Host feces, olfactory beacon guiding aggregation of intestinal parasites Gasterophilus pecorum (Diptera: Gasterophilidae).

The aim of this study was to identify the aggregation sites and transmission characteristics of Gasterophilus pecorum, the dominant pathogen of endangered equines in desert steppe. Therefore, we tested with a four-arm olfactometer the olfactory response of the G. pecorum adults to the odors that have a great impact on their life cycle, and also investigated the occurrence sites of the adults in the area where the Przewalski's horse (Equus przewalskii) roam frequently during the peak period of G. pecorum infection. The results of four-directional olfactory test showed that the fresh horse feces had a stronger attraction rate on both male (50.4%) and female flies (38.2%). Stipa caucasica, the only oviposition plant where G. pecorum lay eggs, had a better attraction effect on females than that on males. And the attraction rates of S. caucasica to G. pecorum females in the early growth stage (Stipa I) and mid-growth stage (Stipa II) were 32.8% and 36.8%, respectively. In addition, the two-directional olfactory test showed that the attraction rate of males to fresh horse feces (68.90%) was higher than that to Stipa II (31.10%), and females also showed similar olfactory responses. Moreover, in our field investigation, 68.29% of G. pecorum adults were collected from around the horse feces. The results of laboratory test and field investigation implied that the location mechanism of G. pecorum aggregation for mating is related to the orientation of horse feces. The horse feces and the vicinity are the key contamination areas of G. pecorum, and it is also the areas where horses are seriously infected with G. pecorum. Those fresh feces, which gather abundant information about the host, naturally had the greatest chance of contacting with the host; G. pecorum adults create the opportunity to enter directly into the host's mouth and infect the host by laying eggs on S. caucasica, which is the most favorite plant of the host in this area. These characteristics are one of the main reasons why G. pecorum has become the dominant species under the condition of sparse vegetation in desert steppe.

Population genetic structure of Gasterophilus pecorum in the Kalamaili Nature Reserve, Xinjiang, based on mitochondrial cytochrome oxidase (COI) gene sequence.

Gasterophilosis is a significant threat to equids in the desert steppe of Xinjiang, China, where Gasterophilus pecorum (Fabricius) (Diptera: Gasterophilidae) is the dominant botfly species. A population analysis was conducted on 195 individual G. pecorum larvae from three host species, Przewalski's horse, the domestic horse and the Asiatic wild ass. The distribution of haplotypes of the maternally inherited mitochondrial cytochrome oxidase subunit I (COI) gene was analysed to assess the population differentiation of G. pecorum. High haplotype diversity was observed among G. pecorum populations from all host species, indicating that the G. pecorum infecting one host had multiple maternal ancestors. A phylogenetic tree showed six clades, suggesting a high degree of genetic differentiation. A constructed haplotype network described both the origin of the haplotypes and the population structure. The findings indicated that G. pecorum infections within Przewalski's horses were mainly transmitted from Asiatic wild asses. Clade 1 was found to be the most primitive group and to have evolved to be highly adaptable to the desert steppe. Clade 2 originated from Clade 1, potentially as a result of the annual migration of domestic horses. Revealing the differentiation of the G. pecorum population is important for elucidating the aetiology of Gasterophilus infection in Xinjiang and for planning appropriate control measures.

Studies on the embryonic development and larval infection potential of the stomach bot flies, Gasterophilus pecorum.

Endangered Przewalski's horses have faced severe infections from Gasterophilus pecorum (Diptera, Gastrophilidae) in Xinjiang's Kalamaili Nature Reserve (KNR). This study examines G. pecorum's development and infection patterns in embryonic and larval stages, crucial for understanding horse botfly disease in desert grasslands. For the incubation of G. pecorum fertilized eggs, we established the six distinct temperature gradients: 16 °C, 20 °C, 24 °C, 28 °C, 30 °C, and 32 °C. Using the least squares method, we calculated the correlation between the developmental threshold temperature of the eggs and their cumulative effective temperature. Furthermore, we meticulously recorded the survival duration of the larvae across a spectrum of temperature gradients (-20 °C, -10 °C, 4 °C, 10 °C, 20 °C, and 30 °C) and under varying conditions (dark and light). This method allows us to analyze and interpret the impact of these environmental factors on larval survival durations. 1) The formula for predicting the embryonic development period of G. pecorum was N = (182.7 ± 12.03)/[T-(3.191 ± 1.48)], where the developmental threshold temperature was 3.191 ± 1.48 °C, and the effective accumulated temperature was 182.7 ± 12.03 d°C 2) The model describing the relationship between the embryonic development rate and temperature was: y = 0.0001x2+0.0007x+0.0378, demonstrating a positive correlation between the development rate and temperature (R-sq = 0.989, p < 0.001). 3) Larvae in the dark group exhibited a longer survival time, with the longest being 9 months at 4 °C. The adaptation of G. pecorum's embryonic development to cold temperature, combined with the extended survival period of larvae in the egg state, significantly increases the infection potential of G. pecorum in colder climates. This discovery offers essential insights into the predominance of G. pecorum in the KNR region and provides a crucial biological basis for the prevention of myiasis and the conservation of vulnerable species, such as Przewalski's horses.

Spatial distribution of Gasterophilus pecorum (Diptera) eggs in the desert steppe of the Kalamaili Nature Reserve (Xinjiang, China).

