Methods for dissecting dry insects and insects preserved in fixative solutions or by refrigeration

Bull. Org. mond. Sant) 1972, 47, 239-244Bull. Wld Hlth Org.J Methods for dissecting dry insects and insects preserved in fixative solutions or by refrigeration ERNEST M. UNGUREANU 1 The methods described in this paper for the dissection of dry and preserved insects have been used for several years on various species, mainly mosquitos. In the past, dry or partially dry mosquitos found in traps or in the laboratory had to be discarded. By soften- ing these insects in a detergent solution, however, it is possible to make most observations in the same way as on fresh material. The preservation of insects in the dry state, in a fixative, or in the refrigerator after collection enables much larger samples to be studied; the whole of the material can be examined and the work can be done when time permits. In addition, material can be sent to central laboratories far from the place of collection, and infected insects can be kept in stock for teaching purposes. This paper is in three sections: the first describes methods of dissection of dry insects and preserva- tion of mosquitos in dry condition; the second deals with preservation in a fixative solution and subse- quent dissection; and the third discusses methods of preserving insects by refrigeration for dissection and examination of internal organs. These methods offer new possibilities for the more complete processing of samples of vector populations collected for different studies. THE DISSECTION OF DRY INSECTS During field and laboratory studies a number of insects become too dry to be dissected for an exami- nation of their internal organs. In many instances some of the insects collected in traps or experi- mental huts, mainly the unfed specimens or those intoxicated by an insecticide, are found dead and partially dry in the morning. A complete processing of the catch, which is particularly important for small samples, is thus impossible. In order to over- come this difficulty, a simple method for examining and dissecting dry insects was developed. The method, which has been used for 3 years in dissect- ing dry Anopheles, Culex, Musca, Stomoxys, Tabanus, Glossina, Erystalis, and Simulium, makes it possible not only to study the abdominal appearance of insects but also to examine the internal organs and parts of the body and to look for external and I Professor of Medical Parasitology, Institute of Medicine, Jassy, Romania. internal parasites. Repeated observations were carried out on different dried wild and laboratory mosquitos. Rehydration The principle of the method is to rehydrate the dry insects. The materials needed are: (1) liquid detergent, without enzymes; (2) physiological saline for invertebrates (0.65 %); (3) tap water. In the absence of a convenient detergent any type of emollient solution can be used: 1 % soap solution, 0.5% sodium benzoate, a weak solution (0.5%Y.) of potassium or sodium hydroxide, or even physiolo- gical saline alone. When physiological saline is used alone the softening time is much longer; the insects should be kept in a refrigerator at about 4°C for 12-24 hours according to their size and the degree of dryness. Diluted solutions of almost all chemicals with detergent or emollient properties can be employed, the time needed for rehydration being established by preliminary trials for each species of insect and softening solution. Household liquid detergents may also be used, the only condition being that they should not contain enzymes. The concentration of the detergent may vary from 0.5% to 20% according to the type used and the speed of softening required. Softening procedure The softening process consists of two steps: (1) The insects are immersed in a diluted deter- gent, one part liquid detergent to 4-9 parts water; they should be kept in this solution for 10-60 min- 2892 - 239- 8 E. M. UNGUREANU utes, according to their dryness and size and the type of solution. (2) The insects are transferred to 0.65% physiolo- gical saline or tap water for a period varying from 20 minutes to several hours according to their size and state of dryness. When only the abdominal appearance of a mosquito is to be studied or only partially dry insects are being dissected, the insects can be prepared for examination in no more than 20 minutes by using a 20% solution of a liquid detergent and