Starling Sturnus vulgaris

Various different species of birds have been shown to regularly introduce fresh green plants into their nests; including starlings S. vulgaris (Clark & Mason, Reference Clark and Mason1985; Gwinner, Reference Gwinner1997; Brouwer & Komdeur, Reference Brouwer and Komdeur2004; Gwinner & Berger, Reference Gwinner and Berger2006), tits Paridae (Cowie & Hinsley, Reference Cowie and Hinsley1988; Banbura et al. Reference Banbura, Blondel, Wilde-Lambrechts and Perret1995; Lambrechts & Dos Santos, Reference Lambrechts and Dos Santos2000; Petit et al. Reference Petit, Hossaert-McKey, Perret, Blondel and Lambrechts2002; Mennerat, Reference Mennerat2008; Mennerat et al. Reference Mennerat, Perret and Lambrechts2009a ; Pires et al. Reference Pires, Belo and Rabaca2012; Tomas et al. Reference Tomas, Merino, Martinez-de la Puente, Moreno, Morales, Lobato and del Cerro2012), sparrows Passer (Sengupta, Reference Sengupta1981; Milton & Dean, Reference Milton and Dean1999), wood storks Mycteria americana (Rodgers et al. Reference Rodgers, Wenner and Schwikert1988), even raptors (Wimberger, Reference Wimberger1984; Malan et al. Reference Malan, Parasram and Marshall2002; Ontiveros et al. Reference Ontiveros, Caro and Pleguezuelos2008; Dykstra et al. Reference Dykstra, Hays and Simon2009; Heinrich, Reference Heinrich2013). The behaviour is most extensively studied in starlings and blue tits, between which there are some major differences.

In starlings the plants are brought exclusively by the males, who are at the same time singing to attract females (Clark & Mason, Reference Clark and Mason1985; Gwinner, Reference Gwinner1997). Male starlings are also often polygynous, competing for multiple females to nest with. This, combined with the fact that amount of greenery brought is a function of the length of courtship and that males carry fresh green plants to the nest in an ‘eye catching manner’, lends more support, at least in the case of starlings, towards the plants serving a role in bond formation or male status signalling (mate hypothesis). Clark & Mason (Reference Clark and Mason1985) also found that the plants preferred by starlings (i.e. found in a higher proportion in nests than in the environment) produced higher concentrations of volatile compounds, than a random subset of plants from their environment.

Later studies have shown that starlings have a seasonal variation in olfactory sensitivity, and that during the breeding season changes suspected to occur in their olfactory bulb allow them to use olfactory cues for plant detection (Clark & Smeraski, Reference Clark and Smeraski1990; De Groof et al. Reference De Groof, Gwinner, Steiger, Kempenaers and Van der Linden2010). Gwinner & Berger (Reference Gwinner and Berger2008) also showed, through the use of aviary choice experiments involving chicks reared in different scented nests, that green nesting material selected through olfaction is reliant on experience-dependant as well as ‘innate’ cues. Blue tits have also been shown to have olfactory abilities sensitive enough to detect small changes in their aromatic environment as a result of the plants they incorporate; either when replacing herbs removed in experiments or reacting to experimental inclusion of herbs into their nests (Petit et al. Reference Petit, Hossaert-McKey, Perret, Blondel and Lambrechts2002; Mennerat et al. Reference Mennerat, Bonadonna, Perret and Lambrechts2005; Mennerat, Reference Mennerat2008).

Blue tit Cyanistes caeruleus

In blue tits the behaviour is performed mainly by females and continues throughout the egg laying and chick stages and actually increases through the breeding cycle (Lambrechts & Dos Santos, Reference Lambrechts and Dos Santos2000). This occurs when parasite load, and therefore threat to chicks in the nest, would be highest (Tripet & Richner, Reference Tripet and Richner1999). Two studies have noted that fresh green plants are brought by females specifically in the evening, prior to roosting (Cowie & Hinsley, Reference Cowie and Hinsley1988; Banbura et al. Reference Banbura, Blondel, Wilde-Lambrechts and Perret1995). However, in these studies the plants were either not accurately identified or were not specifically aromatic, and therefore may serve as insulation. A range of highly aromatic herbs, detectable even by human olfaction, have been recorded being brought to the nest by blue tits, and the behaviour in this species has only been reported in Mediterranean regions of Europe; mainly Corsica (Banbura et al. Reference Banbura, Blondel, Wilde-Lambrechts and Perret1995; Lambrechts & Dos Santos, Reference Lambrechts and Dos Santos2000; Petit et al. Reference Petit, Hossaert-McKey, Perret, Blondel and Lambrechts2002; Mennerat, Reference Mennerat2008) and Portugal (Pires et al. Reference Pires, Belo and Rabaca2012). Mennerat et al. (Reference Mennerat, Perret and Lambrechts2009a ), showed there to be significant variation in herb composition between individuals within a territory and that this variation was conserved between years, suggesting an individual preference by blue tits for different aromatics. The inclusion of these specifically aromatic herbs, and the fact that in monogynous tits there is no obvious involvement in their annual courtship ritual, have lent more support towards the NPH for these species.

