Formulation of pyriproxyfen, a juvenile hormone mimic, for tsetse control

A topical dose, in 1 microliter acetone, of 0.02 microgram-2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine, the juvenile hormone mimic pyriproxyfen (S-31183, Sumitomo Chemical Co.), caused an adult female tsetse, Glossina morsitans morsitans Westwood, to produce non-viable offspring for the whole of her life. Using 14C labelled pyriproxyfen it was determined that as little as 0.001 microgram transferred to the in utero larva was sufficient to arrest development in the pupal stage. A formulation in vegetable oil was prepared for treating black cotton cloth targets which caused females to pick up 0.1 microgram active ingredient (a.i.) by tarsal contact during 1 min of exposure. Males exposed similarly for between 1 and 5 min transferred up to 0.016 microgram a.i. to females if they mated immediately after treatment. Doses as low as 0.01 micrograms in 10 microliters oil cm-2 on black cotton cloth targets caused females to produce non-viable offspring for at least two reproductive cycles following exposure. However, a dose of 0.1 microgram in 10 microliters oil cm-2 was necessary for an exposed male to cause disruption of the reproductive potential of his mate. This juvenile hormone mimic has potential to induce sterility via both sexes of tsetse using treated targets or traps under field conditions.     

Does reproductive investment change with age in tsetse flies, Glossina morsitans morsitans (Diptera: Glossinidae)?

1. Viviparous insects such as tsetse ( Glossina spp.) provide unusual opportunities to compare age-related changes in the proportion of maternal resources transferred to offspring.
2. In laboratory populations of Glossina morsitans morsitans the survival of females was high for the first 60 days of adult life but declined rapidly thereafter.
3. Average longevity did not differ significantly between mated and unmated females (93·6 and 90 days, respectively).
4. Nutritional state in terms of fat content and residual dry mass did not decline with adult female age.
5. The fecundity of mated females was constant for the first 60 days of adult life and declined only slightly thereafter.
6. Offspring size did not change towards the end of the adult female lifespan and there was no evidence of an increase in the allocation of resources to reproduction in older females.
7. Results contrast with those obtained recently for vertebrates and may indicate that age-related changes in offspring size in Glossina are not adaptive, or that so few females reach old age under natural conditions that there is no selection for a strategy of terminal investment.     

Genetics of hybridization of Glossina swynnertoni with Glossina morsitans morsitans and Glossina morsitans centralis

Abstract Reciprocal crosses were performed with Glossina swynnertoni and Glossina morsitans morsitans and with G. swynnertoni and Glossina morsitans centralis , using strains that carried marker genes in all three linkage groups. Glossina swynnertoni males can inseminate, but not fertilize, G.m.morsitans; all other crosses produced some fertile females. Hybridization did not cause sex ratio distortion among F_{ flies. Most F and backcross females were fertile, but all F, males were sterile. Sterility among backcross males was also high (99% in BXj, 85% in Bx₂, and about 50% in Bx₃ to Bx₅). Chromosome transmission by hybrid females usually conformed to Mendelian expectations, but genetic recombination was lower than observed in G.m.morsitans. The reduction in fertility among backcross females was not associated with heterozygosity in any linkage group. Sterility among hybrid and backcross males was associated with heterozygosity of sex chromosomes and probably autosomes. The results support the systematic placement of G.swynnertoni closer to G.mxentralis than to G.m.morsitans.     

An antimicrobial peptide with trypanocidal activity characterized from Glossina morsitans morsitans

Tsetse flies (Diptera:Glossinidae) are vectors of African trypanosomes, the protozoan agents of devastating diseases in humans and animals. Prior studies in trypanosome infected Glossina morsitans morsitans have shown induced expression and synthesis of several antimicrobial peptides in fat body tissue. Here, we have expressed one of these peptides, Attacin (GmAttA1) in Drosophila (S2) cells in vitro. We show that the purified recombinant protein (recGmAttA1) has strong antimicrobial activity against Escherichia coli-K12, but not against the enteric gram-negative symbiont of tsetse, Sodalis glossinidius. The recGmAttA1 also demonstrated inhibitory effects against both the mammalian bloodstream form and the insect stage Trypanosoma brucei in vitro (minimal inhibitory concentration MIC50 0.075 microM). When blood meals were supplemented with purified recGmAttA1 during the course of parasite infection, the prevalence of trypanosome infections in tsetse midgut was significantly reduced. Feeding fertile females GmAttA1 did not affect the fecundity or the longevity of mothers, nor did it affect the hatchability of their offspring. We discuss a paratransgenic strategy, which involves the expression of trypanocidal molecules such as recGmAttA1 in the midgut symbiont Sodalis in vivo to reduce trypanosome transmission.     

