepáticos e musculares, bem como alteração na sensibilidade à insulina, evidenciada pelos testes de OGTT e ITT. Todas as alterações descritas caracterizam o quadro de desnutrição. Após a recuperação nutricional, alguns parâmetros foram normalizados, mas as concentrações de glicose, insulina e IGF-1 permaneceram reduzidas. Da mesma forma, as alterações na concentração de DNA hepático e na sensibilidade à insulina permaneceram nos animais renutridos. A pancitopenia periférica e hipocelularidade da medula óssea e do baço observadas nos animais desnutridos foram revertidas após a renutrição. A avaliação de macrófagos peritoniais mostrou reversão parcial do comprometimento da capacidade e adesão e espraiamento, bem como da atividade fungicida nos animais renutridos. A produção de peróxido de hidrogênio continuou baixa após a recuperação nutricional, enquanto a produção de óxido nítrico voltou a aumentar. O comprometimento da produção de citocinas pró-inflamatórias decorrente da desnutrição não foi completamente revertido, visto que, em camundongos Swiss Webster, somente a produção de TNF-α retornou ao normal, enquanto em camundongos C56BL/6J a produção de nenhuma citocina foi restabelecida. A avaliação da via de sinalização do fator de transcrição NFkB mostrou alteração na expressão de MyD88, TRAF-6, IkKβ e IkBα em animais desnutridos. Após a recuperação nutricional, algumas dessas proteínas não retornaram ao normal. Os animais desnutridos também apresentaram comprometimento da ativação de NFkB, que não foi normalizada após a recuperação nutricional. Sendo assim, é possível afirmar que o retorno a uma dieta normoprotéica não é suficiente para reverter todas as alterações causadas pela desnutrição. / Protein-energy malnutrition (PEM) affects more than 1 billion people worldwide, mainly children, elderly and hospitalized patients. It causes metabolic and hormonal changes, besides affecting hematopoietic tissue. Impaired hematopoiesis causes anemia and leukopenia, modifying innate and acquired immune response of the organism. Thus, it is common the association between malnutrition and infection, leading to impaired treatment and increasing morbidity and mortality in hospitalized patients. After nutritional recovery, it is reported reversal of biochemical and hormonal changes, as well as, reversal of changes in hematopoiesis and immune response. However, many results are controversial, and there are doubts about the reversibility of the changes. Thus, we proposed to evaluate the effects of nutritional recovery biochemical, hormonal, haematological and immunological parameters in a murine model of malnutrition. The malnourished animals showed significant weight loss, reduction in total protein, albumin, glucose, insulin and IGF-1, as well as increased plasma glutamine, corticosterone and muscle glutamine synthetase. There was a reduction in muscle and liver parameters as well as change in insulin sensitivity, evidenced by the tests of OGTT and ITT. All modifications described characterize the malnutrition. After nutritional recovery, there was normalization of some parameters, but the concentrations of glucose, insulin and IGF-1 remained low. Likewise, changes in hepatic DNA concentration and insulin sensitivity remained in renourished animals. Peripheral pancytopenia and hypocellularity in bone marrow and spleen observed in malnourished animals were reversed after refeeding. The evaluation of peritoneal macrophages showed partial reversal of impairment of adhesion and spreading ability, as well as fungicidal activity in animals renourished. The hydrogen peroxide production remained low after nutritional recovery, while nitric oxide production increased again. Impaired production of proinflammatory cytokines due to malnutrition was not completely reversed, whereas in Swiss Webster mice, only the production of TNF-α returned to normal, whereas in C56BL/6J mice no cytokine production was restored. The assessment of the signalling pathway of transcription factor NFkB showed alterations in the expression of MyD88, TRAF-6 IkKβ and IkBα in malnourished animals. After nutritional recovery, some of these proteins didn\'t return to normal. Malnourished animals also showed impaired activation of NFkB, which wasn\'t normalized after nutritional recovery. Therefore, it is possible to say that the return to a normal diet is not enough to reverse all the changes caused by malnutrition.

