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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tcar20 Download by: [179.176.213.72] Date: 19 February 2016, At: 09:47 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: 0008-7114 (Print) 2165-5391 (Online) Journal homepage: http://www.tandfonline.com/loi/tcar20 A Cytogenetic Study of the Effects of X-Irradiation on Aedes Aegypti K. S. Rai To cite this article: K. S. Rai (1963) A Cytogenetic Study of the Effects of X-Irradiation on Aedes Aegypti, Caryologia, 16:3, 595-607, DOI: 10.1080/00087114.1963.10796099 To link to this article: http://dx.doi.org/10.1080/00087114.1963.10796099 Published online: 29 Jan 2014. Submit your article to this journal Article views: 51 View related articles A CYTOGENETIC STUDY OF THE EFFECTS OF X-IRRADIATION ON AEDES AEG YPTI K. S. RAI Department of Biology, University of Notre Dame, Notre Dame, Indiana, U.S.A. Receiced: 27th September 1'963" INTRODUCTION Although a number of species of insects have been used to study the effects of radiation on living systems, most of these studies have centered around Drosophila, Habrobracon and Chortophaga. In a recent review, GROSCH (1962) listed about 1.30 references concerning the effects of radiation on more than 20 insect genera. About two-thirds of these investigations involved Drosophila or H abrobracon. Relatively few studies have been made on the effects of radiation on the yellow fever mosquito, Aedes aegypti. TERZIAN (1953) collected some data on X-ray induced mortality in this species. Later TERZIAN and STAHLER (1958) de- scribed the effects of gamma radiation on the viability of the eggs and on fertility and fecundity. Irradiation has also been used to induce mutations (VANDEHEY and CRAIG 1959) and sterility (McCRAY et al. 1961) in this species. However, no work has been done on the cytogenetic effects of radiation on this species. Some of the recent findings (RAI 1963) mentioned below qualify this species as admirably suited for studies in this field. The normal karyotype is characterised by three pairs of rather large chromosomes which are individually recognisable (Fig. 2). Moreover, the intimate somatic pairing that occurs between homologous chromosomes during mitotic prophase provides a handy tool for the detection of chromosomal aberrations in somatic tissues. Another advanta- geous feature is that in the brain of a developing larva the proportion of mi- totically dividing cells is rather high. In view of the medical importance of Aedes aegypti, an understanding of the response of chromosomes to irradiation is highly desirable. The present stu- dy was undertaken to investigate X-ray-induced changes in mitotic activity, "This work received support from the following sources: (1) The Radiation Laboratory operated by the University of Notre Dame and supported in part under Atomic Energy Ccm- mission Contract AT (11-1)-38; (2) Research Grants No. GM-11753 and AI-2753, National Institute of Health, U.S. Public Health Service; and (3) Research Contract No. DA-18-064- 404-CML 471, U.S. Army Biological Laboratories. 595] [Caryologia, Vol. 16, n. 3, 1963 D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 596 RAI chromosomal aberrations and mortalitv in this species. Another object was to study the effects of relatively low doses of X-irradiation (500 r) on oviposition and hatchability of the eggs. MATERIAL AND METHODS The NIH strain of Aedes aegypti was used in the present study. This strain has been inbred by single-pair, brother-sister matings for more than 20 generations in our laboratory. Details of the rearing methods used are given by CRAIG and VANDE- HEY (1962). Stocks were maintained in a room with a temperature of 80°F (±l'F) and relative humidity of 80% (± 10%). Fresh eggs were hatched in deoxygenated water. The growing larvae were reared in white enamel pans containing tap water. The larvae were fed with commercial dog-food pellets (Gaines). Four batches of 125 larvae were irradiated six days after hatching at a time when most were in the early fourth instar. The source of