BACKGROUND: The dominant Gasterophilus species in the desert steppe (Xinjiang, China) Gasterophilus pecorum poses a serious threat to the reintroduced Przewalski's horses. We investigated the distribution pattern of G. pecorum eggs in June 2017. METHODS: Two sampling methods, transect and grid, were used, and the results were analyzed via geostatistics by semivariance. The nest quadrat was used to determine the optimal quadrat size. RESULTS: Eggs were found in 99 quadrats (63.1%) and 187 clusters (1.5%) of Stipa caucasica on the steppe. The mean oviposition count of a cluster was 3.8 ± 1.6. Three-eggs is the mode of which females oviposit on each ovigerous S. caucasica (22.0%). Semivariogram analysis revealed that the distance of spatial dependence for eggs was 921 m, 1233 m and 1097 m for transect 1, transect 2 and grid methods, respectively, while spatial continuity was 62%, 77% and 57.0% for transect 1, transect 2 and grid, respectively. The eggs showed a patchy, aggregated distribution pattern. This suggested the spherical model is most applicable. The proportion of ovigerous S. caucasica was significantly correlated with the distance from water resources (r = - 0.382, p = 0). CONCLUSION: Our findings indicated that diversification of G. pecorum oviposition was a new adaptative strategy for its survival in the desert steppe ecological niche. This made it more efficient at infecting hosts in the local environment. Areas surrounding water resources, especially around the drinking paths of equids (500 m radius surrounding the water), were concentrated epidemic areas. It is suggested that more attention to be paid to the ecological characteristics of G. pecorum in order to develop control measures that would reduce the infection risk for Przewalski's horses.

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.

Evaluation of the genetic diversity and population structure of Gasterophilus pecorum in Xinjiang Province, China, using fluorescent microsatellites (SSR) markers.

The genetic diversity of Gasterophilus pecorum populations consisting of 192 individuals sampled from Przewalski's horses (Equus ferus przewalskii) in Xinjiang Province, China, was evaluated using 12 microsatellite loci. The genetic variability within populations and genetic differentiation among populations were estimated. A total of 163 alleles were detected and the average value of observed number of alleles at each locus ranged from 7 to 19 (average 13.5625). The expected heterozygosity (He) varied from 0.5933 (GP361) to 0.9208 (GP253) and averaged 0.8426. The effective number of alleles (Ne) of the simple sequence repeat (SSR) markers was 7.1756, and it ranged from 2.4430 to 11.5214. The polymorphism information content (PIC) ranged from 0.5643 (GP361) to 0.9053 (GP253) (average 0.8119). The average Shannon's information index (I) was 2.1272. Parameters of genetic diversity (Ht, Hs and Gst) and F-statistic (Fis, Fit and Fst) were estimated. The mean of total gene diversity (Ht = 0.8798) and the average interspecific genetic diversity (Dst = 0.0110) showed that G. pecorum had a relatively high level of genetic variation, which was mainly within populations. The Fixation indices as Fst ranged from 0.0029 to 0.0950, and the Number of per generation migrants (Nm) among the populations varied from 2.3814 to 85.9745. These results indicate that the heterozygosity and genetic diversity of the G. pecorum populations were very high and that gene exchange was frequent. Correlation analyses demonstrated that the number of alleles and genetic diversity of the populations were not significantly different (p = 0.2). Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering analyses revealed a genetic similarity of 0.0671 - 0.1118. Analysis of molecular variance (AMOVA) demonstrated that the percentage of variation in the intrapopulation was small (0.67%) and the genetic variation in the G. pecorum populations was mainly exist within the species. An understanding of population genetic structure is not only crucial for assessing the actions and interactions of evolutionary forces in natural populations but also helps elucidate parasitic strategies in G. pecorum.

Histopathological Study of Esophageal Infection with Gasterophilus pecorum (Diptera: Oestridae) in Persian Onager (Equus hemionus onager).

BACKGROUND: The larval stages of Gasterophilus are obligate parasites in the gastrointestinal tract of equine accountable for pathologic ulcers in the Persian onager gastrointestinal. The aim of the current report was to study the histopathological change with G. pecorum larvae in the esophagus of a Persian onager. METHODS: This study was performed in Iranian Zebra propagation and breeding site in Khartouran National Park, southeast of Shahrud City, Semnan Province, Iran in 2014. Following a necropsy with specific refer to esophagus of one adult female Persian onager were transmitted to the laboratory. After autopsy, parasites collected from the esophagus were transmitted into 70% alcohol. For histopathological investigation, tissue samples were collected from the esophagus. The tissues were fixed in 10% buffered formalin, and conformity routine processing, there were stained with Hematoxylin and eosin. RESULTS: After clarity by lactophenol parasites were identified as G. pecorum. Microscopic recognition contained hyperemia, inflammatory cell infiltration, epithelial destruction, esophageal gland hyperplasia. CONCLUSION: This is the first survey of G. pecorum and histopathological study in the Persian onager esophagus in the world.

Gasterophilus spp. infections in horses from northern and central Kazakhstan.

A cross-sectional survey was performed to obtain current data on the gastrointestinal myiasis of horses in the provinces of Kostanay, Akmola and Karagandy, northern and central Kazakhstan. The stomach, small intestine and rectum of 148 slaughter horses were examined for Gasterophilus spp. larvae during a 26-month study period. All horses were infected with 2nd and 3rd stage larvae (mean intensity: 803±350), and 22% of them harboured >1000 Gasterophilus spp. larvae each. Four species were identified: G. intestinalis (prevalence: 100%; mean intensity: 361±240 larvae), G. haemorrhoidalis (100%; 353±191), G. nasalis (100%; 73±36) and G. pecorum (91.2%; 18±10). Horses aged<2 years were higher infected with Gasterophilus larvae than 2-4 years old animals. Both the prevalence and extremely high intensity of Gasterophilus infections of horses in these Kazakh regions suggest respective control measurements to improve the health and performance of the animals and to increase the economic income of horse owners.