allowing 10 minutes in the detergent and 10 minutes in water. Dissection methods In insects that have recently been partially dried, the internal organs can be extracted after softening by using methods similar to those for fresh insects. When dealing with insects that are very dry or slightly decomposed before rehydration, the organs must be manipulated with much more care (espe- cially when dealing with small insects) because of the considerable change in their elasticity. Methods of extracting different organs of mosquitos in such situations are illustrated in Fig. 1-3. Extraction of salivary glands (see Fig. 1) (1) Cut offthe head and the anterior legs (A and B). (2) Detach the area of the prothoracic segment, to which the salivary glands remain attached (C and D). (3) Detach the salivary glands with a fine dis- secting needle (E). (4) Add a drop of 0.65% physiological saline containing a little gentian violet, methylene blue, or Giemsa stain in order to colour the tissues slightly, and examine under a cover slip at the appropriate magnification. In some cases extraction of the salivary glands may prove very difficult if not impossible. Extraction of the abdominal organs (see Fig. 2) (1) Separate the abdomen from the thorax (A). Fig. 2. Extraction of the abdominal organs. ,ED 4- WE 29257 240 2- em 32aZ Fig. 1. Extraction of the salivary glands. DISSECTION OF DRY AND PRESERVED INSECTS (2) Fix the extremity of the abdomen with the right-hand needle. Insert the left-hand needle inside the abdomen along the tergal plates, advancing it slowly until it reaches the last segment and touches the slide with its tip (B). (3) Press the right-hand needle against the left one, making a slight movement in order to cut the tergum along the whole length from segments 1 to 8 (C and D). (4) Open the abdomen with the tips of the dis- secting needles; insert one needle under the abdomi- nal organs and remove them from the abdomen (E). If the malpighian tubes or parts of the fat body cells are covering the midgut and cannot be removed with the needle, use a very fine paint brush. Separate the ovaries and, if necessary, dissect them to examine dilatations. (5) Add a drop of physiological saline lightly stained with methylene blue (1: 10 000), gentian violet (1: 10 000), or Giemsa stain (one drop to 4 ml of physiological saline) and examine on a covered slide. Another method of opening the abdomen is illus- trated in Fig. 3. The abdominal pleuron is detached with the tip of the dissecting needle (A and B) and the tergum removed (C). The organs are then separated from the rest of the abdomen (D). Fig. 3. Alternative method of dissecting the abdomen. Material used and results The rehydration method was first used under field conditions in 1968. Since then large numbers of dry mosquitos from different sources have been dissected, including the following species collected in the field: Aedes (O.) communis and Ae. (O.) rusticils (Switzerland and France), Anopheles albi- manus (Haiti), An. annularis and An. balabacensis (Thailand), An. funestus and An. gambiae s.l. (north- ern Nigeria, Senegal, and Togo); An. littoralis (Philippines), An. messae (Romania and Switzer- land), An. minimus minimus (Thailand), An. nili and An. pharoensis (Togo), An. philippinensis (Thailand), and Culex pipiens (Switzerland, France, and Roma- nia). Dry laboratory-bred specimens of the following anopheline species were dissected in Dr G. David- son's laboratory at the Ross Institute of Tropical Hygiene, London School of Hygiene and Tropical Medicine, England: Anopheles albimanus, An. farauti, An. funestus, An. gambiae A, B, C, An. labranchiae atroparvus, An. melas, An. merus, An. pharoensis, and An. stephensi. Dry specimens of An. stephensi infected with Plasmodium berghei were kindly provided by the Ross Institute, the Malaria Reference Centre, Horton Hospital, Epsom, England, and the Department of Parasitology, Liverpool School of Tropical Medi- cine, England. Aedes albopictus infected with P. gal- linaceum oocysts in all stages and sporozoites was provided by the WHO International Malaria Eradi- cation Training Centre, Manila, Philippines. Two types of material have been dissected. The first type consisted of insects found dead and par- tially dried in window and light traps or in the laboratory. Secondly, insects were killed, left to dry, and then dissected after intervals of 1, 2, 3, 7, 14, 21, 30, 45, 60, 