©2017 Institute of Parasitology, SAS, Košice DOI 10.1515/helm-2017-0029 HELMINTHOLOGIA, 54, 3: 225 – 230, 2017 Preimaginal mortality of Musca domestica (Diptera: Muscidae) due to Habronema muscae (Nematoda: Habronematidae) R. K. SCHUSTER Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates, E-mail: [email protected] Article info Summary Received December 29, 2016 In order to study the damage of Habronema muscae (Carter, 1861) on its intermediate host, Mus- Accepted April 24, 2017 ca domestica Linnaeus, 1758, fl y larval feeding experiments were carried out. For this, a defi ned number of praeimaginal stages of M. domestica was transferred in daily intervals (from day 0 to day 10) on faecal samples of a naturally infected horse harboring 269 adult H. muscae in its stomach. The development of M. domestica was monitored until imagines appeared. Harvested pupae were measured and weighted and the success of infection was studied by counting 3rd stage nematode larvae in freshly hatched fl ies. In addition, time of pupation and duration of the whole development of the fl ies was noticed. Pupation, hatching and preimaginal mortality rates were calculated and the number of nematode larvae in freshly hatched fl ies was counted. Adult fl ies harboured up to 60 Habronema larvae. Lower pupal volumes and weights, lower pupation rates and higher preimaginal mortality rates were found in experimental groups with long exposure to parasite eggs compared to experimental groups with short exposure or to the uninfected control groups. Maggots of the former groups pupated earlier and fl y imagines occurred earlier. These fi ndings clearly showed a negative impact of H. muscae on the development of M. domestica. The results are discussed in connection with the destruction of adipose cells in the maggots by developing nematode larvae. Keywords: Habronema muscae; Musca domestica; larval development; parasite host interaction Introduction Previous experimental infections (Schuster & Sivakumar, 2016) of Musca domestica Linnaeus, 1758 larvae with Habronema muscae Equine stomach worms of the family habronematidae have a wide (Carter, 1861) gave fi rst indication that the infection infl uences pu- geographical distribution. While aberrant larvae of these parasites pal sizes and increases the mortality rate of preimaginal stages cause conspicuous cutaneous and conjunctival alterations (sum- of housefl ies. It was noticed that pupae of the uninfected control mer sores) gastric habronematidosis remains usually unrecog- group were smaller compared to those of the experimental group nized and its clinical signifi cance is underestimated. where the insects spent their entire larval development in a dung Although the life cycle with muscid fl ies acting as intermediate sample of naturally infected horse. While in the control group hosts and vectors has been disclosed a century ago (Ransom, 82.0 % of fl y eggs developed into imagines this rate decreased to 1911; Johnston, 1913; Hill, 1918; Johnston & Bancroft, 1920; Rou- 27.0 % in the experimental group. baud & Descazeaux, 1921; Roubaud & Descazeaux, 1922), little The purpose for this study was to investigate the infl uence of rising is known about the impact of parasite larval stages on their averte- expositions of housefl y larvae to H. muscae eggs on pupation rate, brate intermediate hosts. size of pupae and total mortality of preimaginal stages of M. do- 225 mestica for a better understanding of damages caused to averte- their total weight was divided by number of pupae. For all individ- brate hosts by parasites. ual pupae in each group length and width was measured under a stereoscopic microscope. Since the geometrical shape of fl y pu- Materials and Methods pae are close to an elongated ellipsoid (prolate or spheroid) their approximate volume can be calculated best by the formula: V = Musca domestica 4*π/3*a2*c = 4.19a2c, where a and c are half of length and half of A housefl y strain consisting of 300 – 500 individuals kept in a width of the pupae, respectively. screen cage under laboratory conditions at 24 – 30 °C, 60 % rela- tive humidity and light cycle 12 : 12 h (light : dark) was the basis of Examination of fl ies our experiment. The strain originated from 50 housefl ies captured For the examination of fl ies, the following protocol by Schuster and at a camel milk farm (Emirates Industry for Camel Milk Products) Sivakumar (1913, 2017) was used. In brief, hatched fl y imagines in Dubai in 2012. The morphology of the carefully selected insects were separated within 12 h after emergence, immobilized by cold and their development stages matched the description of M. do- (-18 °C for 5 min.), sorted by sex and dissected. Caput, thorax and mestica by Hewitt (1910) and Skidmore (1985). Adult fl ies were fed abdomen were put separately into drops of water on glass slides, with a mixture of