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Anopheles mosquito studies in Pakistan using outlet and inlet traps
14
LABORATORY MEETING
T h e maggots tunnel freely in the treated fabric, as they would do in natural fleece, and mortality counts are taken after 24 hours by unrolling each plug in a petri-dish (with a few drops of lukewarm water) under a binocular microscope. REFERENCES
D u TOXT, R. & FIEDLER, O. G. H. (1953). OnderstepoortJ. vet. Res., 96, 65. GREENWOOD, D. & HARRISON, I. R. (1965). J . Sci. Fd. /lgric., 16, 293. ROXBURGH, N. A. (1972). Abs. 14th fnt. Congr. Entom., Canberra p. 264. I am grateful to Professor J. R. Busvine for guidance in the course of this work, and to the Scholarship Commission of the Association of Commonwealth Universities (U.K.) for financial support.
Effects o f p h a r y n g e a l a r m a t u r e o f m o s q u i t o e s o n m i c r o f i l a r i a e o f Brugia pahangi J. H. BRYAN, P. O O T H M A N , B. ]. A N D R E W S AND P. B. M c G R E E V Y
London School of Hygiene and Tropical Medicine T o invade the haemocoel of their intermediate hosts, microfilariae must first evade the defence mechanism of the vector which operates at the gut level. One of the hazards to microfilariae is damage by the pharyngeal and cibarial armatures. Since COLUZZI and TRABUCCm (1968) first described the danger of mosquito armatures to microfilariae, little effort has been made to assess their effects on vector efficiency. T h e mosquito armature is composed of 2 parts: the cibarial armature, a row of well developed teeth which project into the anterior lumen of the cibarial pump, and the pharyngeal armature, a ring of slender teeth at the posterior end of the pharynx. T h e structure of the armature varies and of those mosquitoes used in the present study Anopheles gambiae A and B and A. farauti No. 1 have both a cibarial and pharyngeal armature, while Aedes aegypti and Ae. togoi have only a pharyngeal armature. T h e armatures damage the microfilariae, causing loss of motility and cuticuiar abrasions. Photographs were exhibited showing the armatures of the above mosquito species and the damaged microfilariae of Brugia pahangi taken from their guts shortly after an infective feed. Graphs were shown to illustrate the effect of the armatures on motility of the microfilariae, as assessed by examining mosquito mid-guts within 10 minutes of an infective feed, using a counting chamber (DENHAM DENNZS, PO~JDURAI, NELSON and GuY, 1971). Graphs also showed the proportion of damaged microfilar/ae determined from stained blood films of engorged mosquito mid-guts. T h e proportion of damaged non-motile microfilariae was much higher in Anopheles spp. than in Aedes spp. These differences may be related to the presence of the cibarial armature in Anopheles and its absence
in Aedes. REFERENGE~
CoLuzzI, M. & TRABUCCHI, R. (1968). Parassitologia, 10, 47. DENHAM, D. A., DENNIS, D. T., PON~-t3DURAI, T., NELSON, G. S. & GuY, F. (1971). Trans. R. Soc. trop. Med. Hyg., 65, 521.
A n o p h e l e s m o s q u i t o s t u d i e s in P a k i s t a n using o u t l e t a n d inlet traps J. A K I Y A M A
Ross Institute, London School of Hygiene and Tropical Medicine Out of 22 anopheline species recorded in Pakistan (ANsARI, 1960), 2 are regarded as malaria vectors of primary importance: Anopheles culicifacies and Anopheles stephensi. These 2 species are widely distributed in the country and are found throughout the year. Generally 2 peaks of seasonal prevalence have been observed: the first during April-May and the second during August-September. Studies were carried out on the behaviour of anopheline mosquitoes using outlet and inlet traps, to obtain base-line data before D D T residual spraying started. Outlet trap observations were continued in several localities after spraying in the operational zones in the National Malaria Eradication Programme, to see if there had been any change in mosquito behaviour following spraying. At first, observations were made using single outlet window traps set up in houses but the traps were not productive. Further observations were therefore carried out using 4 traps fixed into the doorway in village houses. By this method a large number of mosquitoes were caught as they left houses. Several types of inlet traps were used but mosquitoes were only captured in one type: mosquito netting fixed in a doorway forming a funnel-like shape with a rolled up curtain at the entrance. Mosquitoes entering through the doorway were collected every hour after dropping the entrance curtain. Hourly outlet trap collections were made during September-October 1962 at the unsprayed village of Rakh Dala, Lahore District, by changing traps alternately throughout the night from sunset to sunrise. A total of 1,329 females and 2,090 males of A. culicifacies and 3,962 females and 2,890 males of A. stephensi were captured. Most mosquitoes left immediately after sunset and by 21.00 hours 90%. of males and females of both species had left. It is interesting to note that half-gravid females of both species moved
LABORATORY MEETING
15
