3.4 Discussion
4.4.6 Further advice for future work
Due to the distance between the field site and the laboratory there was a delay in getting samples plated. This could affect the results, as there may have been some fungal death during transit. Ideally, a laboratory closer to the study site should be chosen in future studies. However, most NIBK populations are far removed from cities and thus this may reduce the number of populations that may be available for this sort of research.
Fungal identification is notoriously difficult. Currently only 5-10 per cent of fungi species on earth have been named and scientifically described (Carlile et al., 2001). This study is one of the few to identify fungi from the shells of wild bird eggs, yet morphological characteristics only allowed for identification to the genus level. Initially, the Omnilog® fungal identification system was used which has a database of over 297 yeast species and fungi from over 120 genera, yet it failed to identify any of the isolates tested in it. Aspergillus, Penicillium and yeasts are all large groups, with many species within them. The reasons why the Omnilog® may have failed to identify the isolates are as discussed in chapter three, section 3.4.6. DNA sequencing methods for general fungi identification are still inaccurate and expensive (Ferrer et al., 2001) when compared to bacterial identification and this is why I did not attempt it in this study. Further identification is needed, as not all fungal species are pathogenic. However, this remains hindered by the lack of accurate and reliable methods.
A further issue faced in this study was fungi not surviving the glycerol preparation and -80oC storage and therefore this method needs to be improved for any further studies. Higher fungi survival would improve sample size as each isolate is important and contributes significantly to the overall conclusions drawn from the data. Another
storage technique that could be trialled is the use of agar slants with a mineral oil overlay (McGinnis, 1980; Ramirez, 1982). One could also identify the isolates immediately once they have been ensured of purity and remove the reliance on storage. I suggest making multiple glycerols for each isolate to increase the chances of the isolate surviving. In this study most fungi survived the long-term storage method and this means they are available for identification in future work (see chapter seven, section 7.3).
As mentioned, some fungi have the ability to increase bacterial penetration into the egg contents (see chapter two, section 2.3.2) (Board and Halls, 1973; Baggot and Graeme-Cook, 2002; Cook et al., 2003). Future research could be undertaken to investigate if there is a correlation between fungal types on NIBK eggshells and the presence of microbes inside the eggs. This was not possible in this project as different NIBK eggs were used for shell and content analysis due to the restrictions on carrying out microbial work on the contents of NIBK eggs (see chapter five). Future work on the correlation of fungal presence and hatching success of NIBK eggs, with a larger sample size, would also be a beneficial line of research.
Chapter five: Identification of bacteria in the contents
of un-hatched, wild North Island Brown Kiwi eggs and
5.1 Introduction
Bacterial infection inside the eggs of the domestic chicken (Gallus gallus domesticus) has long been known to cause both declines (Bruce and Drysdale, 1991) and increases in hatching success (Ribble and Shinefield, 1967). There is now growing evidence that bacteria could also be important factors reducing the hatching success of domestic ratites (Deeming, 1995a, 1996; Moore, 1996; La´Baque et al., 2003; Chang-Reissig et al., 2004) and wild birds (Kozlowski et al., 1991b; Peralta-Sánchez, 2010).
Avian eggs have a high nutrient content and the optimal temperature for growth of most bacteria is similar to the optimum temperature necessary for avian incubation, which make them highly suitable for microbial growth (Burley and Vadehra, 1989). Avian eggs are not defenceless, possessing several physical and chemical barriers against bacterial attack (see chapter two, section 2.4). In addition, parental behaviours such as incubation and providing specific nesting material may have an anti-microbial benefit (see chapter two, section 2.4.1) (Clark and Mason, 1985; Peralta-Sánchez, 2010). That avian eggs have several, complex defences against microbes suggests that microbes play an important role in avian evolution and supports the theory that microbes significant threat to the avian egg (see chapter two, section 2.4) (Peralta- Sánchez, 2010).
The North Island Brown Kiwi (NIBK, Apteryx mantelli) is endangered and experiencing significant population decline, even with intensive management (Holzapfel et al., 2008) (see chapter one, section 1.4). As discussed in previous chapters (see section 1.3 and 3.1) the main conservation strategies in place focus on predator control to protect NIBK chicks, yet only three out of every ten wild eggs successfully hatch. While predation and infertility are not high enough to explain the high hatching failure of NIBK eggs, previous studies have noted a high level of microbial contamination in contents of wild NIBK eggs (McLennan, 1988; Potter, 1989; McLennan et al., 1996; Ziesemann et al., 2011); and as shown in chapters three and four high levels of bacteria and fungi have been isolated off wild NIBK eggshells
However, having microbes on the shell does not indicate whether those microbes will be able to penetrate into the contents and affect the developing embryo. Bacteria
isolated from inside the egg pose more of a risk to the developing embryo than the bacteria isolated from the shell (Bruce and Drysdale, 1994). Thus, to fully understand the likely effect of microbes to NIBK eggs, it is necessary to investigate whether microorganisms can overcome the various defences of the egg and access the egg contents.
The aim of this study was to determine if bacteria are present inside NIBK eggs that could impact NIBK hatching success, the theory was that these bacteria would have a higher potential to impact the hatching as they have crossed the cuticle, shell and membrane barriers. I would expect higher levels of microbes present inside NIBK eggs than other wild bird’s eggs studied due to the factors that make NIBK susceptible to microbial infection (see chapter one, section 1.2). In light of the results of this study, several suggestions are made in regards to NIBK conservation and egg hatching success.
5.2 Methods
Egg source and laboratory site are listed in chapter one (see section 1.2.2 and 1.2.3).