Starling S. vulgaris

Clark & Mason (Reference Clark and Mason1985) found that the same plants that were preferred by starlings and produced higher concentrations of volatile compounds, were also more effective at preventing Menacanthus (louse) eggs from hatching and at inhibiting bacterial growth in vitro. They also found that wild carrot Daucus carota, naturally used by starlings, can decrease numbers of northern fowl mite Ornithonyssus sylviarum in the nest and that chicks in those nests had higher haemoglobin (Loye et al., Reference Loye, Zuk and Clark1991); a direct factor in flight fitness of fledglings (Kovach & Szasz, Reference Kovach and Szasz1968). Along with Erigeron philadelphicus (Asteraceae), also preferred by starlings, it can also decrease the emergence of feeding instars of the northern fowl mite (Clark & Mason, Reference Clark and Mason1988).

Gwinner et al. (Reference Gwinner, Oltrogge, Trost and Nienaber2000), replaced 148 starling nests over 3 consecutive years with entire man-made nests containing a mix of fresh and dry grass (Brachypodium silvaticum), to mirror the natural starling nests found in their area. Some were then supplemented with a mix of six aromatic herb species in order to investigate the effects of these herbs on chicks and parasite levels. They found no decreases in parasite numbers with herbs, but did find that starling chicks from nests to which herbs were added were heavier and had higher haematocrit, providing support for the drug hypothesis. The number of basophils, eosinophils, lymphocytes and heterophils per hundred leucocyctes were also assessed, after ‘Haema Schnellfaerbung’ staining. They found chicks from herb nests had lower lymphocyte levels; which could suggest they faced less of an immune challenge. This could be correlated with the lower bacterial loads also found in their herb nests. However at the same time the blood smears from herb chicks also contained higher levels of basophils; another white blood cell involved in parasite defence. Gwinner and Berger continued the study and data was collected on fledgling mass for a further 2 years (N = 202), mite load scores for 1 more year (N = 138) alongside bacteria (N = 80) and mosquitoes (N = 51) for a year each. After this increase in sample size still no effect of the plants was found on mite numbers, however fewer bacteria were collected from herb nests and chicks from these nests again had a significantly higher weight (Gwinner & Berger, Reference Gwinner and Berger2005). Fewer mosquitoes were found in herb nests, however, mosquitoes numbers were low and this result was not quite significant (P = 0·055, n = 26/25) (Gwinner & Berger, Reference Gwinner and Berger2006).

Brouwer & Komdeur (Reference Brouwer and Komdeur2004) investigated the NPH and mate hypothesis by emptying half of their starling nest-boxes to provide parasite free nest-boxes, while leaving some infested nest-boxes containing old nests. They also assigned half these nests to green removal or green addition treatments; nests either had a selection of the average species found naturally in their starlings’ nests introduced or had any of the fresh green material added by the starlings themselves removed. This provided a 2 × 2 experimental design investigating the effects of the fresh green plant material added by the starlings and the presence of an old nest, on parasite levels, scabbing caused by mites on chicks and other parameters of chick health. They found no effect of experimentally increasing fresh plants in starling nests on parasite number, scabbing, chick body mass or survival. They also found no preference by starlings for the emptied nest-boxes and that no more greenery was added to the infested nest-boxes; it is difficult to assess how high the parasite loads were that remained in the ‘infested’ nest-boxes and there could also have been multiple other resources, such as time and energy, gained by a starling nesting in a box with an old nest in it. Chicks from cleaned boxes did have significantly lower scab scores but there was no correlation between scabbing and green material manipulations. They did however find that nests from which herbs were experimentally removed produced fewer clutches. Fauth et al. (Reference Fauth, Krementz and Hines1991) showed that scab scores on starling chicks could be lowered when parasite numbers were decreased, through use of insecticide, but again failed to show any effects of experimentally introducing herbs on parasite numbers. Brouwer & Komdeur (Reference Brouwer and Komdeur2004) confirmed earlier findings that fresh green plants are brought to the nest only by males. Female spotless starlings Sturnus unicolor, a similar species with the same plant bringing behaviour have been shown to start removing these green plants while the males supply them; this is unlikely to have evolved for a parasite treatment function, but could relate to extending paternal investment efforts so males do not become polygynous or to avoid signalling to other females a productive male or nest in case of intraspecific brood parasitism (Veiga & Polo, Reference Veiga and Polo2012). Although some of the herbs brought may also function in anti-pathogenic ways, and even be aromatic in nature; perhaps providing an olfactory cue to male quality in a dark nest environment, the most likely evolutionary mechanism for this behaviour to have occurred in starlings is probably through sexual selection.

