Acta Tropica, 48(1991)9-15
9
Elsevier ACTROP 00077
Salivary gland infection: a sex-linked recessive character in tsetse? I. Maudlin 1, S.C. W e l b u r n 1 a n d P. Milligan 2 l Tsetse Research Laboratory, ODA/University o[ Bristol, U.K. and 2Department o["Biological Sciences, UniversiO, of Salford, U.K.
(Received 1 November 1989; accepted 12 January 1990) Male tsetse, when infected in the laboratory with trypanosomes of the subgenus Trypanozoon, usually produce greater salivary gland infection rates than females of the same species. We show that a single sexlinked gene model can be fitted to most recently published data for salivary gland infection rates in tsetse. The maturation of Trypanosoma congolense infections is shown to be independent of fly sex. The possible effects of genetic control of maturation of Trvpanozoon infections in tsetse populations on the transmission of sleeping sickness are considered. Key words: Tsetse; Glossina morsitans morsitans; Trypanosoma brucei rhodesiense: Cyclical transmission; Lectin; Sex linkage
Introduction Early l a b o r a t o r y experiments designed to determine which factors governed the transmission o f sleeping sickness by tsetse suggested that the sex o f the fly was important. Burtt (1946) examined the dissection data from the Tinde experiment and f o u n d that male Glossina morsitans (presumably G.m. centralis) had a salivary gland infection rate (5.4%) m o r e than twice that o f females (2.1%). [In the Tinde experiment, a single strain o f Trypanosoma brucei rhodesiense isolated by C o r s o n (1936) was passaged cyclically t h r o u g h tsetse and various animals continuously for several years.] F a i r b u r n and Culwick (1950), w h o later examined the Tinde data for the effects o f temperature on fly infection rates, also concluded that males were better transmitters o f this t r y p a n o s o m e than females (6.4% and 2.6% salivary gland infections respectively at n o r m a l laboratory temperature). Burtt (1946) noted that differential survival o f male and female flies m a y have affected the infection rates recorded in the Tinde experiment but concluded that females generally survived longer than males which should have benefited infection rates in that sex. The salivary gland infection rates recorded by F a i r b u r n and Culwick (1950) m a y have been imprecise as the flies were proven positive not by dissection but by probing on to slides. Van H o o f (1947) experimented with T.b. gambiense and found that male
Correspondence address." Dr. I. Maudlin, Tsetse Research Laboratory, ODA/University of Bristol,
Langford House, Langford, Bristol BSI8 7DU, U.K. 0001-706X/90/$03.50 (c) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
10
G.p. palpalis produced a greater salivary gland infection rate (32.4%) than females (21.4%); the raw data are not given in this paper to facilitate a genetic analysis. Most recent controlled laboratory experiments have confirmed that male tsetse infected with Trvpanozoon develop a greater proportion of salivary gland infections than females of the same species. We present here an analyis of these recently published data, together with experimental data of our own, designed to test whether the development of salivary gland infections could be a Mendelian character in tsetse.
Materials and Methods
Fly infection Glossina morsitans morsitans were infected as tenerals one day following the day of emergence from the puparium. Flies were kept at 25°C and maintained throughout on defibrinated pig blood fed through an artificial membrane. Flies were infected with either T.b. rhodesiense stock E A T R O 2340 (Cornelissen et al., 1985) or T.b. brucei stock AnTat !.8 (Le Ray et al., 1977), or T. congolense stock SI04/FLY/BE or 1/148 FLY (Young and Godfrey, 1983) using frozen stabilates of mouse blood as previously described (Welburn and Maudlin, 1987). Flies infected with Trypanozoon were dissected 28 days post infection and midguts and salivary glands examined for trypanosomes by phase-contrast microscopy (× 400); for T. congolense infections flies were dissected 21 days post infection when midguts and mouthparts were examined. Data analysis The experimental data given here, together with other recently published results, were tested for goodness of fit to a model which assumed that the development of salivary gland infections is controlled by a single X-linked, recessive, locus. According to the Hardy-Weinberg law, if the proportion of the heterogametic sex in a population carrying a sex-linked recessive character is q then the proportion in the homogametic sex will be q2. Since flies which do not establish a midgut infection cannot ever produce a salivary gland infection whether or not they carry the gene, calculations of q have to be based on the proportion of midgut infected flies producing mature infections. The maximum likelihood estimate of the gene frequency (qrnax) w a s calculated as follows: qmax = ( ( ( n l 2 + n 13) 2 -]- 8 ( n 2 2 ( n 13 -]- 2 n 2 3 ) + n 2 3
"nl 2)) 1/2 --
( h i 2 --
where: nll = total number of males n12 = n u m b e r of males with midgut infections n13 = n u m b e r of males with salivary gland infections n21 = total number of females n22 = number of females with midgut infections n23 = number of females with salivary gland infections