Genetic differentiation of some Glossina morsitans morsitans populations

To study the population structure of Glossina morsitans morsitans Westwood (Diptera: Glossinidae), polymerase chain reaction (PCR) and singlestrand conformational polymorphism (SSCP) methods were used to estimate mitochondrial DNA diversity at four loci in six natural populations from Zambia, Zimbabwe and Mozambique, and in two laboratory cultures. The Zambian and Zimbabwean samples were from a single fly belt. Four alleles were recorded at 12S and 16S1, and five alleles at 16S2 and COI. Nucleotide sequencing confirmed their singularities. Chi-square contingency tests showed that allele frequencies differed significantly among populations. Mean allele diversities in populations averaged over loci varied from 0.14 to 0.61. Little loss in haplotype diversity was detected in the laboratory cultures thereby indicating little inbreeding. Wright's fixation index F(ST) in the natural populations was 0.088+/-0.016, the correlation of haplotypes within populations relative to correlations in the total. A function of its inverse allows an estimate of the mean equivalent number of females exchanged per population per generation, 5.2. No correlation was detected between pairwise genetic distance measures and geographical distances. Drift explains the high degree of differentiation.     

Effect of puparia incubation temperature: increased infection rates of Trypanosoma congolense in Glossina morsitans centralis, G.fuscipes fuscipes and G.brevipalpis

Puparia of Glossina morsitans centralis (Machado), G.fuscipes fuscipes (Newstead) and G.brevipalpis (Newstead) were incubated at 25 +/- 1 degrees C, 28 +/- 1:25 +/- 1 degrees C, day:night or 29 +/- 1 degrees C throughout the puparial period, and maintained at 70-80% relative humidity. Puparial mortality was higher at 29 than at 25 degrees C (optimum temperature) in all three species, particularly in G.f.fuscipes and G.brevipalpis. Adults of G.m.centralis from puparia incubated at 29 degrees C, and those of this subspecies, G.f.fuscipes and G.brevipalpis from puparia incubated at 28:25 degrees C, day:night or 25 degrees C throughout, were infected as tenerals (27 h old) by feeding them at the same time on goats infected with Trypanosoma congolense (Broden) IL 1180 after the parasites were detected in the wet blood film. Infection rates on day 25 post-infected feed were higher in G.m.centralis from puparia incubated at 29 degrees C and in adults of the three different tsetse species from puparia incubated at 28:25 degrees C, day:night, than in those from puparia incubated at 25 degrees C. However, in G.f.fuscipes the labral and hypopharyngeal infection rates were not significantly different from those of the tsetse produced by puparia kept at 25 degrees C.     

Artificial larviposition sites for field collections of the puparia of tsetse flies Glossina pallidipes and G. m. morsitans (Diptera: Glossinidae)