OUt throughout the night. Perhaps they required more blood or they may rest temporarily outdoors during the night, as they have been seen re-entering houses at dawn. Females of A. culicifacies and A. stephensi in the outlet traps were classified into 4 abdominal stages and the percentage of each stage is as follows : - A, culicifacies--unfed 25.8%; freshly fed 0,6%; half gravid 40.8%, gravid 32.8%. A. stephensi--unfed 24.8%; freshly fed 0.7% ; half gravid 38.4% and gravid 35.6%. Morning indoor resting catches usually revealed freshly fed mosquitoes in large numbers. However very few freshly fed females were caught in the traps so the females were remaining indoors until half-gravid or gravid. This means that they rested indoors for at least 12 hours (if they entered at dawn). These mosquitoes were endophilic although they fed outdoors, as the villagers and their domestic animals slept outdoors at the time of the observations. I n spite of these mosquitoes being exophagic, their endophily means that they are amenable to control by residual spraying of houses. However insecticide coverage of all the resting sites inside the houses was difficult to achieve, owing to the complexity of the roof and the large number of items kept inside the houses. T h e structure of houses and objects commonly stored in them in the rural areas of Pakistan were shown in photographs. I n spite of this difficulty the spraying squads achieved good coverage and consequently considerable lowering in density of the vectors occurred after residual spraying with D D T and transmission of malaria was reduced. Two night observations were conducted using inlet traps at the unsprayed village of Lalu Kichi, Lahore District, in May 1962. Only small numbers were captured: 33 females and 1 male A. culicifacies. 66% were freshly fed, 27% half-gravid and 7% gravid. Over 80% of the freshly fed females entered between 23.00 hours and 03.00 hours. REFERENCE
ANsABI, M. A. R. (1960). Government of Pakistan Plan of Action for Malaria Eradication Programme (West).
A s i m p l e field m e t h o d for assessing the proportion o f older parous f e m a l e s in an anopheline
sample (Anopheles forouti)
M A R G A R E T S P E N C E R (Introduced by DR. G. DAVIDSON)
Formerly of the Malaria Service, Territory of Papua, New Guinea T h e quantity of pigment within the ovary and the length of the ovariolar sacs are important aids to detection of the presence of females of epidemiological potential in a biting population. Pigment can readily be seen in the intact ovary b y transmitted light, and these examinations may easily be made up to Christophers' Stage I I I of o6cyte development. Examinations for the length of the ovariolar sacs should be made as soon as possible after oviposition is completed, and for best results should be done at night while the sacs are still fully distended. Slitting of the ovarian sheath will expose the sacs. Short tight ovariolar sacs and little pigment indicate few dilatations and therefore young parous mosquitoes. Long loose sacs and much granular pigment indicate many dilatations and therefore older porous mosquitoes. T h e ovariolar sacs may be very long indeed. I f on dissection the uncontracted sacs prove to be very long and there is much pigment present in a significant number of individuals it can be assumed that the population is likely to be dangerous and further investigations can be planned on that assumption. There are also different handling qualities on dissection-nulliparous ovaries are small, taut, and compact, while older parous ovaries are long, loose, and have great stretchability. T h e pigment shows up as greenish granules when saline is slightly tinted with methylene blue, readily seen in the intact ovary when flattened beneath a coverslip. These points were illustrated by a series of microphotographs, prints and drawings.
Egg m e m b r a n e s o f S i m u l i i d a e T . R. W I L L I A M S (Introduced by DR. W. W. MACDONALD)
Department of Zoology, University of Liverpool Laid as egg-masses simuliid eggs may appear to be embedded in an adhesive gelatinous matrix. Light and electron microscope studies have shown this substance to be the adherent outer membranes of individual eggs. This membrane surrounds the ovarian egg and evidently corresponds to the exochorion of Culicidae (CLEMENTS, 1963) and other Nematocera. T h e exochorion of British Simulium argyreatum consists of an inner lamellar region (further subdivisible) staining for acidic mucopolysaccharides. Its outer lamella is produced into flexible filaments, 3 to 4~ in length, and apparently coated with the surface adhesive. T h e egg surface is irregularly roughened or, at most, indistinctly papillose. Eggs of S. damnosum from Amani, Tanzania, are essentially similar in structure, but have shorter (0.6~), more regularly arranged filaments. A finely divided surface gives the filamentous layer a marked resemblance to a chorionic plastron (HlbrrON, 1969). T h e deeply excavated exochorion of another Tanzanian egg, also thought to be simuliid, is still more strongly suggestive of respiratory adaptation. T h e tough, deeply pigmented "shell" of the simuliid egg thus represents an inner egg membrane or eudochorion. Scanning electron microscopy shows its surface to be either featureless or slightly dimpledj
LABORATORY MEETING
T h e maggots tunnel freely in the treated fabric, as they would do in natural fleece, and mortality counts are taken after 24 hours by unrolling each plug in a petri-dish (with a few drops of lukewarm water) under a binocular microscope. REFERENCES
D u TOXT, R. & FIEDLER, O. G. H. (1953). OnderstepoortJ. vet. Res., 96, 65. GREENWOOD, D. & HARRISON, I. R. (1965). J . Sci. Fd. /lgric., 16, 293. ROXBURGH, N. A. (1972). Abs. 14th fnt. Congr. Entom., Canberra p. 264. I am grateful to Professor J. R. Busvine for guidance in the course of this work, and to the Scholarship Commission of the Association of Commonwealth Universities (U.K.) for financial support.