radiation was a 260 KVP Picker Therapy X-ray machine operated at 250 KVP and 18 rnA. 1.0 mm of AI and 0.25 mm of Cu filteration were used giving a half value layer of 1.05 mm of Cu. The dose rate was approximately 143 r/minute as measured in air with a Victoreen condenser r-meter. The total doses given were 500, 1000, 2000 and 4000 r. An additional batch of 125 larvae was kept as control. Larvae were killed by fixation at regular intervals after irrad"ation. The fixative used was a solution of 6 parts of methanol; 3 parts of chloroform; 2 parts of propionic acid (PIENAAR 1955). This solution acts both as fixative and preservative. From each of the five batches (four irradiated, one control), 5-10 larvae were fixed immediately after irradiation (0 time). The remaining larvae were allowed to develop in the original pans and regular fixations of these were made 1, 5, 6, 12, 18, 36 and 72 hours after irradiation. Mitotic chromosomes were studied from squash preparations of larval brains stained with acetolactic orcein. Methods for making the squashes have been described by ~RELAND (196]) and RAI (1963). In order to study the effects of radiation on mitosis, counts of the total number of dividing cells in a complete larval brain were made. The average of five such counts has been regarded to be a measure of the mitotic activity. Photomicrographs of chromosomal aberrations were taken from temporary slides ringed with finger nail polish. A 35 mm. Zeiss/Ikon camera with Panatomic-X, black and white film was used. Data on pupation, emergence and mortality were collected. The viable adults from unirradiated and 500 r irradiated batches were crossed in single pairs in different combinations. Five single pair crosses were set up in each case. The experiment was repeated using larger samples of 300 larvae. The dosage rate was 53.6 r per minute. However, no fixations for chromosomal study were made. (Data included in Table II is derived from this experiment.) D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 EFFECTS OF X-IRRADIATION ON AEDES AEGYPTI 597 RESULTS A. Effects on mitotic activity. One of the earliest observable effects of all doses of X-rays, was a depres- sion in mitotic activity. In all cases brains fixed 1.5 hours after irradiation showed very little cell division. There were 5-12 dividing cells in the irradiated larvae as compared to 125 cells in mitosis in the unirradiated controls at this time (Table 1). Moreover, this mitotic inhibition occured rather quickly. In the TABLE I Average" number of diciding cells (mitotic activity) per fourth instar larval brain at different time intervals in unirradiated and irradiated larvae. Time (in hours) Average no. of dividing cells in unirradiated and irradiated larvae between U nirradiated irradiation and fixation (control) 500 r 1000 r 2000 r 4000 r larvae 0 113 113 (29)"" 131 (54)"" 83 (87)"" 34 (100)"" 1.5 125 12 (75) 8 (100) 7 (100) 5 (100) 6 159 256 (84) 98 (96) 50 (98) 15 (100) 12 161 249 (77) 297 (94) 129 (98) 21 (100) 18 148 267 (84) 230 (94) 179 (95) 126 (100) 36 139 177 (75) 166 (85) 119 (97) 105 (100) 72 79 79 (52) 76 (72) 40 (92) 35 (100) " Average based on counts made from usually five, rarely 3-4 larval brains. ""Figures in ( ) represent per cent aberrant mitoses. case of larvae exposed to 4000 r, the inhibitory effect was obvious immediately after treatment (time 0). The larvae in this case were under theX-ray beam for 36.22 minutes. The larvae exposed for 8 minutes (1000 r) did not show any decrease in mitotic activity but the batch irradiated for 16.12 minutes (2000 r) did show a slight depression at time 0. It may be deduced from this data that the inhibitory effect of X-rays on mitosis in A. aegypti sets in about 10-15 mi- nutes after the start of irradiation at these dose rates and reaches its highest level approximately 90 minutes after the end of the irradiation. The inhibitory effect was temporary and after a time, which depended on the dose used, it was replaced by a great increase in mitotic activity. Twelve hours after irradiation, for example, the larvae exposed to 500 and 1000 f sho- wed approximately 1Y,.