Tsetse flies Glossina pallidipes Austen and G. morsitans morsitans Westwood deposit their larvae in warthog burrows, in August-November, at Rekomitjie Research Station, Zambezi Valley, Zimbabwe. Artificial burrows, made from 200-l steel drums, were used to sample these flies and to collect their puparia. Sand-filled plastic trays in the burrows served as a substrate for larval deposition. The sand was covered with c. 2 cm of leaf litter after it was shown that only 3% of larvae were deposited on bare sand if both substrates were available. Other burrow modifications - artificially shading the burrow entrance, increasing the relative humidity inside the burrow, or reducing the size of the burrow entrance - significantly decreased deposition rates. The use of burrows in the hot season results in a reduction in the temperature experienced by the puparium towards an assumed optimum level of 26 degrees C. Artificial burrows maintained a mean temperature of 28.5 degrees C during October-November 1998, c. 2.5 degrees C cooler than ambient; earlier work has shown that natural burrows can be c. 5 degrees C cooler than ambient at these times. This may explain why natural burrows in full sunlight were used for larviposition, whereas artificial burrows were used only when they were in deep shade, and why significantly higher proportions of G. pallidipes were found in natural (66%) than in artificial burrows (34%). Better-insulated artificial burrows might produce more puparia with higher proportions of G. pallidipes. Burrows become waterlogged during the rains and may be too cool for optimum puparial development during the rest of the year. The percentages of G. m. morsitans in catches of females from artificial burrows, refuges and odour-baited traps were 34, 26 and < 10% respectively. Traps are biased in favour of G. pallidipes; artificial burrows may show a bias in favour of G. m. morsitans that is a function of temperature. Artificial warthog burrows provide a convenient way of studying the puparial stage in tsetse and for the first time facilitate the capture of females as they deposit their larvae.     

Performance of the tsetse, Glossina morsitans morsitans reared under various feeding regimens in Zambia

Groups of Glossina morsitans morsitans Westwood fed at emergence and thereafter daily, every second or third day respectively, up to the end of their first pregnancy cycle, survived well (73–79%) and produced virtually the same number of puparia/ (0.83–0.85) in the same puparial weight class (23–24 mg). However, adult survival (29%), number of puparia/ (0.30) and puparial weight (19 mg) were much lower in the group consistently fed every 4th day after the initial meal at emergence. It is proposed that tsetse colonies could be fed on Mondays, Wednesdays and Fridays without jeopardising adult survival, puparial production/ and the size (weight) of puparia produced.

---

Performances en Zambie de la mouche Tsé-Tsé (Glossina morsitans morsitans) élevée en utilisant différents régimes alimentaires

Résumé Afin d'estimer si une alimentation quotidienne de G. morsitans morsitans présente un avantage sur une alimentation moins fréquente, nous avons enrigestré la survie des adultes, la production et le poids des pupes chez des individus nourris à l'émergence et, ensuite, tous les jours ou tous les 2, 3 ou 4 jours. Les femelles alimentées quotidiennement, ou tous les 2 ou 3 jours, ont produit le même nombre de pupes (0,83–0,85), avec des poids de même ordre (23–24 mg), et avec une aussi bonne survie (73 à 79%). Alimentées tous les 4 jours, elles ont produit 0,30 pupe/femelle, pesant 19 mg/pupe et avec une plus faible survie (29%). Ces résultats montrent qu'il est inutile de nourrir les mouches chaque jour au lieu de tous les 2 ou 3 jours. Cependant une alimentation à des intervalles supérieurs à 3 jours, a eu des conséquences clairement défavorables. Ainsi, peuvent être considérablement réduits et le travail et le coût des élevages, sans porter préjudice à la production, en alimentant les mouches 3 fois par semaine, par ex. les lundis, mercredis et vendredis.

     

Feeding frequency in relation to reproduction in Glossina morsitans morsitans and G.pallidipes

Abstract Measurements of residual haematin in males of Glossina morsitans morsitans Westwood reared in the laboratory at 25^oC suggest that blood meal digestion is completed in 4 or 5 days after feeding. However, a high proportion of haematin is present as faecal matter 2 days after feeding and it is concluded that digestion is completed sooner than indicated by the regression of logio haematin on time. Therefore, low levels of residual haematin in field-caught tsetse provide no indication of the frequency with which they feed. For this reason the effects of feeding frequency upon various reproductive parameters in the laboratory have been examined. It is concluded that the best performance is achieved by G.m. morsitans females which ingest four blood meals per inter-larval period and that for a similar performance in G.pallidipes five blood meals are required. The extent to which such feeding frequencies are a reflection of feeding activity in the field are discussed in terms of the biochemical requirements to maintain a reproductive adult female tsetse in positive energy balance.     