wetted milk powder (Aptamil®) and sucrose (2:1). were dismembered by preparation needles and checked for the A cylinder with moist fi lter paper served as drinker. The parasite presence of nematode larvae under a stereoscopic microscope. free medium for rearing fl y larvae consisted of a moist mixture of Prior to examination, the slides were put for 10 min on a heated wheat bran and alfalfa hay powder (9:1). plate (40 °C) to stimulate the movement of Habronema larvae to actively to leave the tissues of the fl ies. The slides were then put Habronema muscae for 5 min on an ice pack to slow down the active movement for a Adults of H. muscae in numbers of 112 male and 157 female sto- better counting. The separate examination of caput, thorax and mach worms was a secondary fi nding in a horse necropsied in abdomen was primarily done to determine the larval burden of the July 2016. Pieces of dung taken from the rectum in aliquots of 30 parasite more exactly. g were put into glass cylinders, covered with parafi lm and stored until use for up to 10 days at 4 °C in the refrigerator. Statistical treatment of the data Prevalence and mean intensity of Habronema larvae in fl y imagi- Experimental design nes at their 95 % confi dence intervals were calculated using the Housefl y eggs were harvested by putting an open 90 mm Petri software package Quantitative Parasitology 3.0 (QP WEB) (Rozsa dish fi lled with larval rearing medium into the screen cage. After et al., 2000). The same software was applied to calculate average 5 h the dish was removed and checked for the presence of fl y and 95 % confi dence intervals for pupation and total survival rates eggs. The mixture of larval rearing medium and Musca eggs was as well as for the pupal volumes. then transferred into in a large glass Petri dish (diameter: 200 mm) at a constant temperature of 26 °C. At day 0, 100 eggs were Table 1. Time of larval pupation and fl y hatching of Musca domestica under various exposure of their larvae to Habronema muscae eggs. transferred directly from the larval rearing medium onto the fi rst Group Average Pupation Hatching dung sample (substrate) of the naturally H. muscae infected horse exposure (d post oviposition) (d post oviposition) st (group D0). At day 1, 100 1 Musca instars were collected from the (d) rearing medium and were transferred onto the next faecal sample (group D1). From day 2 to day 10 each 50 fl y larvae were used D0 8.5 7 – 10 11 – 14 (groups D2 – D10). Two uninfected control groups starting with D1 7.5 7 – 10 13 – 14 50 fl y eggs each were reared on a sample of larval rearing me- D2 6.5 7 – 10 11 – 13 dium (group K0 medium) and on faeces of an uninfected horse D3 5.5 7 – 10 12 – 15 (group K0 faeces), respectively. To avoid contamination all glass D4 6 9 – 11 12 – 13 cylinders were kept in plastic containers covered with paper tissue D5 4 8 – 10 13 – 14 secured by rubber rings and were stored at 26 °C. Depending on food consumption of the growing larvae of experimental groups D6 3 8 – 10 15 – 16 further dung pieces of the infected horse were added. At the end D7 2 8 – 10 14 – 15 of their larval development maggots started to leave the breeding D8 3.5 10 – 13 14 – 18 substrate to pupate. Starting at day 6 containers were checked for D9 3 11 – 13 16 – 21 the presence of pupae. Remaining fl y larvae were put back into the D10 2 11 – 13 17 – 22 substrate and the examination was repeated the following days. To establish the average weight, all pupae that were present at K0medium 0 11 – 13 17 – 20 the second day of pupation were put on a laboratory scale and k0faeces 0 10 – 13 14 – 17 226 Table 2. Development of Musca domestica, larvae of which were exposed to Habronema muscae eggs in comparison to uninfected controls (K0). Figures in brackets show the 95 % confi dence intervals. (* these samples were inoculated with Musca eggs). Group Larvae Pupae Pupation rate Flies hatched Hatching rate Preimaginal mortality rate used developed (%) (n) (%) (%) (n) (n) D0 100* 44 44.0 37 84.1 63.0 <34.1;54.3> <52.8;72.4> D1 100 45 45.0 38 84.4 62.0 <35.0;55.3> <51.7;71.5> D2 50 24 48.0 24 100.0 52.0 <33.7;62.6> <337.4;66.3> D3 50 26 52.0 22 84.6 56.0 <37.4;66.3> <41.3;70.0> D4 50 33 66.0 33 100.0 34.0 <51.2;78.8> <21.2;48.8> D5 50 38 76.0 36 94.7 28.0 <61.8;86.9> <16.2;42.5> D6 50 39 78.0 30 76.9 40.0 <64.0;88.5> <26.4;54.8> D7 50 43 86.0 41 95.3 18.0 <73.3;94.2> <8.6;31.4> D8 50 45 90.0 40 88.9 20.0 <78.2;96.7> <10.0;33.7> D9 50 45 90.0 37 82.2 26.0 <78.2;96.7> <14.6;40.3> D10 50 47 94.0 41 87.2 18.0 <83.5;98.7> <8.6;31.4> K0medium 50* 42 84.0 41 97.6 18.0 <70.9;92.8> <8.6;31.4> K0faeces 50* 41 82.0 39 95.1 22.0 <68.6;91.4> <11.5;31.4> Results Smallest pupae of lengths below 5 mm and width smaller 2 mm and a subsequent volume of less than 10.4 mm3 were present in Most of the fl y larvae left the substrate prior to pupation and pupae groups D0 to D5.