-2 times as many mitotic figures in their brains cells as D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 598 RAI did the unirradiated material. The increase in mitotic activity at higher doses was less extreme and took longer to occur (Fig. 1). In the case of larvae expo- sed to 4000 r, the average number of dividing cells per larval brain never rea- ched the control values. Seventy-two hours after irradiation, larvae exposed to 500 and 1000 r had the same average number of dividing cells as the unirradiated control (F'ig. 1). However, at higher doses the mitotic activity by this time had fallen to a value below that of the control. Data on per cent aberrant division figures in the irradiated larval brains are included in Table I. Because of technical difficulties in distinguishing between certain normal and aberrant division figures, these values should be regarded as approximations rather than absolute. Beyond 36 hours, there was a noticeable decrease in the mitotic activity in the unirradiated control larvae. This corresponds with the beginning of pupa- tion and agrees with earlier reports (BRELAND 1961, RAI 1963). B. Effects on chromosomes. It is well known that X-rays bring about alterations in chromosomes. X- ray-induced chromosomal changes are usually referred to as of two types: (1) physiological and (2) structural. Aberrations belonging to both these types were observed in A. aegypti as follows: Physiological aberrations. - The physiological effect of radiation on the mitotic chromosomes of this species was noticeable in some cases immediately after irradiation. Chromosomes, especially at higher doses, lost their discreteness and individuality. They appeared to be clumped and vacuolated (Fig. 3). Some TABLE II X-ray-induced mortality in " Aedes aegypti " . Number and Larvae Pupae Treatment No. % Unsexed 0 9 Total ol 0 ,o 500 r 3 1.0 0 4 0 4 1.3 3 1000 r 4 1.3 0 2 5 "1 2.3 4 ' 2000 r 3 1.0 103 89 39 231 77.0 45 4000 r 175 58.3 109 16 0 125 41.7 0 None (control) 0 0 3 1 1 5 1.7 2 ·------ D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 EFFECTS OF X-IRRADIATION ON AEDES AEGYPTI 599 of them could not separate completely at anaphase due to stickiness. This gave rise to anaphase bridges (Fig; 4). Some of the observed anaphase bridges in the irradiated larvae may have resulted from some of the chromosome ends sticking to each other at metaphase. Structural aberrations. - LEA (1947) has pointed out that for the detailed study of structural changes caused by radiation " it is necessary to use nuclei in which chromosomes are large and few in number. n Both these conditions are realized in A. aegypti. Depending on whether a chromosome at the time of irradiation was single or double, chromosomal or chromatid aberrations were observed. The following structural changes were seen in the architecture of the chromosomes in the irradiated larvae: Terminal deletions. - Simple breakages resulting in terminal deletions were among the commonest aberrations encountered. Figure 5 shows a large chromatid deletion in a metaphase chromosome. In Figure 6, chromosomal de- letions at early anaphase are shown. Two chromosome pairs rather than single chromosomes were involved in this case. Most of the observed fragments were acentric and were observed at all mitotic stages and in the cells of the larvae fixed at different time intervals. At anaphase, the acentric fragments (Fig. 7) lagged behind the centric chromosomes (Fig. 8). At the end of the mitotic di- vision, therefore, these fragments would not be included in the daughter nuclei. However, they may get attached with the centric chromosomes (Fig. 9) and thus undergo anaphasic movement. As a consequence, one or more chromoso- mes with greatly unequal arms were seen. The normal chromosome II is only slightly submetacentric (RAI 1963 and Fig. 2). percentage of induced mortality Emerging adults Adults Total 9 Total % 0 '? Total % No. % 0 3 1.0 6 3 9 3.0 19 6.3 5 9 3.0 114 24 138 46.0 158 52.7 9 54 18.0 6 4 10 3.0 298 99.3 0 0 0 0 0 0 0 300 100.0 3 5 1.7 0 0 0 0 10 3 . .'3 D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 60) RAI Interstitial deletions. - These require two independent breaks in a chro- mosome or chromatid. If the two broken parts rejoin leaving behind the inter- stitial piece, an interstitial deletion results. Interestingly, when two homologous chromosomes, one of which carried an interstitial deletion, paired somatically during prophase, a typical deletion loop was observed (Fig. 10). A large part c ~ -= - "' ... "' C> -·e -C> c:) z: 300 250 200 150 50 1\ I ' I ' I ' I ' / .:-....