A dehidroepiandrosterona (DHEA) tem sido considerada como o esteróide de múltiplas ações e vem interessando pesquisadores da área médica. Trabalhos demonstram a estimulação imunológica induzida pelo DHEA em animais de laboratório e humanos, como terapia alternativa e imunomoestimuladora nas infecções virais, bacterianas e parasitárias. O presente projeto avaliou os efeitos terapêuticos da administração de DHEA em ratos Wistar machos e fêmeas infectados com a cepa Y de Trypanosoma cruzi durante a fase aguda da infecção chagásica. Os parâmetros analizados foram o número de parasitas sangüíneos e teciduais, bem como produção de citocinas (TNF-, IFN-, IL-2, IL-12, IL-10, IL-4 e TGF-) e populações celulares (CD3+, CD4+ e CD8+), análise de glicose, colesterol e triglicérides, produção de anticorpos líticos, óxido nítrico, contagem global de leucócitos e macrófagos. Através dos resultados obtidos podemos concluir que o DHEA quando utilizado como tratamento na infecção chagásica experimental é parcilamente eficaz, pois reduz o número de parasitas sanguíneos e teciduais em ratos machos e fêmeas. Os diferentes parâmetros imunológicos analisados na vigência da terapia com DHEA revelou uma ação parcialmente efetiva sobre a resposta imunológica, o que favoreceu o controle da evolução da fase aguda da infecção experimental, deve ser levado em conta que a utilização deste hormônio não assume caráter curativo, apenas propicia melhores condições ao hospedeiro de direcionar sua resposta imunológica de forma a controlar a replicação parasitária, sem causar no hospedeiro os efeitos colaterais danosos ocasionados pelas drogas tripanossomicidas disponíveis atualmente no mercado. / Dehidroepiandrosterone (DHEA) has been considered for many researchers as the steroid of multiple functions. The immunologic stimulation triggered by DHEA has been demonstrated not only in animal models but also in humans. DHEA has been used as an alternative therapy to up-modulate immune response in host bearing viral, bacterial and parasitic infections. The present work evaluate the therapeutic effects of DHEA administration in male and female Wistar rats infected with the Y strain of T. cruzi during the acute phase of infection. The evaluated parameters were the blood and tecidual parasitic intensity, cytokines and cellular population, glucose analysis, cholesterol and triglycerides, percentage of lytic antibodies, nitric oxide, global counting of leukocytes and macrophages. DHEA, when used as a treatment of experimental T. cruzi infection is efficient; reducing the number of blood and tissue parasites of both genders. The therapy with DHEA demonstrated cellular and humoral immune stimulation for the control of the evolution of the acute phase. It has to be emphasized that DHEA therapy is not curative, but improves to the host immunological response to control the parasitic burden, without the harmful collateral effects caused by the available T. cruzi drugs in the market.

Glicocorticóides modulam a resposta imune de forma complexa em vertebrados expostos a diferentes estressores. Dado que as populações naturais têm estado expostas a uma multiplicidade de estressores, uma melhor compreensão da associação funcional entre a duração e a intensidade da resposta ao estresse, as mudanças resultantes nos níveis dos hormônios esteróides e seu impacto sobre os diferentes aspectos da imunocompetência emergem como um ponto chave para as estratégias de conservação dos vertebrados. Nós investigamos as relações entre os níveis plasmáticos de hormônios esteróides e a imunocompetência inata em anfíbios anuros, incorporando as metodologias de desafio de contenção, elevação experimental dos níveis de corticosterona por aplicação transdérmica, capacidade bactericida por espectrofotometria e o desafio imunológico com fitohemaglutinina. Nossos resultados demonstram que a capacidade bactericida plasmática (CBP) medida por espectrofotometria é um método confiável e preciso para estimar a imunocompetência de anfíbios anuros, além disso, mostramos a existência de uma grande diversidade interespecífica na CBP de anuros machos. Quando quatro diferentes espécies de Bufonídeos foram submetidas a um desafio de contenção, as respostas gerais incluíram aumento dos níveis plasmáticos de corticosterona (CORT) e da relação neutrófilo/linfócito (N:L) e diminuição dos níveis plasmáticos de testosterona (T). As respostas da CBP à contenção foram muito mais variáveis, com R. ictérica mostrando diminuição e R. marina mostrando aumento dos valores de CBP. Adicionalmente, CORT e N:L tenderam a aumentar mais em resposta à contenção com restrição de movimento do que à contenção sem restrição