75, 180, and 300 days. After being killed the insects were transferred to small wide- mouthed bottles or to large glass or plastic tubes containing a layer of silica gel or calcium chloride D 3-WHO 2010 241 E. M. UNGUREANU covered with a layer of cotton wool (Fig. 4). The containers were then carefully closed and sealed with solid paraffin. In a warm and dry climate the insects dry in a very short time, but to preserve them in good condition they should be placed in tubes containing silica gel or calcium chloride or in a desiccator. In a hot and humid climate, their internal organs may be rapidly disintegrated by bacteria, though the ovaries are more resistant than the diges- tive tract. At the Ross Institute in April 1970, mosquitos dried in paper cups near an electric heater for 1-2 hours after killing were successfully dissected and examined for Sella's and Christophers' stages and for parity and nulliparity up to 180 days. An. fune- stus, An. gambiae A, and An. melas from Togo were kept for 45 days in containers with silica gel and then examined for abdominal appearance (Sella's stages), parity rates, stomach infections (in empty ones), condition of salivary glands, Christophers' stages, retained eggs, and refeeding by gravid females. Agamodistomum sp. was found in An. funestus from Togo and An. m. minimus from Thailand. After a batch of dried An. funestus and An. gam- biae from Kano, Nigeria, had been kept at room temperature in Geneva for 2 years, it proved pos- sible to examine the insects for abdominal appear- ance, Christophers' stages, refeeding by gravid females, retained eggs, and Coelomomyces. The reactions produced by aquatic mites and by black spores were also clearly identifiable. The stomachs of unfed mosquitos can be satisfactorily examined for the presence of oocysts in all stages if well 4 -WFP -w WHO 20289 Fig. 4. Tubes for drying mosquitos. M: mosquitos; CW: cotton wool; FP: filter paper. preserved in the dry state. When An. stephensi infected with P. berghei sporoblasts and sporocysts was rehydrated and dissected after being kept dry for 14 days, the fine internal structure of unstained sporoblasts could be seen and the sporozoites were visible. Apart from being dried in tubes containing silica gel or calcium chloride, mosquitos may be lyophy- lized under vacuum in a test-tube which is then sealed by heat, or they can be dried in a refrigerator at about 0°C. Dry mosquitos kept in the deep- freeze compartment of a refrigerator can be held for months before rehydration for dissection. When rehydrated mosquitos cannot be dissected the same day, they can be kept in a refrigerator at a temperature ranging from -4°C to +4°C; a disinfectant (0.5% sodium benzoate) may be added to the physiological saline if a refrigerator is not available. This method enabled dry mosquitos to be exam- ined for the following: abdominal appearance (Sel- la's stages); Christophers' stages; parous and nulli- parous females; sacs and dilatations in the ovarioles (mainly in recently dried specimens); refeeding of gravid females; number of mature eggs per batch; retained eggs; state of spermatheca; presence of Coelomomyces spores in the ovaries; mermithid larvae; filaria larvae (Dirofilaria, Microfilaria); encysted forms of trematode larvae; Crithidia; young and old Plasmodium oocysts in unfed mos- quitos and, on a few occasions, sporozoites in the glands; and aquatic mite larvae and the reactions produced by them and by black spores. EXAMINATION AND DISSECTION OF MOSQUITOS PRESERVED IN DIFFERENT FIXATIVE SOLUTIONS The use of formalin or ethanol for preserving specimens of insects for identification is widespread. However, the method has not so far been employed to preserve adult mosquitos for subsequent examina- tion of abdominal appearance and physiological stages or for the study of internal organs for various purposes (mainly in unfed mosquitos). Method offixation Recently killed or anaesthetized mosquitos are dipped in one of the following solutions. formula (a) formalin (neutralized) . . . . . . . . . . .10 ml glycerol . . . . . . . . . . . . . . . . 4 ml 242 DISSECTION OF DRY ANI) PRESERVED INSECTS acetic acid, glacial .... . . . . . .. . physiological saline 0.65% (or distilled water) sodium tetraborate (10 H20) . formula (b) ethanol, 75%. glycerol formula (c) (Bouin's solution) picric acid, saturated aqueous solution formalin. acetic acid, glacial . . . . . . . .. .. . 