Helminth gut parasites of black grouse (Lyrurus tetrix) in northern England, their impact on productivity and possible sources of infection

Black grouse (Lyrurus tetrix) declines continue throughout much of temperate Europe, following habitat loss and population isolation, associated with reduced productivity. In northern England, only 1,437 lekking males remained in 2014, genetically isolated from other UK populations, and typified by low productivity but high adult survival. Their distribution overlaps with that of red grouse (Lagopus scotica) on moorland managed for red grouse shooting. Here, quasi-cyclical fluctuations in red grouse numbers are driven by the parasite Trichostrongylus tenuis. Whilst management for red grouse may benefit black grouse survival through reducing predators, we hypothesised that parasite transmission from red to black grouse may contribute to low black grouse productivity. We measured T. tenuis prevalence and mean intensity from 186 black grouse carcasses (40 radio-tagged birds, 30 shot birds, the remainder found by chance) and compared them with equivalent measures in red grouse. T. tenuis occurred in 63% of black grouse at a mean intensity of 390 worms per bird, and in 95% of red grouse at 1,535 worms per bird. Seventeen black grouse contained Heterakis spp., and two contained the tapeworm Parionella urogalli. T. tenuis intensity in black grouse was 2-4 times higher in predated birds than in ones that died from other causes. Body condition and productivity were both lower when T. tenuis abundance in black grouse was higher, supporting the hypothesis that parasites contributed to low productivity. Despite T. tenuis abundances varying temporally by approximately 15-fold in both grouse species, patterns of worm abundance between the species were not correlated, nor did we detect any relationships between black grouse breeding success and red grouse worm abundance. Despite the lack of direct evidence for parasite cross-transmission, we suggest ongoing precautionary surveillance of parasitic worms in areas where red and black grouse overlap.

Methods
Data collection

Overall, 186 black grouse carcasses were collected between 1998 and 2022. Of these, 40 were radio-tagged birds from published studies of population dynamics in the North Pennine Dales (Warren and Baines 2002, Baines and Richardson 2007). A further 30 birds had been shot and provided by hunters, and the remaining 116 were found by staff, gamekeepers, farmers, and reserve wardens whilst conducting other tasks and provided for examination.

Of the 186 carcasses, 160 (86%) were found in the North Pennines Dales (109 in Teesdale, 29 in Weardale, 20 in Tynedale and one in each of Allendale and Derwentdale), 25 (13%) in the Yorkshire Dales, comprising 14 in the Yorkshire Dales National Park (six in Wensleydale, five in Swaledale, two in Coverdale, and one in Arkengarthdale), nine in the Nidderdale National Landscape, and one from an unspecified location in northern England (Fig. 1). Carcasses were available in 22 of the 25 years, when they averaged eight per year, varying annually from one to 23 per year. The breakdown of the sample by age, sex, and region is provided in Table 1.

Carcasses were aged as either adult or juvenile based on primary feather structure and pigmentation (Helminen 1963); sex was confirmed at the time of post-mortem examination. Weight (to the nearest 10 g) and wing length (flattened to the nearest mm) were measured from 135 fresh and intact carcasses. The season of death was recorded as either spring (March to May, n=41), summer (June to August, n=37), autumn (September to November, n=54), or winter (December to February, n=54). Cause of death was assigned for all but one of the 186 carcasses to one of four categories: collision, when the carcass was found adjacent to a fence, underneath electric wires, or in the immediate proximity of a road, and there was physical damage consistent with a collision; predation, when feathers had been either plucked or bitten from the carcass consistent with predation by raptors and mammals respectively (Thirgood et al. 1998), the carcass partially eaten, and, in the case of predation by stoat Mustela erminea, small, paired incisor holes were found, usually in the neck; shot, when the carcass was provided freshly killed by a hunter, or where skinning a carcass revealed pellet holes in the flesh; “other” causes were when the above criteria were not met, often where the carcass was thin, or the bird was unable to fly. This category probably included birds dying from disease.

Adult T. tenuis abundance was estimated following Wilson (1983), whereby worms were collected from one of the two caeca by cutting it open lengthways and flushing its contents with water through an 850-μm sieve into a 212-μm sieve. The sieved contents were washed with 300 ml of water into a beaker and mixed thoroughly. Three 10-ml subsamples were placed into separate petri dishes and the total number of adult worms in each were counted using a binocular microscope with 25x magnification. Given the negligible variation between sub-samples, the sum of these counts was multiplied by 10 to estimate the total number of adults worms in a caecum and then doubled to give the total number per carcass. Other helminth worms observed during this procedure were recorded as present / absent.

Following Bush et al. (1997), we define prevalence, mean intensity, and mean abundance of a parasite species as: prevalence = (number of carcasses infected with that species) / (total number of carcasses examined), mean intensity = (total number of parasites) / (total number of infected carcasses) and mean abundance = (total number of parasites) / (total number of carcasses).