de movimento, indicando que os sapos demostraram um aumento da resposta ao estresse quando submetidos ao estressor mais intenso. Todas as variáveis estudadas mostraram variação interespecífica. Rhinella ornata apresentou os maiores níveis basais de CORT quando comparado com as outras espécies, enquanto R. ornata e R. ictérica mostraram os maiores valores basais de CBP. Entretanto, as mudanças na relação N:L, nos níveis de T e na CBP, não foram correlacionadas com o aumento em CORT, dentro ou entre espécies. A aplicação transdérmica de corticosterona eficientemente simulou eventos repetidos de resposta ao estresse agudo em Rhinella ictérica, sem alterar os parâmetros imunitários, mesmo após treze dias de tratamento. Curiosamente, o cativeiro a longo prazo não atenuou a resposta ao estresse, uma vez que estes sapos mantiveram um aumento de três vezes em CORT mesmo depois de três meses sob estas mesmas condições. Além disso, a manutenção em cativeiro a longo prazo, nas mesmas condições, aumentou a contagem total de leucócitos (TLC) e gerou uma diminuição ainda maior na CBP, sugerindo que as consequências da resposta ao estresse podem ser agravadas pelo tempo em cativeiro. Com base em nossos resultados, consideramos que uma avaliação cuidadosa é necessária para compreender a modulação da resposta imunitária pelo estresse a nível intra e interespecífico. A inclusão de diferentes segmentos da resposta imune é desejável, e a padronização da coleta de dados para todas as espécies sob o mesmo período (em geral, dentro ou fora da época reprodutiva) e mesma atividade (em geral, vocalizando ou forrageando) se faz obrigatória / Glucocorticoids modulate the immune response in complex ways in vertebrates exposed to different stressors. Given that natural populations have been exposed to a multitude of stressors, a better understanding of the functional association between duration and intensity of the stress response, the resulting changes in steroid hormone levels and their impact on different aspects of immunocompetence emerges as a cornerstone for vertebrate conservation strategies. We investigated the relationships between steroids levels and innate immunocompetence in anuran amphibians, incorporating the methodology of restraint challenge, experimental elevation of corticosterone levels by transdermal application, bacterial killing ability by spectrophotometry and the immune challenge with phytohemagglutinin. Our results demonstrate that the bacterial killing ability (BKA) measured by spectrophotometry is a reliable and accurate method to estimate the immunocompetence of anuran amphibians, additionally showed the existence of a large interspecific diversity in BKA from male anurans. When four different species of Bufonids were submitted to a restraint challenge, the general responses included increased in corticosterone plasma levels (CORT) and neutrophil/lymphocyte ratio (N:L) and decreased in testosterone plasma levels (T). The responses of BKA to restraint were much more variable, with R. icterica showing decreased and R. marina showing increased values. Additionally, CORT and N:L tended to increase more in response to restraint with movement restriction than to restraint without movement restriction, indicating that toads showed an increased stress response to the more intense stressor. All variables studied show interspecific variation. Rhinella ornate showed higher baseline CORT when compared to other species, while R. ornate and R. icterica showed the highest baseline BKA values. However, changes in N:L ratio, T levels and BKA, were not correlated to increased CORT within or between species. Transdermal application of corticosterone efficiently mimics repeated acute stress response events in Rhinella icterica, without changing the immune parameters even after thirteen days of treatment. Interestingly, long-term captivity did not mitigate the stress response, since the toads maintained three fold increased CORT even after three months under these conditions. Moreover, long-term captivity in the same condition increased total leukocyte count (TLC) and generated an even greater decrease in BKA, suggesting that consequences of the stress response can be aggravated by time in captivity. Based on our results, we consider that a careful evaluation is necessary in order to understand the modulation of the immune response by stress at intra and interspecific levels. The inclusion of different segments of the immune response is desirable, and a standardized data collection for all the species under the same period (e.g. inside or outside of breeding season) and same activity (e.g. calling or foraging) is mandatory