1 ml utes in tap water. The internal organs of preserved 100 ml mosquitos cannot be extracted by the methods used 0.5 g to dissect fresh mosquitos; a procedure similar to those illustrated in Fig. 1 and 2 for dissecting dried insects after rehydration should be used. Inter- 95 ml nal organs can be stained with haemalum or haema- 5 ml toxylin and mounted as a permanent preparation in Canada balsam or euparol. Stomachs infected with Plasmodium oocysts can be prepared in paraffin for 30 ml microscopical section. 10 ml 2 ml Bouin's solution is particularly useful for histologi- cal studies as the cell nuclei are well fixed and their structure appears clearly. formula (d) (Carnoy's solution) chloroform . . . . . . . . . . . . . . . 30 ml ethanol, absolute . . . . . . . . . . . . . 60 ml acetic acid, glacial . . . . . . . . . . . . 10 ml Carnoy's solution is particularly useful for fixing the chromosomes of larval salivary glands, the ova- ries or malpighian tubes of adults, or malaria parasites, since it fixes the nucleus and its con- stituents. When fixing larval glands or adult abdo- minal organs, the thorax of larvae or the abdomen of adults should be separated from the rest of the body to permit rapid penetration of the fixative; alternatively, the organs should be dissected before fixing. For longer preservation, the material should be placed in solution (e) after fixation for 3-4 hours in Carnoy's solution. formula (e) formalin (neutralized) . . . . . . . . . . . 6 m physiological saline (0.65 %) . . . . . . . . 94 m formula (f) formalin . . . . . . . . . . . . . . . . 10 ml water . . . . . . . . . . . . . . . . . 140 ml sodium tetraborate (10 H2O) . . . . . . . . 0.5 g Before dipping the mosquitos in solutions (a), (c), (e), or (f), they should first be dipped for 1-2 seconds in 90o% ethanol in order to reduce the surface tension of the cuticula and help the fixative to pene- trate. Many other fixative solutions may be used if desired. Method of dissection Before mosquitos preserved in formalin or ethanol are dissected, they should be washed for a few min- Mosquitos may be kept for long periods in these solutions, and can be examined even after several years for the characteristics and infections already mentioned. Plasmodium sporozoites can be detected in the salivary glands of these insects when stained; however, they are not as clear as in fresh specimens because the sporozoites are already fixed inside the cells of the glands. DISSECTION OF FROZEN MOSQUITOS Methods ofpreservation and dissection Mosquitos recently collected by pyrethrum spray and live mosquitos anaesthetized by ether may be immersed in a mixture of water and glycerol in the proportion of 8: 3 by volume in small glass or plastic bottles or tubes. This mixture has a low freezing point and mosquitos can be frozen at between - 150 and - 180 C while the mixture remains liquid. They should be immersed only after the mixture has reached these temperatures. The mosquitos can be extracted one by one from the mixture while they are still frozen without defreezing the whole batch of mosquitos. After some days, however, the organs may be hardened by the glycerol. A preferable method is to dip recently killed mos- quitos for 1 second in 80% ethanol, immerse them in water in small plastic tubes, and freeze them at -18° to -25°C. The frozen mosquitos can be dissected in the same way as fresh ones, but their organs are less elastic than when fresh. Before dis- section the mosquitos must be dipped in 0.65% saline. All internal organs have been extracted in good condition after freezing for 1 month and oocysts and sporozoites can be easily detected. After 90 days, ovaries of Aedes rusticus were easily dissected for dilatations of the ovarioles. Observations on the practical uses of this method are continuing. 