The role of sex pheromones in initiation of copulatory behaviour by male tsetse flies, Glossina morsitans morsitans

ABSTRACT. Copulatory responses to a variety of decoy objects baited with synthetic 15,19,23-trimethylheptatriacontane were induced in adult male Glossina morsitans morsitans Westw., provided the object was of similar shape and size to a female fly. Chemical and configurational (shape and texture) stimuli were important in eliciting and maintaining copulatory behaviour of males. A dose/response relationship was obtained from 7-day-old test males, with an ED₉₅ of 2 μg using as decoys solvent-washed dead males baited with 15,19,23-trimethylheptatriacontane. The quantity of this compound in 5-day-old adult females is more than 4.0 μg; in males, only 0.6 μg. It is considered to have the characteristics of a true contact sex pheromone for the species, and does not act synergistically with other less stimulatory synthetic compounds. Gas chromatographic analysis of adult female cuticular paraffins of G.m.morsitans showed the presence of two large peaks between C₃₇ and C₃₈ which were virtually absent in males. Young adults of both sexes possessed a series of hydrocarbons below C₂₉ which disappeared during the first few days of adult life. The natural pheromone appears on the pharate adult female about 2 days before emergence from the puparium and is present throughout life. This suggests it is a component of the surface cuticular waxes. Responsiveness of males to pheromone-baited decoys increased to a maximum 3–4 days after emergence in regularly fed flies and only declined during the 24 h prior to death from starvation.     

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria

Mortality in adult tsetse, Glossina morsitans morsitans, caused by Pseudomonas aeruginosa, Serratia marcescens, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis H-14, B. thuringiensis 1, B. thuringiensis 5, B. thuringiensis var. insraelensis, and Providentia rettgeri was determined. When bacteria were smeared on rabbit skin and tsetse allowed to feed only once on the contaminated area, mortality 8 days postingestion was significantly higher (P less than 0.01) in tsetse fed on P. aeruginosa, S. marcescens, B. thuringiensis 1, and P. rettgeri and increased when tsetse were allowed to feed for the second time on the contaminated skin. With this smear technique, however, mortalities were generally not remarkable. In artificial membrane feeding experiments using low concentrations of bacteria (-10(6)/ml of blood), the B. thuringiensis strains caused low mortality, except B. thuringiensis H-14, which caused 59% mortality. However, at this concentration, P. aeruginosa, S. marcescens, B. cereus, and P. rettgeri caused highly significant (P less than 0.01) mortality (64-96%). When higher concentrations of bacteria (10(7)/ml) were used, all the bacteria tested, except B. sphaericus, caused high mortality ranging from 70 to 98%. Thus, mortality depended on the species of bacteria, the dose ingested, and time postingestion.     

Suppressive action of Samorin on the cyclical development of pathogenic trypanosomes in Glossina morsitans centralis

Male Glossina sexually sterilized by gamma-irradiation are as efficient vectors of trypanosomiasis as fertile males. An attempt was made, using isometamidium chloride (Samorin), to interfere with the cyclical development of trypanosomes in sterile males, destined for use in the sterile insect release (SIR) method of tsetse eradication. The infection rate with mature Trypanosoma congolense Broden was effectively reduced in sterile male Glossina morsitans centralis Machado, when the flies were fed on an infected goat 2 days after they were fed as tenerals on an in vitro bloodmeal containing 8 micrograms Samorin/ml blood. The infection rates with mature T.vivax Ziemann and T.brucei brucei Plimmer & Bradford were completely suppressed at this drug dose. Whensterile teneral males were fed on a bloodmeal containing 12 micrograms/ml Samorin and given the infected bloodmeal 10 days later, infections by mature T.vivax, T.congolense and T.b.brucei were completely suppressed. Hence in the management of a tsetse eradication programme utilizing the SIR method, it is recommended that the sterile teneral male tsetse should, prior to release, be given a bloodmeal containing Samorin at 12-15 micrograms/ml blood. This will effectively suppress future disease transmission.     

Determining the age of adult male and female Glossina morsitans morsitans using a new technique

Abstract. 1. Pteridines accumulated linearly with age up to at least 63 days in male and 140 days in female Glossina morsitans morsitans Westwood kept under laboratory conditions. Time is the major factor influencing total pteridine accumulation levels with the accumulation rate being modulated by temperature.
2. It is suggested that pteridine accumulation may be used to determine the age of male and female tsetse flies in the field.     