Effects o f p h a r y n g e a l a r m a t u r e o f m o s q u i t o e s o n m i c r o f i l a r i a e o f Brugia pahangi J. H. BRYAN, P. O O T H M A N , B. ]. A N D R E W S AND P. B. M c G R E E V Y
London School of Hygiene and Tropical Medicine T o invade the haemocoel of their intermediate hosts, microfilariae must first evade the defence mechanism of the vector which operates at the gut level. One of the hazards to microfilariae is damage by the pharyngeal and cibarial armatures. Since COLUZZI and TRABUCCm (1968) first described the danger of mosquito armatures to microfilariae, little effort has been made to assess their effects on vector efficiency. T h e mosquito armature is composed of 2 parts: the cibarial armature, a row of well developed teeth which project into the anterior lumen of the cibarial pump, and the pharyngeal armature, a ring of slender teeth at the posterior end of the pharynx. T h e structure of the armature varies and of those mosquitoes used in the present study Anopheles gambiae A and B and A. farauti No. 1 have both a cibarial and pharyngeal armature, while Aedes aegypti and Ae. togoi have only a pharyngeal armature. T h e armatures damage the microfilariae, causing loss of motility and cuticuiar abrasions. Photographs were exhibited showing the armatures of the above mosquito species and the damaged microfilariae of Brugia pahangi taken from their guts shortly after an infective feed. Graphs were shown to illustrate the effect of the armatures on motility of the microfilariae, as assessed by examining mosquito mid-guts within 10 minutes of an infective feed, using a counting chamber (DENHAM DENNZS, PO~JDURAI, NELSON and GuY, 1971). Graphs also showed the proportion of damaged microfilar/ae determined from stained blood films of engorged mosquito mid-guts. T h e proportion of damaged non-motile microfilariae was much higher in Anopheles spp. than in Aedes spp. These differences may be related to the presence of the cibarial armature in Anopheles and its absence
in Aedes. REFERENGE~
CoLuzzI, M. & TRABUCCHI, R. (1968). Parassitologia, 10, 47. DENHAM, D. A., DENNIS, D. T., PON~-t3DURAI, T., NELSON, G. S. & GuY, F. (1971). Trans. R. Soc. trop. Med. Hyg., 65, 521.
A n o p h e l e s m o s q u i t o s t u d i e s in P a k i s t a n using o u t l e t a n d inlet traps J. A K I Y A M A
Ross Institute, London School of Hygiene and Tropical Medicine Out of 22 anopheline species recorded in Pakistan (ANsARI, 1960), 2 are regarded as malaria vectors of primary importance: Anopheles culicifacies and Anopheles stephensi. These 2 species are widely distributed in the country and are found throughout the year. Generally 2 peaks of seasonal prevalence have been observed: the first during April-May and the second during August-September. Studies were carried out on the behaviour of anopheline mosquitoes using outlet and inlet traps, to obtain base-line data before D D T residual spraying started. Outlet trap observations were continued in several localities after spraying in the operational zones in the National Malaria Eradication Programme, to see if there had been any change in mosquito behaviour following spraying. At first, observations were made using single outlet window traps set up in houses but the traps were not productive. Further observations were therefore carried out using 4 traps fixed into the doorway in village houses. By this method a large number of mosquitoes were caught as they left houses. Several types of inlet traps were used but mosquitoes were only captured in one type: mosquito netting fixed in a doorway forming a funnel-like shape with a rolled up curtain at the entrance. Mosquitoes entering through the doorway were collected every hour after dropping the entrance curtain. Hourly outlet trap collections were made during September-October 1962 at the unsprayed village of Rakh Dala, Lahore District, by changing traps alternately throughout the night from sunset to sunrise. A total of 1,329 females and 2,090 males of A. culicifacies and 3,962 females and 2,890 males of A. stephensi were captured. Most mosquitoes left immediately after sunset and by 21.00 hours 90%. of males and females of both species had left. It is interesting to note that half-gravid females of both species moved
LABORATORY MEETING
15