---·""-.. r----- .. __, ---- ' ' . ~-- ' . i I ', ·, I ' •• I ......... '· I ', ·, I ', .. . / ........... '·. I ..._,, ; ',~ I '~·-,....._ I I ._,.~~ .. --------~~---- ~~ .... --Control ---- 500 r -----· 1000 r ---2000 r ---- --4000 r I I I I . / I I ............. .... , I I I I , I I I I I I 'I ' I \~ ! ,'/ \,'I ·. "·''//' .. ·.l.!,/ .-- ...... . 1.- , .. ,, '~~ ~-......... ':---......_ "' .... :"\.. · .. _ .............. ·--:~ ·-.·.-:-........_ .. ---"'>-. 0 1.5 6 12 18 36 72 Time (in) hours after irradiatioh Fig. 1. - Effect of different doses of X-rays on the mitotic activity of fourth lnstar larval brains of Aedes aegypti. of the smallest chromosome pair shown in Fig. 10 illegitimately joined with the pair showing the deletion loop. Chromatid deletion loops were also obser- ved during mitotic prophase (Fig. 11). The behaviour of the fragments resulting from either one or two breaks, was similar. Until observed at prophase when the homologous chromosomes are somatically paired, it was not possible to tell whether a particular chromo- somal fragment was terminal or interstitial. Due, perhaps to partial breaks or incomplete fusions, some pairs appeared constricted (Fig. 12). Although no counts were made, the number of fragments per nucleus was D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 EFFECTS OF X-IRRADIATION ON AEDES AEGYPTI 601 apparently higher at higher dosages than at lower. Also, chromosomal deletions were more commonly observed than the chromatid deletions. Ring chromosomes. - These were observed at all division stages. A large proportion of the ring chromosomes must have resulted from the interstitial de- letions by a rejoining of the two broken ands (Fig. 13). The ring in Fig. 14 appears to have been formed by two homologous chromosomes. It may be assumed that deletions from both ends of a pair of chromosomes left four bro- ken ends.If the ends nearest to each other joined, a ring seen in Fig. 14 would result. Inversions. - Sometimes, when a chromosome is broken at two points, the interstitial piece instead of being deleted as a fragment, may be inverted and reinserted in the original chromosome. Depending on whether the two breaks are in the same or different arms of the chromosome, a paracentric or a pericentric inversion results. Some cases of dicentric bridges accompanied by acentric fragments were observed (Figs. 15 and 16). The acentric fragment shown in Fig. 17 must have been attached to another chromosome and thus carried to one of the daughter nuclei. The pulling apart at two points in a dicentric bridge results in its brea- kage (Fig. 18). Although, alternative explanations (LEA 1947, pages 194-95) can not be ruled out, the dicentric bridges accompanied by acentric fragments, may have resulted from heterozygous paracentric inversions. Chromosome exchanges. - When two independent breaks are induced by radiation in the same chromosome or in different chromosomes, the breaks instead of restituting, may rejoin illegitimately. These exchanges may either be symmetrical or asymmetrical. In the present study mostly asymmetrical exchanges were observed. Sometimes two centric pieces united, forming a dicentric chromosome at metaphase (Fig. 19). In Fig. 20 all the three pairs have been involved in breakages and reunions. Since the nucleus in this case belonged to a larva fixed 6 hours ofter irradiation, the particular appearance may be attri- buted to both physiological and structural changes in the chromosomes. Miscellaneous effects. 