Tree swallow Tachycineta bicolor

In an interesting attempt to separate the mate and nest protection hypotheses, Shutler & Campbell (Reference Shutler and Campbell2007) experimentally introduced yarrow Achillea millefolium into the nests of tree swallow T. bicolor during laying; it is a species that does not naturally add fresh greenery to its nests. They found flea numbers to be twice as high in the control nests compared with the herb nests, and with no effect on chicks’ size, number or leucocyte levels, provide more support for the NPH than for the drug hypothesis. Although one would expect the reduction in parasite load to have indirect improvements on chick health, the burden may not have been high enough to cause detectable detrimental effects in the first place. However in a similar study 3 years earlier involving tree swallows, yarrow instead was associated with higher parasite numbers, but also increased hatching success and lowered reproductive failure (Dawson, Reference Dawson2004).

Blue tit C. caeruleus

Mennerat et al. (Reference Mennerat, Perret, Bourgault, Blondel, Gimenez, Thomas and Lambrechts2009c ) experimentally enlarged blue tit broods, by moving chicks between nests at 2–3 days old, as well as repeatedly adding five species of aromatics post hatching. They showed that body mass was significantly increased by the aromatics in enlarged groups only, suggesting a possible condition-dependent positive effect. Feather development was also significantly increased by the presence of the aromatics, while decreased in the enlarged broods. In this experiment they removed all nidiculous parasites, by removing and microwaving nests, in order to show that the effect of aromatics occurred independently of them. Both of these studies provide strong support towards either the ‘drug hypothesis’ or a non-parasite related NPH, involving inhibition of other nest pathogens.

Tomas et al. (Reference Tomas, Merino, Martinez-de la Puente, Moreno, Morales, Lobato and del Cerro2012), found that experimental introduction of French lavender Lavandula stoechas and cotton lavender Santolina chamaecyparissus into blue tit nests, two herbs commonly added by blue tits in nature, significantly decreased the abundance of parasites in the nests of yearling females only. The authors suggested that this could be because older females reduce parasites through other techniques such as preening and nest sanitation; behaviours which have been shown to increase in more heavily parasitized blue tit nests (Hurtrez-Boussès et al. Reference Hurtrez-Boussès, Renaud, Blondel, Perret and Galan2000). Yearling females also had significantly higher numbers of blackfly and biting midges in their nests, but these parasites were unaffected by herbs. The plants were introduced every 3 days, so as to maintain the aromatic environment in the nest, but ceased at egg laying. It has been argued that this lack of any fresh plants for over 2 weeks during the hatchling stages and when parasite load is highest (Tripet & Richner, Reference Tripet and Richner1999), could partly explain the apparent ineffectiveness of the herbs. However, it has been shown that the amount of volatiles in the head space air of starling nests increases from the incubation to hatchling stages, despite no more fresh plants being added after laying (Gwinner, Reference Gwinner, East and Dehnhard2013). Suggestions were made that this is due to the increased humidity and movement of hatchlings breaking up the plants.

Lafuma et al. (Reference Lafuma, Lambrechts and Raymond2001) showed the repellent, and possibly also masking, effects of a mixture of these commonly used aromatic herbs against mosquitoes Culex pipiens. They showed that a mix of herbs (as well as Lavandula species by themselves) had significant repellent effects on mosquitoes. They also had a repellent, or possibly masking, effect when mosquitoes were introduced to live chicks in a choice experiment. Over a 14 h period mosquitoes had access to two boxes, one containing a live chick and the following four aromatic herbs; yarrow Achillea ligustica, curry plant Helichrysum italicum, French lavender Lavandula stoechas and pink rock-rose Cistus Creticus, and one simply containing a chick. This shows the possibility of overlooking the effects of flying insects, parasites that are rarely measured in these studies, instead focussing on the more easily detected nidiculous parasites. Bacteria are also often overlooked; Mennerat et al. (Reference Mennerat, Perret, Caro, Heeb and Lambrechts2008, Reference Mennerat, Mirleau, Blondel, Perret, Lambrechts and Heeb2009b ), when introducing two herbs commonly found in blue tit nests, failed to show any effect on blowfly Protocalliphora numbers, but found decreases in bacterial richness and diversity.

Table 1 displays all the studies found by literature review, which experimentally tested the NPH in the field, by artificially adding different aromatic herbs to birds’ nests and assessing parasite or pathogen loads as well as various parameters of chick health.

