The impact of temperature on the life cycle of Gasterophilus pecorum in northwest China

Background The departure of the mature larvae of the horse stomach bot fly from the host indicates the beginning of a new infection period. Gasterophilus pecorum is the dominant bot fly species in the desert steppe of the Kalamaili Nature Reserve (KNR) of northwest China as a result of its particular biological characteristics. The population dynamics of G. pecorum were studied to elucidate the population development of this species in the arid desert steppe. Methods Larvae in the freshly excreted feces of tracked Przewalski’s horses (Equus przewalskii) were collected and recorded. The larval pupation experiments were carried out under natural conditions. Results There was a positive correlation between the survival rate and the number of larvae expelled (r = 0.630, p < 0.01); the correlation indicated that the species had characteristic peaks of occurrence. The main periods during which mature larvae were expelled in the feces were from early April to early May (peak I) and from mid-August to early September (peak II); the larval population curve showed a sudden increase and gradual decrease at both peaks. Under the higher temperatures of peak II, the adults developing from the larvae had a higher survival rate, higher pupation rate, higher emergence rate and shorter eclosion period than those developing from peak I larvae. Although G. pecorum has only one generation per year, its occurrence peaked twice annually, i.e. the studied population has a bimodal distribution, which doubles parasitic pressure on the local host. This phenomenon is very rarely recorded in studies on insect life history, and especially in those on parasite epidemiology. Conclusion The period during which G. pecorum larvae are naturally expelled from the host exceeds 7 months in KNR, which indicates that there is potentially a long period during which hosts can become infected with this parasite. The phenomenon of two annual peaks of larvae expelled in feces is important as it provides one explanation for the high rate of equine myiasis in KNR.


Background
Horse stomach bot flies (Gasterophilus spp.) are common obligate parasites in equids [1,2]. Gasterophilus larvae parasitize the digestive tract of equids and cause inflammation, ulceration, herniation and other symptoms [3][4][5]. The larvae absorb nutrients from the host and secrete toxins, and may lead to host death when the level of infection is severe [6,7]. The genus Gasterophilus comprises 9 species globally [8,9], of which 6 have been recorded in China [10]. The infection rate of bot flies in the equid population is 100% in Kalamaili Nature Reserve (KNR), Xinjiang, which is located in desert grassland [11]. Among the 6 species of horse stomach bot flies found in KNR, the rate of infection with G. pecorum is extremely high, accounting for 89-98% of all bot fly infections [11,12]. In other regions of China, Gasterophilus intestinalis and Gasterophilus nasalis are the dominant horse stomach bot fly species [13,14].
Horse stomach bot flies undergo complete metamorphosis; they have four developmental stages, i.e. egg, larva, pupa and adult, one generation per year, and the larvae take 9-10 months to develop in the digestive tract of the host [8]. The larvae develop in the digestive tract of equids until they are mature, then leave the host and develop into flies and start a new life cycle. The mature larva is the first stage of the host examined in vitro, and the population dynamics of these larvae determine subsequent population growth. The most significant feature of the life cycle of G. pecorum is its "unusual" reproductive strategy of laying eggs on grass [8]. Most studies on G. pecorum follow on from the early biological observations of Chereshnev in Kazakhstan [15], who found that the mature larvae mainly appear in early August to early September. However, a study designed to examine the development period of pupae reported a significant number of G. pecorum larvae collected in KNR in spring, thus the highest incidence of mature larvae was considered to be spring [16]. Based on the differences between some recent findings and existing life history records of G. pecorum, a systematic study of the population dynamics and growth of local G. pecorum was carried out in vitro to understand the development of this species in the desert steppe.
Insect are poikilotherms, whose metabolism, life cycle and lifespan are influenced by the ambient temperature [17,18]. Horse stomach bot flies are exposed to a relatively stable environment in the digestive tract of the host, but once expelled from the host will be affected by the external temperature [19]. Horse stomach bot flies are thermophilic insects that tend to live at high temperatures [20]. Higher temperatures within a preferential temperature range accelerate the development rate of insects [21,22]. In addition, the survival rate of insects metamorphosing between different states is also affected by temperature [23][24][25]. Knowledge of the developmental response of insects to temperature is important for an understanding of their ecology and life history [26]. Thus, we conducted an experiment to investigate the effect of different temperatures on the developmental rate, survival, pupation and emergence of G. pecorum to determine parameters that can be used to predict and manage horse stomach bot fly populations in KNR.