nl 3 ) ) / 4 n 2 2
+ 2nl 2
11
Results Table 1 shows the infection rates obtained in the present study with different trypanosomes species and stocks. In Trypanozoon infected flies males had significantly greater salivary gland infection rates than females (T.b. rhodesiense x2 = 15.2, p < 0.001; T.b. brucei x2 = 4.9, 0.05 >p > 0.01). T. congolense infection rates by contrast showed no significant differences between male and female hypopharyngeal infection rates in either trypanosome stock. There were no significant differences between the sexes in the proportions of flies with midgut infections in any of the four experiments. The data obtained in these Trypanozoon experiments (Table 1) are presented together with recently published data from other workers in Fig. 1 in which observed male and female salivary gland infection rates are shown together with expected values calculated using qmax.The values for qmax(used to calculate expected numbers in Fig. 1) are given in Table 2. As can be seen from the x2 values in Table 2, the only significant departure from the expectations of a sex-linkage model was found in experiment (e) (Makumyaviri et al., 1984b) but this was only significant at the 5% level.
Discussion The results presented here show that male tsetse of different species when infected experimentally with Trypanozoon produced salivary gland infection rates greater than females of the same species infected under the same conditions. Sex-linked recessive genes can be detected with a fair degree of certainty without breeding experiments simply by observing the frequency of a character in the two sexes of a population (Sheppard, 1967). Analysis of the results from eight experiments involv-
TABLE Infection
1 rates in male and female G.m. morsitans
Trypanosome/ Stock
Males
infected
with four different
stocks of trypanosomes
Females
P
n
%G
%M
n
%G
%M
143
73
27
114
75
7
T.b. brucei AnTat 1.8
44
71
32
64
77
13
T. congolense Sl04/FLY/BE
51
55
31
75
65
40
N.S.
T. congolense l/148 FLY
36
53
53
41
61
61
N.S.
T.6. rhodesiense EATRO 2340
9
Elsevier ACTROP 00077
Salivary gland infection: a sex-linked recessive character in tsetse? I. Maudlin 1, S.C. W e l b u r n 1 a n d P. Milligan 2 l Tsetse Research Laboratory, ODA/University o[ Bristol, U.K. and 2Department o["Biological Sciences, UniversiO, of Salford, U.K.
(Received 1 November 1989; accepted 12 January 1990) Male tsetse, when infected in the laboratory with trypanosomes of the subgenus Trypanozoon, usually produce greater salivary gland infection rates than females of the same species. We show that a single sexlinked gene model can be fitted to most recently published data for salivary gland infection rates in tsetse. The maturation of Trypanosoma congolense infections is shown to be independent of fly sex. The possible effects of genetic control of maturation of Trvpanozoon infections in tsetse populations on the transmission of sleeping sickness are considered. Key words: Tsetse; Glossina morsitans morsitans; Trypanosoma brucei rhodesiense: Cyclical transmission; Lectin; Sex linkage
Introduction Early l a b o r a t o r y experiments designed to determine which factors governed the transmission o f sleeping sickness by tsetse suggested that the sex o f the fly was important. Burtt (1946) examined the dissection data from the Tinde experiment and f o u n d that male Glossina morsitans (presumably G.m. centralis) had a salivary gland infection rate (5.4%) m o r e than twice that o f females (2.1%). [In the Tinde experiment, a single strain o f Trypanosoma brucei rhodesiense isolated by C o r s o n (1936) was passaged cyclically t h r o u g h tsetse and various animals continuously for several years.] F a i r b u r n and Culwick (1950), w h o later examined the Tinde data for the effects o f temperature on fly infection rates, also concluded that males were better transmitters o f this t r y p a n o s o m e than females (6.4% and 2.6% salivary gland infections respectively at n o r m a l laboratory temperature). Burtt (1946) noted that differential survival o f male and female flies m a y have affected the infection rates recorded in the Tinde experiment but concluded that females generally survived longer than males which should have benefited infection rates in that sex. The salivary gland infection rates recorded by F a i r b u r n and Culwick (1950) m a y have been imprecise as the flies were proven positive not by dissection but by probing on to slides. Van H o o f (1947) experimented with T.b. gambiense and found that male
Correspondence address." Dr. I. Maudlin, Tsetse Research Laboratory, ODA/University of Bristol,
Langford House, Langford, Bristol BSI8 7DU, U.K. 0001-706X/90/$03.50 (c) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
10
G.p. palpalis produced a greater salivary gland infection rate (32.4%) than females (21.4%); the raw data are not given in this paper to facilitate a genetic analysis. Most recent controlled laboratory experiments have confirmed that male tsetse infected with Trvpanozoon develop a greater proportion of salivary gland infections than females of the same species. We present here an analyis of these recently published data, together with experimental data of our own, designed to test whether the development of salivary gland infections could be a Mendelian character in tsetse.
