The Effects of a Tsetse DNA Virus Infection on the Functions of the Male Accessory Reproductive Gland in the Host Fly Glossina morsitans centralis (Diptera; Glossinidae)

Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies. The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0.0001) than those of the controls. Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Χ² = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates. Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells. Received: 18 November 1998 / Accepted: 10 December 1998     

Reproductive disorders in Glossina palpalis palpalis (Diptera: Glossinidae) in forested areas of Ivory Coast

Study of reproductive disorders were carried out through the dissection of 11,012 tsetse flies caught over a period of one year in forested different habitats of Glossina palpalis palpalis of Daloa in Ivory Coast. The proportion of females with reproductive disorders was very low and estimated at 0.79%. Out of 87 tsetse files with reproductive disorders, 93.10% were abortions, 5.77% were ovular blockage and 1.13% was uterine pupaison. Reproductive disorders were recorded from all age groups: 0.78% in young parous (out of 6,398 tsetse flies examined) and 0.80% in old parous (out of 4,614 tsetse flies dissequed). Our results show that reproductive disorders occur at any stage of the female pregnancy cycle. Amplifying these reproductive disorders using chemical compounds is proposed as a way of improving the efficacy of insecticide-impregnated targets (pour-on, traps and screens) of tsetse control in rain-forest areas.     

Respiratory pattern transitions in three species of Glossina (Diptera, Glossinidae)

Glossina exhibit cyclic (^CYCGE) or continuous gas exchange (^CONGE) patterns at rest. However, the factors influencing the transition from one pattern to another are not well understood for these or other insect species. This study examines which factors could aid in predicting the presence or absence of ^CYCGE in adults of three Glossina species: G. palpalis, G. brevipalpis and G. austeni. We report the results of temperature effects on VCO₂, pattern type and the proportion of a population showing ^CYCGE, and the prediction of ^CYCGE versus ^CONGE in Glossina. First, we investigated the influence of temperature on VCO₂ and found significant elevation in resting metabolic rate (RMR) with higher temperature in all three species (P < 0.001). Temperature-induced increases in VCO₂ were modulated by increased burst volume and by cycle frequency, except in G. brevipalpis which only appeared to modulate burst volume. These results are largely in keeping with VCO₂ modulation reported for other Glossina species previously. Second, elevating temperature resulted in significantly reduced numbers of individuals showing ^CYCGE (P < 0.001 for all three species) contrary to previous reports for other Glossing species. Finally, we examined a range of variables as potential predictors of presence or absence of ^CYCGE in these three species. Using an information theoretic approach (Akaike weights) to select the best explanatory combination of variables which predicts likelihood of ^CYCGE, we found that results varied among species. When species were pooled, the simplest, best-fit model (ΔAIC < 2 from the best model, 44.4% probability of being the best model) for predicting pattern type variation was RMR. Overall these results suggest that RMR is a key variable driving pattern type and that elevated temperature reduces the number of individuals showing cyclic patterns through elevation of RMR in these species. This study supports the idea that an interaction between cellular metabolic demand, morphological features of the gas exchange system (e.g. tracheal and spiracular conductances), and CO₂ buffer capacity likely determine gas exchange pattern variation over short time-scales.     

Macrogeographic population structure of the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)

Tsetse flies are confined to sub-Saharan Africa where they occupy discontinuous habitats. In anticipation of area-wide control programmes, estimates of gene flow among tsetse populations are necessary. Genetic diversities were partitioned at eight microsatellite loci and five mitochondrial loci in 21 Glossina pallidipes Austin populations. At microsatellite loci, Nei's unbiased gene diversity averaged over loci was 0.659 and the total number of alleles was 214, only four of which were shared among all populations. The mean number of alleles per locus was 26.8. Random mating was observed within but not among populations (fixation index FST=0.18) and 81% of the genetic variance was within populations. Thirty-nine mitochondrial variants were detected. Mitochondrial diversities in populations varied from 0 to 0.85 and averaged 0.42, and FST=0.51. High levels of genetic differentiation were characteristic, extending even to subpopulations separated by tens and hundreds of kilometres, and indicating low rates of gene flow.     