Study area
KNR is located in the desert subregion of northwest China (44°36′ ~ 46°00′N, 88°30′ ~ 90°03′E). KNR has an area of 18,000 km 2 , an altitude of 600-1464 m, an average annual temperature of 2.4 ℃, average annual precipitation of 159 mm and annual evaporation of 2090 mm. Winter in the desert steppe is cold and long, lasting from late October to early March [27,28]. Protected animals in KNR include the reintroduced species Equus przewalskii (EN) and the endangered species Equus hemionus (NT), as well as Gazella subgutturosa (VU) and Ovis ammon (NT). Domestic livestock such as horses graze in KNR seasonally [29].

Materials
Based on the climate of KNR and the life history of stomach bot flies in the area, the population survey of G. pecorum larvae was carried out from early March to late September 2018. The feces of Przewalski's horses were inspected for this study. As the horses defecated one distinct pile in a single defecation event, we could count the number of piles of feces for the survey (Additional file 1). Larvae were collected from fresh feces 4-6 days/week, and the number of feces (piles of feces) and larvae were statistically analyzed on a weekly basis. We investigated fresh feces from 50-100 piles/week within 5 min after the horses defecated, and used tweezers to separate larvae from the feces. The larvae of G. pecorum were collected and third-instar stomach bot fly larvae identified according to Li et al. 's method [30].
Transparent plastic cups (8 cm in diameter, 6 cm in height) were used as the pupation containers for G. pecorum larvae. Five larvae were placed in each cup, the cup mouth sealed with gauze and the larvae cultured in outdoor shade (low light intensity, where the photoperiod was that of the natural environment) in KNR. The pupation and eclosion behavior of G. pecorum were observed daily. The number of insects in each phase was recorded, and the survival rate, pupation rate and eclosion rate calculated. The temperature determined by thermometer at the larvae culture site was recorded at 2:00, 8:00, 14:00, 20:00 hours daily. (2) Number of larvae per fece (NL)

Data analysis
To evaluate the survival, pupation and eclosion rates of the stomach bot flies, the following formulas were utilized: Spearman's correlation analysis was used to analyze the relationship between the number of mature larvae and their survival rate; the significance level was set as α = 0.05. Data analysis was performed in SPSS 20.0, and the graphs were drawn by using Sigmaplot 12.0 and Graphpad prism 7.