Relationships between host blood factors and proteases in Glossina morsitans subspecies infected with Trypanosoma congolense

Abstract. Host blood effects on Trypanosoma congolense establishment in Glossina morsitans morsitans and Glossina morsitans centralis were investigated using goat, rabbit, cow and rhinoceros blood. Meals containing goat erythrocytes facilitated infection in G. m. morsitans , whereas meals containing goat plasma facilitated infection in G. m. centralis. Goat blood effects were not observed in the presence of complementary rabbit blood components. N-acetyl-glucosamine (a midguMectin inhibitor) increased infection rates in some, but not all, blood manipulations. Cholesterol increased infection rates in G. m. centralis only. Both compounds together added to cow blood produced superinfection in G. m. centralis , but not in G. m. morsitans. Midgut protease levels did not differ 6 days post-infection in flies maintaining infections versus flies clearing infections. Protease levels were weakly correlated with patterns of infection, but only in G. m. morsitans. These results suggest that physiological mechanisms responsible for variation in infection rates are only superficially similar in these closely-related tsetse.     

The control of receptivity and ovulation in the tsetse fly, Glossina morsitans

ABSTRACT. A rapid decline in receptivity of mated female Glossina morsitans morsitans Westwood is shown to depend on both physical and chemical stimuli associated with copulation. Radiolabelling revealed the transfer of substances from the male to the haemolymph of the female during copulation. Implantation of male tissues or their injection as homogenates into virgin females showed the chemical stimulus to come from the male accessory glands. Receptivity decreased in females mated to males with ejaculatory ducts severed or testes removed and also in females which had a glass bead inserted into their uterus and/or the tip of their abdomen covered with wax, suggesting that a physical stimulus inducing refractoriness is provided by distension of the uterus and/or stimulation of their terminalial setae. Exposing virgin females to daily short matings in which no male materials were transferred, confirmed this. Receptivity also declined slowly with age in unexposed virgin females. Transfusion of haemolymph from mated females (up to 11 days old) into virgins did not indicate the existence of a haemolymph-borne ovulation-inducing factor; apparently only physical stimuli from mating are involved in the induction of ovulation, and somehow prime the ovarian tissue so that it responds appropriately later when the egg has matured. Whether the stimulus is transmitted to the ovary neurally or hormonally is unknown.     

Activity patterns in pregnant tsetse flies, Glossina morsitans

ABSTRACT. Actograph measurements of the spontaneous flight activity of isolated female Glossina morsitans morsitans Westw. were made through their first two pregnancy cycles in constant conditions under LD 12:12. Activity fell from a mean of c. 20 units/day to near zero during the 24 h preceding the day on which larvipositionoccurred. Activity on the day of larviposition rose significantly, to c. 35 units/day, then fell back to the baseline 20 units on subsequent days. The circadian pattern of activity was changed markedly by larviposition: 48 h before it, activity in the morning peak of the usual V-shaped diel pattern was twice normal; over 70% of the flies then fed, and the evening arm of their V disappeared; 24 h before larviposition, most flies were totally inactive, the few that did fly performing only 1 or 2 units/day, and only in the first and last hours of the photophase; on the day of larviposition, the morning peak of the V was reduced by c. 70%, and activity peaked instead during the post-noon 2–3 h, before and during the act of parturition. Almost all larvipositions occurred between noon and lights-out. It is suggested that the c. 48 h of pre-larviposition quiescence may play a role in the fact that pregnant G. morsitans females often survive non-residual aerosol insecticide campaigns.     

Persistent courtship reduces male and female longevity in captive tsetse flies Glossina morsitans morsitans Westwood (Diptera: Glossinidae)

Where males can increase their mating success by harassing femalesuntil they accept copulation, harassing tactics can be expectedto evolve to a point where they have costs to the longevityof both sexes. By experimentally manipulating the sex ratioin captive groups of tsetse flies Glossina morsitans morsitans,we demonstrated that the longevity of females declines wheresex ratios are biased toward males, while the longevity of malesdeclines where the sex ratio is biased toward females. Neitherirradiation of males nor prevention of copulation by blockingor damaging the external male genitalia increased the longevityof females caged with them, suggesting that female longevitywas reduced by the physical aspects of male harassment ratherthan by components of the ejaculate