Table 1. Studies investigating the NPH through artificial manipulation of aromatic herbs in wild birds’ nests

^a Plant species include: Daucus carota, Achillea millefolia, Lavandula stoechas, Helichrysum italicum, Santolina chamaecyparissus, Calamintha nepeta, Achillea ligustica, Pulicaria odora, Bromus inermis, Aegopodium podagraria, Heracleum sphondylium, Sambucus niger, Anthriscus sylvestris, Salix alba, Lamium purpureum, Agrimonia parviflora, Solidago rugosa, Solidago ulmifolia, Conium maculatum, Polemonium reptans, Glechoma hederacea, Geum virginianum, Geum canadense, Taraxacum officinale, Lonicera japonica, Alliaria officinalis, Senecio obovatus

Limitations of studies

There is great heterogeneity in the results of experimental tests of the NPH, and there are multiple potential reasons for this. Firstly the NPH itself is fairly broad and various nest ‘pathogens’ have been shown to be affected by herbs, including fleas, mites, lice, mosquitoes and bacteria. Therefore studies may fail to show any effect by not accounting for the pathogen affected by the plant in the bird species used in their study. There is also the possibility that the effects of the plants are condition-dependent and are only measurable when the health of the birds is compromised, either due to pathogen load or environmental conditions. Food provisioning and begging have been shown to increase in chicks affected by parasites; in profitable years this increased feeding by parents could compensate any negative effects of pathogens on young, transferring negative effects to parents instead, and making any positive effect of the herbs on chicks undetectable (Christe et al. Reference Christe, Richner and Oppliger1996; Tripet & Richner, Reference Tripet and Richner1997). Goodenough et al. (Reference Goodenough, Elliot and Hart2011) suggests that birds nesting in man-made nest boxes, which are cleaned out annually between seasons, typically have to deal with lower parasite loads compared with those in natural cavities, and are therefore perhaps less likely to produce measurable effects on pathogen load.

Sample size of studies also varies widely and could account for some of the variation in results. Several studies which failed to detect a change in nest pathogens have had relatively small sample sizes: Gwinner et al. (Reference Gwinner, Oltrogge, Trost and Nienaber2000) had 7 herb and 8 control nests, Dawson (Reference Dawson2004) had 8 herb and 5 control nests. Studies that did produce a measurable effect of the herbs generally had much greater sample sizes: Tomas et al. (Reference Tomas, Merino, Martinez-de la Puente, Moreno, Morales, Lobato and del Cerro2012) had 32 herb and 27 control nests, Mennerat et al. (Reference Mennerat, Mirleau, Blondel, Perret, Lambrechts and Heeb2009b ) had 20 of each, and Gwinner & Berger (Reference Gwinner and Berger2005) had 79 nests in total.

With so many confounding variables, which are often impossible to eliminate in field trials like these, sample size is likely to be a limiting factor. If for instance parasite or pathogen load in the nest is to be assessed, studies have already shown that this can be confounded by variables such as fledging date (Goodenough et al. Reference Goodenough, Elliot and Hart2011), brood size (James F. Scott-Baumann, unpublished 2013), between season cleaning of nest-boxes (Rendell & Verbeek, Reference Rendell and Verbeek1996) and the exact species of bird nesting (Goodenough & Stallwood, Reference Goodenough and Stallwood2012). As more variables are measured and included in statistical models in order to control them, the sample size must also increase. Many different methods can be used to try to control for these factors; type of nest-box and specifically hole diameter can be altered and used to attract certain species of birds, brood manipulation could be performed to control for varying clutch size, trials could also be repeated across years or nest box sites to increase sample size, however, this adds yet more confounders. All these alterations are labour intensive and can be invasive for the birds, and with a very large sample size, studies like this become impractical. In the south west of the UK it is hard to find single sites at all with 100+ nest-boxes, and even once all the nest-boxes are recruited for a study, birds may not choose to nest in all of them.

Given these complexities, it might be efficient to test putative anti-parasitic properties of phytochemicals such as essential oils using in vitro using bioassays, to complement field studies of nest protection. Gwinner & Berger (Reference Gwinner and Berger2006) identified candidate bioactive compounds in the field by sampling head-space air from starling nests; they found that although no more plants were added to the nest after egg laying, volatile substances including sabinene, myrcene, limonene, phellandrene and ocimene, which are all cyclic or mono-terpenes, were all still present during the hatchling period. Methods for testing the activity of such compounds against ectoparasites are well established (Perrucci et al. Reference Perrucci, Macchioni, Cioni, Flamini and Morelli1995; Yang et al. Reference Yang, Lee, Lee, Choi and Ahn2003; Kim et al. Reference Kim, Yi, Tak and Ahn2004; Bakkali et al. Reference Bakkali, Averbeck, Averbeck and Idaomar2008; George et al. Reference George, Callaghan, Guy and Sparagano2008), and could be allied to field studies in future.