1. Presumed suppression in anaphasic move- ment. - In two dividing cells (Figs. 21 and 22) the smallest choromosome pair (chromosome I) was unable to enter an aphasic movement synchronously with the other two pairs. In both these cases the two larger pairs were in early and late anaphase respectively, while the small pair was still at metaphase. 2. Appearance of unusual structures in the cells. - Immediately after irradiation (time 0), the larvae exposed to higher doses, particularly 4000 r, showed structures resembling greatly elongated spindle fibers in their brain cells. On the equatorial plane of these lay a mass of chromatic material (Fig. 23). D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 602 RAI C. Effects on viability and fertility. Data on X-ray-induced mortality at different developmental stages of Aedes aegypti are included in Table II. A dose of 2000 r or more resulted in almost complete mortality. Furthermore, the lower the total dose, the later in development did the death occur and vice versa. At 1000 r, for example, most of the irradiated larvae developed to normal appearing adults. However, they were sluggish and were unable to fly away from the water surface. At 2000 r, 77% died as pupae and 18% died between the pupal and the adult stage. The latter failed to emerge completely out of the pupal case. Finally none of the larvae irradiated to 4000 r developed beyond the early pupal stage. Since the unirradiated control larvae also showed some mortality (3.3%), a small part of the total mortality in the irradiated batches may be due to causes other than radiation. Doses of 2000 r and 4000 r greatly arrested the rate of growth and deve- lopment. The larvae took much longer to pupate. Most of them died as untanned Figs. 2-24. - X-ray-induced chromosomal aberrations in the brain cells of fourth instar larvae of Aedes aegypti, x 2(J.J0. First entry in ( ) indicates the X-ray close to which a larva was irradiated and the second indicates the time in hours, between irradiation and fixation of the larva for cytological examination. Fig. 2. - Shows normal karyotype in an unirracliatecl larva. Fig. 3 and 4. - (2000 r, 18 hrs.) shows «physiological n effects. Note the anaphase bridge resulting from chromosome stickiness in Fig. 4. Fig. 5. - (2000 r, 18 hrs.) shows a large chromatid deletion in a metaphase chromosome. Terminal chromosomal deletions from two pairs at early anaphase are seen in Fig. 6 (1000 r, 12 hrs.). An acentric fragment lies at the equator of a dividing ,cell (1000 r, 12 hrs.) in Fig. 7. Many such fragments appear as « laggards » in a late anaphase nucleus in Fig. 8 (2000 r, 18 hrs.). In Figs. 9 and 10. - (2000 r, 18 hrs.) asymmetric chromosome exchanges have occurred. Note greatly unequal arms of two metaphase chromosomes in Fig. 9 and a typical interstial deletion loop in Fig. 10. A large part of the smallest chromosome pair in Fig. 10 has been attached to the largest pair. Note the interstial chromatid deletion loops in Fig. 11 (2000 r, 12 hrs.). Fig. 12. - (2000 r, 120 hrs.) shows an excessive fragmentation of all the chromosome pairs. Chromosomal material appears to be disorganizing in this case. A large ring formed from part of a prophase chromosome is shown in Fig. 13 (2000 r, 12 hrs.). Fig. 14 (2000 r, 18 hrs.) shows a ring formed from a pair of metaphase chromosomes. Note some deletions also in both these figures. Dicentric bridges accompanied by acentric fragments are shown in Fig. 15 and 16 (2000 r, 18 hrs.). The acentric fragment has almost been included in one of the two telophase nuclei in Fig. 17 (1000 r, 18 Ius.). Two divided fragments and a broken clicentric bridge are seen in Fig. 18 (500 r, 12 hrs.). Fig. 19. - Shows a clicentric chromosome besides a large number of fragments (1000 r, 18 hrs.). All the three pairs are involved in breakages and reunions in Fig. 20 (2000 r, 6 hrs.). Fig. 21 (2000 r, 12 hrs.) and Fig. 22 (2000 r, 18 hrs.). - Shows only two pairs in anaphasic movement. The smallest pair, still at metaphase seems unable to move. Structures resembling long spindle fibers