Population dynamics of mature larvae of G. pecorum
A total of 2,021 piles of equine feces were examined, of which 443 (21.92%) contained G. pecorum larvae ( Table 1). The proportion of feces containing larvae (PF) in early April and mid-to-late August was 56.03% and 53.23%, respectively. There were two obvious larval peaks with a significant range. In May and September, the PF gradually decreased and remained low. The PF was lower than 20% during three periods: March; mid-to-late May to the end of July; and mid-to-late September.
A total of 704 larvae of G. pecorum were collected in this study. The average number of larvae per pile of feces (NL) was 0.35 during the entire investigation period; the highest number was in early April with an average of 1.40, followed by mid-to-late April and mid-to-late August, with averages of 0.79 and 0.84, respectively. The NL showed the same trend as the PF (Fig. 1).
The percentages of larvae found in April and August were 42.47% and 28.55%, respectively. The percentages of larvae were 27.98% and 14.79% in early and mid-to-late April, respectively, and 23.86% in mid-to-late August; these percentages were significantly higher than those recorded for the other periods (p < 0.05). The number of larvae collected from early April to early May and from mid-August to early September accounted for 48.72% and 32.52% of the total number of larvae, respectively, and the cumulative proportion of the two phases accounted for 81.24%. Thus, the main periods of mature larvae emergence in KNR were from early April to early May (peak I) and from mid-August to early September (peak II) (Fig. 1), although larvae were expelled in feces continuously throughout March to September. The NL was derived from observations made on 10 wild horses in temporary captivity that showed that an adult Przewalski's horse defecates an average of 10.1 piles of feces per day. It was estimated that 749 larvae of G. pecorum were discharged from each horse, which was close to the figure calculated from the annual average number of infective G. pecorum expelled locally [11].
The plot of the cumulative number of larvae collected during the survey period showed a double S curve (Fig. 2). There was one spike in April and one in August, and the slopes of the curves showed that in the first phase the increase in the number of total larvae (Fig. 2a) and survival of larvae (Fig. 2b) were highest on 5 April, and in the second phase (Fig. 2c, d) they were both highest on 23 August.
The average pupal period of peak I and peak II lasted for 34.05 days and 20.2 days, respectively (p < 0.001, t = 15.513) (Fig. 3a). The longest and shortest development periods of peak I were 39 days and 26 days, respectively, and the longest and shortest development periods of peak II were 24 days and 18 days, respectively. There was a positive correlation between the survival rate and the number of larvae (r = 0.630, p < 0.01), i.e. the survival rate of G. pecorum larvae was higher during the two peak periods.
Population decreases in the three stages from the development of larvae (N) to adults (N') was as follows: N' = αβγN, where α is survival rate, β is pupation rate and γ is eclosion rate. The number of adults that developed from larvae during peak I and peak II are given by N 1 ' and N 2 ' , respectively; N 1 ' = 15.22% N, N 2 ' = 23.98% N.
Larvae collected during peak I that developed into adults accounted for 15.22% of the total larvae, and larvae collected in peak II that developed into adults accounted for 23.98% of the total larvae. The number of larvae collected in peak I was 1.48 times higher than that collected in peak II, but the higher survival rate, pupation rate and emergence rate of peak II resulted in 1.32 times more adults compared to peak I. There were differences in the sex ratio between the two peak periods. The ratio of males to females in peak I was 0.73, which was lower than that in peak II (0.76) ( Table 2). The proportion of female adults in relation to the total number of larvae was 0.10% in peak II, which was slightly higher than that in peak I (0.09%); this indicated that peak II was of greater significance in terms of potential G. pecorum infections of equids in KNR.

Temperature characteristics during the periods that larva were expelled
The ambient temperature rose rapidly from March to April, showed a slower increase from April to July, and began to decline after reaching the highest temperature of 38 ℃ on 20 July (Fig. 4a). The maximum daily temperature difference was 20 ℃, and the minimum and average daily temperature differences were 3 ℃ and 12.78 ℃, respectively. The temperature curve was parabolic, which is characteristic of an initially increasing then decreasing temperature (y = 0.8570 + 3.7449x + 0.1031x 2 -0.0240x 3 ,R 2 = 91.18%) (Fig. 4b). The maximum temperature, as shown by the fitted curve, was on 19 July (Fig. 4).