T. tenuis intensities in autumn-shot red grouse were measured each year at three driven grouse moors in Teesdale, the North Pennine Dale from where 59% of black grouse carcasses were also recovered. Here, spring densities of red grouse averaged 57 pairs km-2 during the period 2000-22, synchronously fluctuating three- to four-fold between a mean low of 27 pairs and a mean high of 92 pairs km-2. For five consecutive years, worms in juvenile red grouse, which have fewer worms than adults (Shaw and Moss 1989), were not sampled owing to extremely low worm intensities in previous years. Numbers of adult red grouse sampled (both sexes combined) varied per year, with a mean of 194 (range 14 - 546). Their worm burdens were estimated as described above for black grouse.

Black grouse breeding success was estimated each August at up to three sites in Teesdale using pointing dogs to find and flush females and their broods (see Baines et al. 2007). During the period 1998-2022, 890 females were found at an average of 36 per annum (range 9-74) and occurred at a mean annual density of 3.5 km-2 (range 1.6-6.0). Counts were combined across sites to give one annual value. Three measures of annual breeding success were calculated: the proportion of females with broods (number of broods / number of females), brood size (number of chicks / number of broods) and chicks reared per female (number of chicks / number of females).

Statistical analysis

The 186 carcasses were unlikely to be a random sample of the black grouse population. To consider biases amongst carcasses, chi-squared contingency tests were used to compare causes of death between radio-tagged birds and those non-tagged birds found by chance.

      Whether T. tenuis prevalence and intensity in black grouse carcasses differed in relation to region, season, age, sex and cause of death was considered in two parts. First, for worm prevalence we used a generalised linear mixed model (GLMM) with binomial error and logit link function, with a binary response variable (T. tenuis present (1) or absent (0)), and region (North Pennines or Yorkshire Dales), season (spring, summer, autumn, or winter), age (adult or juvenile) sex (male or female), and cause of death (collision, predation, shot or other) as fixed effects. Two-way interactions between season, age, and cause of death were also included as fixed effects. Second, to consider worm intensity amongst infected carcasses, we used a GLMM with Poisson error adjusted for overdispersion and a logarithmic link function (Wilson & Grenfell 1997), with the T. tenuis count per bird as the response variable and incorporating the same fixed effects. The year in which a black grouse carcass was collected was incorporated as a random effect in both models.

To determine whether observed levels of  T. tenuis infection impacted body condition in black grouse, we modelled loge(weight) as the dependent variable, with loge(wing length), region, age, sex, season, and loge(T. tenuis abundance + 1) as fixed effects and year as a random effect in a GLMM with a normal distribution and identity link function (cf. Packard & Boardman 1988). The model was equivalent to investigating factors affecting a body condition index defined as loge(weight/(wing length)b) where the exponent b accounted for the allometric relationship between weight and wing length. One obvious outlier (a juvenile male with long wing and low weight, not yet fully grown) was omitted from the analysis.

Annual T. tenuis intensities in black grouse carcasses collected throughout the calendar year in the North Pennine Dales, where 86% of the sample was collected, were compared with those in red grouse shot in the autumn at three moors in Teesdale, one of the North Pennine Dales. Data were not collected from all moors in all years. To overcome missing moor-years, we fitted a GLM with logarithmic link function and overdispersed Poisson error, using total worms on a moor as the dependent variable and loge(number of grouse sampled from the same moor) as an offset, and moor and year as explanatory variables. We used the predictions from the model for year, setting offset = 0, to give annual values for the mean abundance of T. tenuis in  shot red grouse in Teesdale. Annual mean T. tenuis abundances (loge(x+1)-transformed) for each grouse species were compared in a general linear model (GLM), with worms in black grouse as the response variable, weighted by the number of carcasses in each year, and T. tenuis in red grouse as a covariate, first in the same year, then in the previous year, to help account for differences in timing of data collection between grouse species and any lag in response.

Measures of annual black grouse breeding success in Teesdale were related to annual measures of worms in black grouse in all of the North Pennine Dales for 22 years during the period 1998-2022. The proportion of females with broods was modelled in a GLM with a binomial error and logit link function, with broods found per annum as the response variable and females observed per annum as the binomial total. Both brood size and chicks reared per hen were modelled with a Poisson error and logarithmic link function, loge(broods) and loge(females) respectively as offsets, and black grouse annual worm mean abundance (loge-transformed to linearize the relationship), weighted by the number of carcasses, as the explanatory variable. Insufficient black grouse carcasses and brood counts were available from the Yorkshire Dales to enable a similar comparison. The analysis was then repeated using red grouse loge(annual worm mean abundance) in the same year and in the previous one as an explanatory variable to consider whether worm abundance in red grouse influenced black grouse breeding success.

For GLM and GLMM models, we reviewed standardised residuals for normality and bias using a Shapiro-Wilk test (Shapiro & Wilk 1965) coupled with visual examination of their frequency distribution, Normal plots, half-Normal plots and residuals versus fitted values. We adjusted Poisson and binomial errors for overdispersion where the residual deviance was significant when treated as a chi-square value with degrees of freedom equal to the residual degrees of freedom. The significance of main effects and interactions were assessed using Wald statistics, obtaining significance levels from a chi-square distribution if standardised residuals were normally distributed, and from random permutation tests (Edgington and Onghena 2007) if not. Interactions were dropped if not significant at P<0.05. Analyses were conducted using Genstat 24th Edition (VSN International, Hemel Hempstead, UK).