OUt throughout the night. Perhaps they required more blood or they may rest temporarily outdoors during the night, as they have been seen re-entering houses at dawn. Females of A. culicifacies and A. stephensi in the outlet traps were classified into 4 abdominal stages and the percentage of each stage is as follows : - A, culicifacies--unfed 25.8%; freshly fed 0,6%; half gravid 40.8%, gravid 32.8%. A. stephensi--unfed 24.8%; freshly fed 0.7% ; half gravid 38.4% and gravid 35.6%. Morning indoor resting catches usually revealed freshly fed mosquitoes in large numbers. However very few freshly fed females were caught in the traps so the females were remaining indoors until half-gravid or gravid. This means that they rested indoors for at least 12 hours (if they entered at dawn). These mosquitoes were endophilic although they fed outdoors, as the villagers and their domestic animals slept outdoors at the time of the observations. I n spite of these mosquitoes being exophagic, their endophily means that they are amenable to control by residual spraying of houses. However insecticide coverage of all the resting sites inside the houses was difficult to achieve, owing to the complexity of the roof and the large number of items kept inside the houses. T h e structure of houses and objects commonly stored in them in the rural areas of Pakistan were shown in photographs. I n spite of this difficulty the spraying squads achieved good coverage and consequently considerable lowering in density of the vectors occurred after residual spraying with D D T and transmission of malaria was reduced. Two night observations were conducted using inlet traps at the unsprayed village of Lalu Kichi, Lahore District, in May 1962. Only small numbers were captured: 33 females and 1 male A. culicifacies. 66% were freshly fed, 27% half-gravid and 7% gravid. Over 80% of the freshly fed females entered between 23.00 hours and 03.00 hours. REFERENCE
ANsABI, M. A. R. (1960). Government of Pakistan Plan of Action for Malaria Eradication Programme (West).
A s i m p l e field m e t h o d for assessing the proportion o f older parous f e m a l e s in an anopheline
sample (Anopheles forouti)
M A R G A R E T S P E N C E R (Introduced by DR. G. DAVIDSON)
Formerly of the Malaria Service, Territory of Papua, New Guinea T h e quantity of pigment within the ovary and the length of the ovariolar sacs are important aids to detection of the presence of females of epidemiological potential in a biting population. Pigment can readily be seen in the intact ovary b y transmitted light, and these examinations may easily be made up to Christophers' Stage I I I of o6cyte development. Examinations for the length of the ovariolar sacs should be made as soon as possible after oviposition is completed, and for best results should be done at night while the sacs are still fully distended. Slitting of the ovarian sheath will expose the sacs. Short tight ovariolar sacs and little pigment indicate few dilatations and therefore young parous mosquitoes. Long loose sacs and much granular pigment indicate many dilatations and therefore older porous mosquitoes. T h e ovariolar sacs may be very long indeed. I f on dissection the uncontracted sacs prove to be very long and there is much pigment present in a significant number of individuals it can be assumed that the population is likely to be dangerous and further investigations can be planned on that assumption. There are also different handling qualities on dissection-nulliparous ovaries are small, taut, and compact, while older parous ovaries are long, loose, and have great stretchability. T h e pigment shows up as greenish granules when saline is slightly tinted with methylene blue, readily seen in the intact ovary when flattened beneath a coverslip. These points were illustrated by a series of microphotographs, prints and drawings.
Egg m e m b r a n e s o f S i m u l i i d a e T . R. W I L L I A M S (Introduced by DR. W. W. MACDONALD)
Department of Zoology, University of Liverpool Laid as egg-masses simuliid eggs may appear to be embedded in an adhesive gelatinous matrix. Light and electron microscope studies have shown this substance to be the adherent outer membranes of individual eggs. This membrane surrounds the ovarian egg and evidently corresponds to the exochorion of Culicidae (CLEMENTS, 1963) and other Nematocera. T h e exochorion of British Simulium argyreatum consists of an inner lamellar region (further subdivisible) staining for acidic mucopolysaccharides. Its outer lamella is produced into flexible filaments, 3 to 4~ in length, and apparently coated with the surface adhesive. T h e egg surface is irregularly roughened or, at most, indistinctly papillose. Eggs of S. damnosum from Amani, Tanzania, are essentially similar in structure, but have shorter (0.6~), more regularly arranged filaments. A finely divided surface gives the filamentous layer a marked resemblance to a chorionic plastron (HlbrrON, 1969). T h e deeply excavated exochorion of another Tanzanian egg, also thought to be simuliid, is still more strongly suggestive of respiratory adaptation. T h e tough, deeply pigmented "shell" of the simuliid egg thus represents an inner egg membrane or eudochorion. Scanning electron microscopy shows its surface to be either featureless or slightly dimpledj