Discussion
The study of insect population dynamics is an important part of insect ecological research [31,32]. The prevalence of G. pecorum has been reported to be low in most countries and regions [2,13,33]. However, G. pecorum is common in the digestive tract of equids in the Mongolia-Xinjiang and Qinghai-Tibet regions of China [12,34], central and northern Kazakhstan [14], the Republic of Mongolia [35] and the Yakut Republic of Russia [36]. There also tends to be a higher incidence of this species in certain regions. A study carried out on the period of pupal development of G. pecorum reported a model that can be used to predict the period of adult occurrence [16]. A study on the egg development period and the survival period of first-instar larvae of G. pecorum has also been completed (in submission). The results from these studies will further improve our understanding of this species and contribute to a better understanding of the epidemiology of disease caused by this parasite. These parasites can adapt to their environment, and have different performance characteristics in different environments [37][38][39]. The KNR is a desert grassland with high temperatures and drought in summer, a severe and long winter with low temperatures, and low annual precipitation. The unique environmental conditions of the KNR have enabled the specific occurrence of G. pecorum there. These special conditions differ from those of Kazakhstan, where an earlier study of G. pecorum showed that the larvae were only excreted there in August [15]. The findings reported here for KNR also differ from those reported for southern Italy, where larvae of G. pecorum were found in January, March and July [33], and for South Africa, where adult G. pecorum only appeared from February to May and in August [8].
The phenology of an insect affects its development dynamics [40,41]. The development rhythm of some insects that produce one generation per year is closely related to their phenology [42,43]. With the change of plant growth period, the population dynamics of Uroleucon rudbeckiae showed a peak period of correlation [44]. In the present study, the larval population of G. pecorum showed two peaks annually, which were closely related to the particularly arid climate of the desert steppe. As water is the most crucial factor for life in the desert steppe [45,46], many of the plants that grow there are ephemeral. Due to the characteristics of local precipitation [47], some Stipa sp. showed a special secondary growth phenomenon due to adaptation to the environment [48]. We found that the locally dominant Stipa began to resume growth at the end of March and early April, with seed heads developing in May, and began to grow new leaves in late August, all of which confirmed the phenomenon of secondary growth. Some studies have shown that once water conditions are suitable, many desert plants develop rapidly and have a faster life cycle [49][50][51]; the effects of this are reflected in the KNR ecosystem by the phenomenon of the simultaneous occurrence of G. pecorum and vector plants. In contrast to the other 5 species of Gasterophilus found in this region, which lay eggs directly on the horse host, G. pecorum lays eggs on Stipa [52]. The parasite's population dynamics were found to match perfectly with the growth of vector plants and phenological changes, which also led to the local bimodal population distribution phenomenon of G. pecorum seen here. This phenomenon indicates that there are two main infection periods per year, which differs from the annual infection of animals that occurs with linear host-parasite phenomena [53,54], and also differs from the particular characteristics of infection associated with phenology seen with most arthropod parasite infections [55][56][57].  Table 2 The sex ratio of G. pecorum adults in the two peak periods Proportion of female adults that developed from the total number of larvae in peak I was 0.09%, and in peak II 0.10% In an appropriate temperature range, the higher the temperature, the more beneficial it is to the development of insects [58]. The average temperature of peak I (11.3 ± 5.3 ℃) was significantly lower than that of peak II (24.4 ± 2.7 ℃) (t = − 11.083, p < 0.001), which affected the survival rate of mature larvae. The environmental temperature during peak II was more beneficial for the subsequent pupal development stage. This resulted in a higher survival rate (survival rate, pupation rate and eclosion rate) of mature larvae from peak II, and a shorter pupal stage, which lead to the higher number of adults produced from peak II than from peak I larvae.

Conclusions
The natural period in which G. pecorum larvae are expelled in horse feces in KNR exceeds 7 months. The close relationship between the bimodal population distribution of G. pecorum and the secondary growth of Stipa in the desert steppe of KNR is important as it can explain the high number of infective G. pecorum, high infection rate and dominance of Gasterophilus spp. in equids in the reserve. The larval population from peak II had a higher survival rate than that from peak I because of the suitable conditions for the development of the former. The results of this study demonstrate the highly co-evolutionary nature of the phenomenon described here in the desert steppe ecosystem, and reveal the high adaptability of organisms under adverse conditions.
Additional file 1: Figure S1. Method of larvae collection and definition of piles of feces.

Abbreviations
KNR: Kalamaili Nature Reserve; PF: Proportion of feces containing larvae; NL: Number of larvae per pile of feces.