with chromatic material on their equatorial plane appear in Fig. 23 (4000 r, 6 hrs,). Some ends of anaphase chromosomes arc bipartite in Fig. 24 (2000 r, 6 Ius,). D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 1 0 ll D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 604 RAI pupae. Doses of 500 r or 1000 r did not have any significant effect on overall rates of development, pupation or emergence. After exposure to cOO r, irradiated females (mated to either irradiated or unirradiated males) laid slightly reduced number of eggs (Table III). Irradiation of the female parent had no effect on the hatchability of these eggs. However, fewer larvae hatched from eggs produced by females mated with irradiated males. DISCUSSION The initial inhibitory effect of X-irradiation on the mitotic activity of both the animal and plant materials has been observed by a number of workers. The mitotic activity of the animal cells reaches a minimum point approximately Y,.- 6 hours after irradiation (CARLSON 1954). In the case of the brain tissue of Aedes aegypti this point was reached approximately 1Y,. hours after irradiation. The duration of the arrested mitotic activity depended on the dose. The higher the dose used, the more extended was the time interval between irradiation and the period of minimum mitotic activity. STRANGEWAYS and HoPWOOD (1926) working on in vitro fibroblasts of chick embryos reported that although there was a temporary inhibition in the onset of mitotic division, cells undergoing mitosis remained unaffected. This does not appear to be so in Aedes aegypti. The inhibition in the mitotic activity in this species is due perhaps to (1) an X-ray-induced delay in the onset of mitosis and (2) a reversion of some of the dividing nuclei (especially at early prophase) at the time of irradiation to a nondividinginterphase state following irradiation. This is evidenced by a comparison of the total number of dividing cells in the unirradiated and the irradiated larvae at time 0. Although the time interval for the completion of mitosis for this species is not yet computed, it is highly unlikely that the observed difference between the unirradiated controls and the larvae exposed to 2000 r or 4000 r at time 0 (Table I) represents the number of nuclei that in the absence of radiation would have completed the mitotic division within about 16-36 minutes (approx. time for which the two batches were irradiated). Evidence from neuroblasts of Chortophaga (CARLSON 1954) indicating a radiation-induced delay in the completion of mitosis strengthens this argument. Furthermore, by using selected grasshopper neuroblasts in hanging drop preparations before and after irradiation, CARLSON (1941) has shown mitotic reversion from early prophase stages. CARLSON et al. (1953) have attributed the increased mitotic activity after irradiation to a simultaneous release from an inhibition of a population of cells which accumulated in prophase. They have pointed out that the increase above the normal after recovery just about compensates for the initial fall below normal. Broadly this same relationship applies to the present study at doses D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 EFFECTS OF X-IRRADIATION ON AEDES AEGYPTI 605 of 500 r and 1000 r. However, with a dose of 2000 r the previous deficit was not compensated and in the ca~e of larvae irradiated to 4000 r the number of mitoses never even reached the control values. In inversion heterozygotes, normally the dicentric bridges accompanied by acentric fragments arise from exchanges within the inversion loops in meiotic tissues. In the present case, somatic exchanges facilitated by intimate pairing· in the inversion loops may have occurred either spontaneously or more probably may have been X-ray-induced. That X-rays both induce and increase the inci- dence of crossing over is well known. STERN (1936) assumed that many cases of mosaic formation in X-rayed Drosophila melanogaster were due to induced crossing over in somatic tissues. FRIESSEN (1933) and PATTERSON and SuCRE (1934) have shown