Characterization of microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)

Glossina pallidipes is a vector of African trypanosomiasis. Here we characterize eight new polymorphic microsatellite loci in 288 G. pallidipes sampled from 12 Kenya populations. The number of alleles per locus ranged from four to 36 with a mean of 20.5 +/- 10.1. Expected single locus heterozygosities varied from 0.044 to 0.829. Heterozygosity averaged 0.616 +/- 0.246. No linkage disequilibrium was found. We also report results in eight other tsetse species estimated by using the primers developed in G. pallidipes. The primers worked best in G. swynnertoni and G. austeni and worst in G. m. morsitans and G. m. submorsitans.     

Anatomical relations during sperm transfer in Glossina austeni Newstead (Glossinidae,Diptera)

SYNOPSIS An account is given of the uterine anatomy of virgin Glossina austeni Newstead and this is compared with the anatomy of the female tract both during mating and after the mating act has been completed. Notes on the functional morphology of the male genitalia are included.     

Functional Morphology of the Phallosome in Glossina (Diptera, Glossinidae) and Its Evolutionary Implications

Current views on the phylo-genetjc relations of the three species groups within the genus Glossina are critically reviewed. In respect of the male genitalia, the fusca group is here regarded as more specialized than the morsitans group. The detailed phallosome morphology of G. austeni is compared with that of G. fuscipes , on the basis of their mode of action in living flies. The comparison is extended to fusca group species, using dead material only. The presumed phylogeny of Glossina is presented in outline; the evolution of the genus has involved a progressive invasion of riverine and later of forest habitats, from an original semi-arid grassy woodland habitat.     

Electron Microscopic (Energy Dispersive X-ray Analysis) Study of Human Male Reproductive Organs and Semen

Recently, technological advancement helped to improve our knowledge on trace elements in human male reproductive organs and its secretion, semen. In this study, employing energy dispersive x-ray analysis facilities on electron microscope, presence of different elements in human male reproductive organs-??testis, epididymis, caput, corpus and cauda, prostate gland, seminal vesicle, Cowper??s gland and vas deferens??seminal plasma and spermatozoa pellet was studied. Several elements were observed. Gold was one among them that was present in seminal plasma and spermatozoa. It was also present in epididymis caput. Authors consider epididymis caput as the source of gold in semen.     

Prediction of the distribution of Glossina tachinoides (Diptera: Glossinidae) in the Volta basin of northern Ghana

The classification of a Landsat Thematic Mapper satellite image helped demonstrate prevailing habitat types and land use intensity in the Volta basin of the Northern Region of Ghana. A geo-referenced data layer comprising the capture results of a cross-sectional survey of Glossina tachinoides Westwood was over-laid on a data layer of habitat types within 500 m of either bank of the Volta river and its tributaries. An evaluation of the relationship between habitat types and the capture results of G. tachinoides suggested a strong preference of G. tachinoides for woodland, followed by shrubland, grassland and flood plains. The findings were used to classify the suitability of habitat types for G. tachinoides as 'high', 'medium' and 'low' and a prediction map for the distribution of G. tachinoides in the entire river network was produced. The usefulness of this method in estimating the potential distribution of G. tachinoides in an area of increasing agricultural expansion is discussed.     

A cytogenetical study of Glossina fuscipes fuscipes including a comparison of the polytene chromosome maps with those of Glossina austeni (Diptera,Glossinidae)

The male meiotic sequence is described for the tsetse fly Glossina fuscipes fuscipes together with the polytene chromosome maps and all principal cytological markers. The diploid chromosome number is 2n=6 and includes a pair of large submetacentric autosomes (L₁), a shorter pair of metacentric autosomes (L₂), and an X and Y which constitute a heteromorphic pair. Male meiosis is normally achiasmate although evidence is presented which suggests that chiasmata do form in about 1% of males. A detailed comparison between the polytene chromosomes of this species and Glossina austeni indicates that although they must have had a common ancestor, G. austeni is genetically more closely related to morsitans group tsetses.     

Ultrastructural evidence for trans-ovum transmission of the DNA virus of tsetse,Glossina pallidipes (Diptera: Glossinidae)

The occurrence of vertical transmission of the DNA virus of tsetse was studied in virus-infected, femaleGlossina pallidipes with hypertrophied salivary glands (HSG). Ultrastructural examination of tissue components of ovaries of these females revealed virus particles within both germ cell cystocyte clusters and in the follicles, sparsely distributed within nurse cells and in the oocyte cytoplasm. The presence of the virus particles within the ooplasm demonstrates the ovum as a vehicle through which theG. pallidipes virus is disseminated in nature.     

