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Fig. 4 in Mosquito identification and haemosporidian parasites detection in the enclosure of the African penguins (Spheniscus demersus) at the SANBI zoological garden

Fig. 4. Maximum likelihood tree showing the clustering of Leucocytozoon sp. (Clade I) and Plasmodium sp. (Clade II) with Haemoproteus sp. as outgroup. Sequences from this study are highlighted with red circles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Maximum likelihood tree for Culex species showing the 5 clades representing 5 subgroups. Clade I in Mosquito identification and haemosporidian parasites detection in the enclosure of the African penguins (Spheniscus demersus) at the SANBI zoological garden

Fig. 3. Maximum likelihood tree for Culex species showing the 5 clades representing 5 subgroups. Clade I is the Trifilatus Subgroup (Mattingly and Rageau, 1958) for Cx. torrentium; Clade II and III are the Pipiens Complex; Clade IV the Theileri Subgroup (Sirivanakarn, 1976) for Cx. theileri; and Clade V is the Tarsalis (Edwards, 1932) for Cx. declaratory and Apicinus Subgroups (Edwards, 1932) for Cx. mollis. Lutzia sp. used as outgroups. Sequences from this study are indicated by asterisks (*).

Fig. 1 in Mosquito identification and haemosporidian parasites detection in the enclosure of the African penguins (Spheniscus demersus) at the SANBI zoological garden

Fig. 1. Map of South Africa showing the National Zoological Gardens (NZG). The red star indicates where the African penguin enclosure is located and where mosquito samples were collected (Labuschagne et al., 2008). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6 in Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus)

Fig. 6. Analysis of the sensitivity and specificity of the P. relictum MSP-1 capture antigen-based ELISA. Serial dilutions of normal chicken serum and three sera samples from P. relictum-infected penguins (8776, 8783 and 8784) were used for coating the assay wells. Each dilution was performed in triplicate, and the data shown represent means of three independent experiments with standard error bars and levels of statistical significance (****: P <0.0001).

Fig. 4 in Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus)

Fig. 4. Validation of ELISA. The ELISA developed using P. relictum MSP-1 protein was tested with known P. relictum positive penguin serum collected from penguin # 8790 at week 26. Black column represents positive penguin serum. Hatched and white columns represent negative controls containing normal chicken serum and PBS, respectively. Grey column represents the positive reaction containing streptavidin alkaline-phosphatase and its substrate pnitrophenyl phosphate. Each reaction was performed in triplicate, and the data shown represent means of three independent experiments with standard error bars and levels of statistical significance (****: P <0.0001).

Fig. 2 in Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus)

Fig. 2. Verification of the biotinylation of recombinant P. relictum MSP-1. Purified recombinant P. relictum MSP-1 protein was labeled with biotin. Biotinylation of MSP-1 was confirmed using an enzyme (streptavidin alkaline phosphatase)-linking assay and absorbance read at 405 nm indicated the presence of biotinylated MSP-1. Black column represents biotinylated MSP-1 protein. Hatched column and white column represent negative controls containing non-biotinylated MSP-1 and PBS, respectively. Grey column represents the positive control containing streptavidin alkaline-phosphatase (SAP) and its substrate p-nitrophenyl phosphate (PNPP). Each reaction was performed in triplicate, and the data shown represent means of three independent experiments with standard error bars and levels of statistical significance (****: P <0.0001).

Fig. 3 in Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus)

Fig. 3. Biotin-labeled MSP-1 protein titration curve. Serially diluted (300 ng/ μL to 0.003 ng/μL) biotinylated MSP-1 was used for coating the surfaces of the reaction wells overnight at 4 ◦C. The amount of biotinylated MSP-1 immobilized on the surface of the well was proportional to the intensity of the colored product generated which in turn was proportional to the absorbance value measured at 405 nm wavelength. Reactions were performed in triplicate, and the data shown represent means of three independent experiments with standard error bars.

Fig. 7. MSP-1 in Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus)

Fig. 7. MSP-1 capture antigen-based ELISA analysis of 370 sera samples collected from 11 penguins during three consecutive seasons of Spring (March to May), Summer (June to August) and Fall (September to November). (A) ELISA absorbance readings for the 370 sera samples collected from Spring to Fall are arranged in ascending order. Time of collection is indicated by color. Light grey represents Spring, dark grey represents Summer, and black represents Fall. Black dashed line indicates the single cut-off point (0.488) determined by change-point analysis. (B) Left: percentage of positive (A405 ≥ 0.488) and negative (A405 <0.488) sera samples; Right: distribution of positive samples in Spring (March to May), Summer (June to August), and Fall (September to November). (C) Percentage of penguin sera samples that tested positive (A405> = 0.488) in each month from March to November.

Fig. 5. P. relictum MSP-1 in Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus)

Fig. 5. P. relictum MSP-1 capture antigen-based ELISA analysis of test sera from penguins. Sera samples (370 total) from eleven penguins collected from Spring through Fall season were used as test samples in the assay to determine anti-P. relictum antibodies level. Light grey, dark grey, and black columns represent penguin sera samples collected in Spring, Summer, and Fall, respectively. Panel A–K represents ELISA results for individual penguins' sera collected at different time points from Spring to Fall. Panel L represents average ELISA absorbances for all 11 penguins at different sampling points. Each sample was assayed in triplicate, and the data shown represent means of three independent experiments with standard error bars.

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Intercolony variation in reproductive skipping in the African penguin mark-recapture data

In long-lived species, reproductive skipping is a common strategy whereby sexually mature animals skip a breeding season, potentially reducing population growth. This may be an adaptive decision to protect survival or a non-adaptive decision driven by individual-specific constraints. Understanding the presence and drivers of reproductive skipping behaviour can be important for effective population management, yet in many species such as the endangered African penguin (Spheniscus demersus), these factors remain unknown. This study uses multistate mark-recapture methods to estimate African penguin survival and breeding probabilities at two colonies between 2013 and 2020. Overall, survival (mean ± SE) was higher at Stony Point (0.82 ± 0.01) than Robben Island (0.77 ± 0.02). Inter-colony differences were linked to food availability; under decreasing sardine (Sardinops sagax) abundance, survival decreased at Robben Island and increased at Stony Point. Additionally, reproductive skipping was evident across both colonies; at Robben Island, the probability of a breeder becoming a nonbreeder was ~0.22, versus ~0.1 at Stony Point. Penguins skipping reproduction had a lower probability of future breeding than breeding individuals; this lack of adaptive benefit suggests reproductive skipping is driven by individual-specific constraints. Lower survival and breeding propensity at Robben Island places this colony in greater need of conservation action. However, further research on the drivers of inter-colony differences is needed.

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Fig. 2 in Mosquito identification and haemosporidian parasites detection in the enclosure of the African penguins (Spheniscus demersus) at the SANBI zoological garden

Fig. 2. The single UV-light trap used to collect mosquito samples.