that crossing over in the males of Drosophila can be induced by X-rays. The frequency of genetic exchanges in the females may also be si- milarly increased (WHITTINGHILL 1938). RAI (1963) has presented evidence that in an unirradiated, normal cell all the three pairs of chromosomes enter anaphase at the same time. Furthermore anaphasic separation also occurs at the same or similar rates in such cells. The apparent suppression in anaphasic movement of chromosome I in the irradiated cells (Figs. 21 and 22) may be due to either the centromere being rendered ineffective or due to its deletion, in part or whole. Since no deleted parts were observed in both these cells, the former explanation appears more likely. It may be that a single ' hit' or multiple ' hits ' in the region of the centromere render it incapable of anaphasic movement. The exact number of chromatids in a chromosome has long been debated. In Aedes aegypti, available evidence indicates that a prophase or a metaphase chromosome may be four stranded rather than two stranded. A longitudinal split at the ends of some of the anaphase chromosomes was observed lFig. 24). Whether this condition is radiation-induced or is a normal feature of this species is hard to decide. VoN BoRSTEL (1961) has provided excellent insights to the nature of X-ray- induced dominant lethality in Habrobracon and Drosophila. He has distin- guished five types of nuclear damage causing death. Furthermore, he has emphasized that the manner in which death is induced changes in type and frequency "from organism to organism, from tissue to tissue and from cell to cell". The mortality observed at larval, pupal and adult stages in A. aegypti must be due to a variety of causes. A considerable proportion may result from the induction of structural chromosomal aberrations especially those that result in the loss of chromosomes or chromosome parts. The latter were observed very frequently and would be lethal in a basic diploid species such as A. aegypti. Chromosomal rearrangements also must have contributed their share. Besides these aberrations, inability to complete mitosis is probably also involved (Fig. D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 606 RAJ 12). The chromosomal material shown in this seems at the verge of disorgani- sation 120 hours after an irradiation of 2000 r. " Possible inhibition of an energy pathway" (.34), cytoplasmic damage and other causes may also have played some part. Available evidence indicates that irradiation of the males results in the induction of dominant lethals in the spermatozoa. When these spermatozoa fertilize the eggs, the resulting zygotes fail to hatch (Table III). It should be TABLE III Effects of 50J r X-irradiation on the fertility of " Aedes aegypti ". Cross and treatment" Eggs hatched Eggs laid"" Female Male No. % u X u 574 531 92.5 u X I 640 574 89.7 I X u 515 475 92.2 I X I 518 464 89.6 "U unirradiated; I = irradiated. "" Data pooled from five females. mentioned that fertilization precludes egg laying in A. aegypti. In the female, radiation appears to interfere with normal oogenesis. If a certain number of egg chambers in an irradiated female degenerate or do not develop normally, a corresponding decrease in the number of eggs laid by this female would be expected. This explanation has been shown to account for apholate-induced changes in fecundity in this species (RAr 196.'3, unpubl.) and gamma radiation and apholate-induced changes in Drosophilg, rnelanogas~er (CANTWELL and Acknowledgments. - The author wishes to thank his colleagues George B. CRAIG, Jr. for his interest in this investigation and Brother Raphael WrLso:-~ for providing the radiation facilities. REFERENCES BRELAND 0. P., 1961. - Studies Oil the chromosomes of mosquitoes. Ann. Entomol. Soc. Am., 54: 360-.'375. CANTWELL G. E. and HEN!'iEBERRY T. J., 1963. - The effects of gamma radiation and apho- late on the reproductive tissues of Drosophila melanogaster. Meigen. Journ. Insect Pathol., 5: 251-264. CARLSON J. G., 1941. - Effect of X-radiation on grasshopper chromosomes. Cold