Assessing mating performance of male Glossina pallidipes (Diptera: Glossinidae) using a walk-in field cage

To monitor the quality of male tsetse for use in the sterile insect technique (SIT), a field cage test was developed and evaluated. Mating competitiveness was tested with male Glossina pallidipes Austen that emerged from pupae stored for different periods at 15 degrees C. Control males emerged from pupae stored at 23-24 degrees C and emerged at 26.5 degrees C. Each sample of test males was divided into two groups with one group being irradiated at 120 Gy; the other group was not irradiated. More than 70% of the maximum possible number of mating pairs occurred in all tests. Males emerged from pupae kept at low temperature and then irradiated formed a greater proportion of mating pairs than the controls. Males emerged from pupae kept at 15 degrees C generally started mating more quickly than the standard colony males although there was no significant difference. Insemination rates were above 99%. Pooled data indicated that mean spermathecal values for females mated with irradiated males were significantly lower than for control males. The duration of copulation varied significantly between treatment groups and was significantly longer for irradiated male flies; there was no correlation between duration of copulation and mean spermathecal value.     

Metabolic rate variation in Glossina pallidipes (Diptera: Glossinidae): gender, ageing and repeatability

Despite the importance of metabolic rate in determining flight time of tsetse and in mediating the influence of abiotic variables on life history parameters (and hence abundance and distribution), metabolic rate measurements and their repeatability have not been widely assessed in these flies. We investigate age-related changes in standard metabolic rate (SMR) and its repeatability, using flow-through respirometry, for a variety of feeding, gender and pregnancy classes during early adult development in laboratory-reared individuals of the tsetse fly, Glossina pallidipes. Standard metabolic rate (144-635 microW) was generally within 22% of previous estimates, though lower than the values found using closed system respirometry. There was no significant difference between the genders, but metabolic rate increased consistently with age, probably owing to flight muscle development. Repeatability of metabolic rate was generally high (r=0.6-.09), but not in younger teneral adults and pregnant females (r approximately equal to 0.05-0.4). In these individuals, low repeatability values are a consequence of muscle or in utero larval development. Tsetse and other flies generally have a much higher metabolic rate, for a given size, than do other insect species investigated to date.     

Morphometric diagnosis of <Emphasis Type="Italic">Glossina palpalis</Emphasis> (Diptera: Glossinidae) population structure in Ghana

Objective

This study aimed to identify isolated population(s) of Glossina palpalis in Ghana using geometric morphometrics to evaluate variations in wing-shape and size between populations of the fly from three regions.

Results

Wing shape of G. palpalis tsetse flies from the Northern, Western and Eastern Regions varied significantly between each other. Populations from the Northern and Western Regions varied the most (Mahalanobis Distance = 54.20). The least variation was noticed between populations from the Western and Eastern Regions (MD = 1.99). On morphospace, the Northern population clearly separated from the Eastern and Western populations both of which overlapped. Wing centroid size also significantly varied among populations. Reclassification scores were satisfactory reaching 100% for the Northern population. The Northern population of G. palpalis is possibly isolated from the Western and Eastern Region populations. Meanwhile, a panmictic relationship could be on-going between the Western and Eastern populations. We speculate that geographical distance and subspecific difference between populations are among factors responsible for observed pattern of wing shape variations among the studied populations. The implications of results regarding choice of control strategy and limitations of the study are discussed.

     

Identification and properties of microsatellite markers in tsetse flies Glossina morsitans sensu lato (Diptera: Glossinidae)

Genomic libraries enriched for simple sequence repeats were constructed for Glossina morsitans morsitans, G. m. submorsitans, and G. m. centralis. Sixteen microsatellite markers were isolated from the libraries and evaluated on flies from natural G. m. morsitans populations and other Glossina species in the Morsitans and Palpalis species groups. The primers amplified appropriate sized DNA fragments in the Morsitans and Palpalis groups. In G. morsitans s.l., eight of 12 dinucleotide repeats and four of 12 trinucleotide repeats were polymorphic. The polymorphic loci showed a mean 7.5 +/- 4.8 alleles per locus and their mean heterozygosity was 55.8 +/- 7.7%.     

The salivary secretome of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) infected by salivary gland hypertrophy virus

Background

The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly''s salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.

Methodology/Principal Findings

After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.

Conclusions/Significance

SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.