Materials and Methods
Fly infection Glossina morsitans morsitans were infected as tenerals one day following the day of emergence from the puparium. Flies were kept at 25°C and maintained throughout on defibrinated pig blood fed through an artificial membrane. Flies were infected with either T.b. rhodesiense stock E A T R O 2340 (Cornelissen et al., 1985) or T.b. brucei stock AnTat !.8 (Le Ray et al., 1977), or T. congolense stock SI04/FLY/BE or 1/148 FLY (Young and Godfrey, 1983) using frozen stabilates of mouse blood as previously described (Welburn and Maudlin, 1987). Flies infected with Trypanozoon were dissected 28 days post infection and midguts and salivary glands examined for trypanosomes by phase-contrast microscopy (× 400); for T. congolense infections flies were dissected 21 days post infection when midguts and mouthparts were examined. Data analysis The experimental data given here, together with other recently published results, were tested for goodness of fit to a model which assumed that the development of salivary gland infections is controlled by a single X-linked, recessive, locus. According to the Hardy-Weinberg law, if the proportion of the heterogametic sex in a population carrying a sex-linked recessive character is q then the proportion in the homogametic sex will be q2. Since flies which do not establish a midgut infection cannot ever produce a salivary gland infection whether or not they carry the gene, calculations of q have to be based on the proportion of midgut infected flies producing mature infections. The maximum likelihood estimate of the gene frequency (qrnax) w a s calculated as follows: qmax = ( ( ( n l 2 + n 13) 2 -]- 8 ( n 2 2 ( n 13 -]- 2 n 2 3 ) + n 2 3
"nl 2)) 1/2 --
( h i 2 --
where: nll = total number of males n12 = n u m b e r of males with midgut infections n13 = n u m b e r of males with salivary gland infections n21 = total number of females n22 = number of females with midgut infections n23 = number of females with salivary gland infections
nl 3 ) ) / 4 n 2 2
+ 2nl 2
11
Results Table 1 shows the infection rates obtained in the present study with different trypanosomes species and stocks. In Trypanozoon infected flies males had significantly greater salivary gland infection rates than females (T.b. rhodesiense x2 = 15.2, p < 0.001; T.b. brucei x2 = 4.9, 0.05 >p > 0.01). T. congolense infection rates by contrast showed no significant differences between male and female hypopharyngeal infection rates in either trypanosome stock. There were no significant differences between the sexes in the proportions of flies with midgut infections in any of the four experiments. The data obtained in these Trypanozoon experiments (Table 1) are presented together with recently published data from other workers in Fig. 1 in which observed male and female salivary gland infection rates are shown together with expected values calculated using qmax.The values for qmax(used to calculate expected numbers in Fig. 1) are given in Table 2. As can be seen from the x2 values in Table 2, the only significant departure from the expectations of a sex-linkage model was found in experiment (e) (Makumyaviri et al., 1984b) but this was only significant at the 5% level.
Discussion The results presented here show that male tsetse of different species when infected experimentally with Trypanozoon produced salivary gland infection rates greater than females of the same species infected under the same conditions. Sex-linked recessive genes can be detected with a fair degree of certainty without breeding experiments simply by observing the frequency of a character in the two sexes of a population (Sheppard, 1967). Analysis of the results from eight experiments involv-
TABLE Infection
1 rates in male and female G.m. morsitans
Trypanosome/ Stock
Males
infected
with four different
stocks of trypanosomes
Females
P
n
%G
%M
n
%G
%M
143
73
27
114
75
7
T.b. brucei AnTat 1.8
44
71
32
64
77
13
T. congolense Sl04/FLY/BE
51
55
31
75
65
40
N.S.
T. congolense l/148 FLY
36
53
53
41
61
61
N.S.
T.6. rhodesiense EATRO 2340