Spring Harbor Symp. Quant. Bioi., 9: 104-112. -, 1954. - in "Radiation Biology n, Vol. I, Part 2, A. HoLLAENDER. eel., McGraw Hill, New York. D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16 EFFECTS OF X-IRRADIATION ON AEDES AEGYPTI 607 CARLSON J. G., HARRII"GTON N. G. and GAuLDEN M. E., 1953. - Mitotic effects of prolonged irradiation with low intensity gamma rays on the Chortophaga neuroblast. Bioi. Bull., 104: 31.'3-323. CRAIG G. B. Jr. and VANDEHEY R. C., 1962. -Genetic variability in Aedes aegypti (Diptera: Culicidae). I. Mutations affecting color pattern. Ann. Entomol. Soc. Am., 55: 47-58. FRIESSEN H., 1933. - Artificially induced crossing over in males of Drosophila melanogaster. Science, 78: 513-514. GROSCH D. S., 1962. - Entomological aspects of radiation as related to genetics and phy- siology. Ann. Rev. Entomol., 7: 81-106. LEA D. E., 1947. -Action of radiatiom on living cells. The Macmillan Company, New York. McCRAY E. M., JENSON J. A. and SCHOOF H. F., 1961. - Cobalt-00 sterilization studies with Aedes aegypti. Proc. N. ]. Mosq. Ext. Ass. 48th Ann. Mtg., 110-115. PATTERSON J. T. and SucHE M. L., 1934. - Crossing over induced by X-rays in Drosophilamales. Genetics, 19: 223-236. PIENAAR R. DE V., 1955. - Combinations and variations for improved chromosome studies in the Gramineae. Journ. South African Bot., 21: 1-8. RAI K. S., 1963. - A comparative study of mosquito karyotypes. Ann. Entomol. Soc. Am., 56: 160-170. STERN C., 1936. - Somatic crossing over and segregation in Drosophila melanogaster. Gene- tics, 21: 625-730. STRANGEWAYS T. S. P. and HoPWOOD F. L., 1926. - The effects of X-rays upon mitotic cell division in tissue cultures in vitro. Proc. Roy. Soc. (London), series B, 102: 9-29. TERZIAN L. A., 1953. - The effect of X-irradiation on the immunity of mosquitoes to malarial infection. Journ. Immunol., 72: 202-206. TERZIAN L. A. and STAHLER N., 1958. - A study of some effects of gamma radiation on the adults and eggs of Aedes aegypti. Bioi. Bull., 115: 536-550. VANDEHEY R. C. and CRAIG G. H. Jr., 1959. - Radiation-induced mutations in Aedes aegypti. Bull. Entomol. Soc. Am., 5: 113. VoN BoRSTEL R. C., 1961. - in "Progress in Photobiology», B. CHR. CHRISTENSEN and B. BucHMANN editors, Elsevier Publishing Company, Amsterdam. WHITTINGHILL M., 1938. - The induction of oogonial crossing over in Drosophila melano- gaster. Genetics, 23: 300-306. SUMMARY Cytogenetic effects of X-rays (500, 1000, 2003, 4000 r) on the NIH strain of Aedes aegypti L. have been studied. Larvae were reared under controlled conditions and were irradiated six clays after hatching at a time when most were in the early fourth instar. Five to ten larvae were fixed for cytological examination at intervals of 0, 1%, 6, 12, 11:!, 36 and 72 hours afte; irracliation. Mitotic chromosomes were studied from squash preparations of larval brains stained with acetolactic orcein. Mitotic activity was measured in terms of the total number of dividing cells per brain. Initially X-irradiation inhibited cell division. Mitotic activity was almost completely sup- pressed Ph hours after irradiation. After a time (depending on dose used), this effect was replaced by a great increase in mitotic activity. Twelve hours after irradiation, the larvae exposed to 500 and 1000 r showed about twice as many mitotic figures as did the unirradiated matedal. The increase in mitotic activity at higher doses was less extreme and took longer to occur. Among the chromosomal aberrations noted were deletions, inversions, exchanges, rings, dicentrics and anaphase bridges. Explanations for the induction of these aberrations habe been discussed. A relationship existed between the close received and the developmental stage at which an individual died. The higher the dose, the earlier the death occurred. Furthermore 2000 r or more resulted in almost 100% mortality. Some possible causes of X-ray-induced mortality and changes in fertility have been suggested. D ow nl oa de d by [1 79 .17 6.2 13 .72 ] a t 0 9:4 7 1 9 F eb ru ary 20 16
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