243 E. M. UNGUREANU CONCLUSION The methods described above are not intended to replace those now in use for the processing of fresh material. However, they offer the possibility in spe- cial situations of processing samples more complete- ly. Dry insects that were previously discarded as useless, with the result that information was lost, can be dissected and examined. Mosquitos can also be preserved in a dry state, in fixative solution, or by deep freezing for later examination when lack of time and staff prevents complete processing of very large samples when fresh. The methods permit preserved material to be sent to different laboratories for demonstration pur- poses and can assist in controlling the quality of work carried out by field teams. Indeed, certain aspects of the work may be undertaken at leisure, at a convenient time under proper laboratory con- ditions. ACKNOWLEDGEMENTS The author thanks Professor L. J. Bruce-Chwatt, Director, and Dr G. Davidson, Ross Institute of Tropical Hygiene, London School of Hygiene and Tropical Medi- cine, England; Professor W. Peters, Liverpool School of Tropical Medicine, England; Mr P. G. Shute and Miss M. Maryon, Malaria Reference Laboratory, Horton Hospital, Epsom, England; and the WHO field staff who provided much of the material used for this study. RESUME METHODES DE DISSECTION D'INSECTES DESSJtCHIS ET D'INSECTES CONSERVES DANS DES SOLUTIONS FIXATIVES OU PAR REFRIGtRATION L'auteur decrit des methodes de dissection d'insectes partiellement ou entierement desseches ou conserves dans differentes solutions fixatives. Des insectes congeles a - 18°C ont egalement ete disseques soit pour etudier les organes internes soit pour constater une infection par un Plasmodium ou par d'autres endoparasites. La dissection des insectes desseches est rendue ais&e apres rehydratation. Des detergents liquides courants ont et utilises, mais les detergents renfermant des en- zymes ne devraient pas etre employes. Le procedd de rehydratation comporte deux etapes: a) immersion des insectes dans une solution de detergent (1 partie pour 4-9 parties d'eau) ou ils sont gardes pendant 10 a 60 min. La duree d'immersion depend de l'etat de dessechement et de la taille des insectes, ainsi que du type de deter- gent; b) on place ensuite les insectes dans l'eau (ordinaire) et, apres un lavage de 20 A 30 min, on les met dans une solution physiologique a 0,65%. (Le transfert des insectes dans des solutions physiologiques n'est pas necessaire s'ils ne sont pas gardes plus d'une demi-heure a une heure dans l'eau). Le temps requis pour une rehydratation satisfaisante est facilement determine par chaque chercheur. Cette methode de rehydratation peut etre appliqu6e soit aux insectes partiellement ou entierement desseches, collectes lors de differentes observations, soit aux insectes collectes vivants et intentionnellement desseches pour etre disseques plus tard. A cette fin, il est conseille de placer les insectes dans des tubes en verre ou en plastique, remplis au tiers de Silicagel ou de chlorure de calcium, et soigneusement refermes apres l'operation. Il faut se souvenir que, dans un climat chaud et humide, les organes internes des insectes se decomposent assez rapidement. Au laboratoire, les insectes peuvent etre conserves dans un dessiccateur. En utilisant la methode de rehydratation, il est possible d'effectuer pratiquement tous les examens realises sur les insectes frais. L'examen des glandes salivaires des moustiques pour la recherche des sporo- zoltes est un peu plus difficile A r6aliser sur des insectes desseches que sur des insectes A l'etat frais. Des dissec- tions de moustiques conserves dans la formaline ou dans l'alcool ont ete effectuees en utilisant les methodes illustrees par les figures 1, 2 et 3. Presque tous les examens effectues sur des moustiques a l'etat frais ont pu l'etre egalement sur des moustiques conserves dans une solu- tion fixative, exception faite de l'examen du nombre des dilatations des ovarioles; l'identification des sporozoltes dans les glandes salivaires est moins facile A effectuer sur des moustiques fixes que sur des preparations fraiches. Les meme dissections et les memes examens sont pos- sibles sur des moustiques congeles A - 18°C et sur des moustiques frais. Les methodes decrites offrent de larges possibilites pour l'utilisation des echantillons d'insectes, les insectes partiellement ou totalement dess&ehs pouvant etre utilises pour differents examens. La conservation des insectes a l'etat sec ou dans la formaline ou dans l'alcool, etc., offre la possibilite d'examiner plus tard des specimens qui ne peuvent pas etre examines sur place, en raison du manque de temps ou d'un echantillonnage extremement abondant. Ces methodes offrent aussi la possibilite d'exercer un meilleur contr6le de la qualite du travail des equipes isolees qui travaillent sans surveillance effective, ou d'examiner le materiel a une date plus convenable, ou dans un laboratoire tres eloigne du lieu de collecte des specimens. 244