How monoxenous trypanosomatids revealed hidden feeding habits of their tsetse fly hosts.

Tsetse flies are well-known vectors of trypanosomes pathogenic for humans and livestock. For these strictly blood-feeding viviparous flies, the host blood should be the only source of nutrients and liquids, as well as any exogenous microorganisms colonising their intestine. Here we describe the unexpected finding of several monoxenous trypanosomatids in their gut. In a total of 564 individually examined Glossina (Austenia) tabaniformis (Westwood) (436 specimens) and Glossina (Nemorhina) fuscipes fuscipes (Newstead) (128 specimens) captured in the Dzanga-Sangha Protected Areas, Central African Republic, 24 (4.3%) individuals were infected with monoxenous trypanosomatids belonging to the genera Crithidia Léger, 1902; Kentomonas Votýpka, Yurchenko, Kostygov et Lukes, 2014; Novymonas Kostygov et Yurchenko, 2020; Obscuromonas Votýpka et Lukes, 2021; and Wallacemonas Kostygov et Yurchenko, 2014. Moreover, additional 20 (3.5%) inspected tsetse flies harboured free-living bodonids affiliated with the genera Dimastigella Sandon, 1928; Neobodo Vickerman, 2004; Parabodo Skuja, 1939; and Rhynchomonas Klebs, 1892. In the context of the recently described feeding behaviour of these dipterans, we propose that they become infected while taking sugar meals and water, providing indirect evidence that blood is not their only source of food and liquids.

The analysis and interpretation of critical temperatures.

Critical temperatures are widely used to quantify the upper and lower thermal limits of organisms. But measured critical temperatures often vary with methodological details, leading to spirited discussions about the potential consequences of stress and acclimation during the experiments. We review a model based on the simple assumption that failure rate increases with increasing temperature, independent of previous temperature exposure, water loss or metabolism during the experiment. The model predicts that mean critical thermal maximal temperature (CTmax) increases non-linearly with starting temperature and ramping rate, a pattern frequently observed in empirical studies. We then develop a statistical model that estimates a failure rate function (the relationship between failure rate and current temperature) using maximum likelihood; the best model accounts for 58% of the variation in CTmax in an exemplary dataset for tsetse flies. We then extend the model to incorporate potential effects of stress and acclimation on the failure rate function; the results show how stress accumulation at low ramping rate may increase the failure rate and reduce observed values of CTmax We also applied the model to an acclimation experiment with hornworm larvae that used a single starting temperature and ramping rate; the analyses show that increasing acclimation temperature significantly reduced the slope of the failure rate function, increasing the temperature at which failure occurred. The model directly applies to critical thermal minima, and can utilize data from both ramping and constant-temperature assays. Our model provides a new approach to analyzing and interpreting critical temperatures.

Evaluation of radiation sensitivity and mating performance of Glossina brevipalpis males.

BACKGROUND: Area-wide integrated pest management strategies that include a sterile insect technique component have been successfully used to eradicate tsetse fly populations in the past. To ensure the success of the sterile insect technique, the released males must be adequately sterile and be able to compete with their native counterparts in the wild. METHODOLOGY/PRINCIPAL FINDINGS: In the present study the radiation sensitivity of colonised Glossina brevipalpis Newstead (Diptera; Glossinidae) males, treated either as adults or pupae, was assessed. The mating performance of the irradiated G. brevipalpis males was assessed in walk-in field cages. Glossina brevipalpis adults and pupae were highly sensitive to irradiation, and a dose of 40 Gy and 80 Gy induced 93% and 99% sterility respectively in untreated females that mated with males irradiated as adults. When 37 to 41 day old pupae were exposed to a dose of 40 Gy, more than 97% sterility was induced in untreated females that mated with males derived from irradiated pupae. Males treated as adults with a dose up to 80 Gy were able to compete successfully with untreated fertile males for untreated females in walk-in field cages. CONCLUSIONS/SIGNIFICANCE: The data emanating from this field cage study indicates that, sterile male flies derived from the colony of G. brevipalpis maintained at the Agricultural Research Council-Onderstepoort Veterinary Institute in South Africa are potential good candidates for a campaign that includes a sterile insect technique component. This would need to be confirmed by open field studies.

Symbiont-induced odorant binding proteins mediate insect host hematopoiesis.

Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies (Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host's immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia, up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila's indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems.

Improving the Diet for the Rearing of Glossina brevipalpis Newstead and Glossina austeni Newstead: Blood Source and Collection - Processing - Feeding Procedures.

One of the challenges to maintain tsetse fly (Diptera: Glossinidae) colonies is the sustainable supply of high quality blood meals. The effect of using anticoagulants during collection of the blood, the addition of phagostimulants to the blood meals as well as using mixtures of bovine and porcine blood in different proportions for feeding on colony productivity was assessed. Defibrinated bovine blood was found to be suitable to maintain both the Glossina brevipalpis Newstead and Glossina austeni Newstead colonies. Blood collected with the anticoagulants sodium citrate, citric sodium combination, citrate phosphate dextrose adenine and citric acid did not affect colony performance of both species. Defibrinated bovine and porcine blood in a 1:1 ratio or the feeding of either bovine or porcine blood on alternating days improved pupae production of G. austeni and can be used to enhance colony growth. Bovine blood is appropriate to maintain G. brevipalpis colonies, however, feeding either bovine or porcine blood on alternating days did improve productivity. Adding the phagostimulants inosine tri-phosphate, cytosine mono-phosphate and guanosine mono-phosphate to the blood at a concentration of 10-4 M improved pupae production of the G. brevipalpis colony. The addition of adenosine tri-phosphate and inosine tri-phosphate improved the performance of the G. austeni colony. Decisions on the most suitable rearing diet and feeding protocols will not only depend on the biological requirements of the species but also on the continuous supply of a suitable blood source that can be collected and processed in a cost-effective way.

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species.

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse's ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.

Relationship between Neutrophil-To-Lymphocyte Ratio and Electrocardiographic Ischemia Grade in STEMI / Associação entre a Relação Neutrófilos/Linfócitos e o Grau de Isquemia Eletrocardiográfica no IAMEST

Background: Neutrophil-to-lymphocyte ratio (NLR) has been found to be a good predictor of future adverse cardiovascular outcomes in patients with ST-segment elevation myocardial infarction (STEMI). Changes in the QRS terminal portion have also been associated with adverse outcomes following STEMI. Objective: To investigate the relationship between ECG ischemia grade and NLR in patients presenting with STEMI, in order to determine additional conventional risk factors for early risk stratification. Methods: Patients with STEMI were investigated. The grade of ischemia was analyzed from the ECG performed on admission. White blood cells and subtypes were measured as part of the automated complete blood count (CBC) analysis. Patients were classified into two groups according to the ischemia grade presented on the admission ECG, as grade 2 ischemia (G2I) and grade 3 ischemia (G3I). Results: Patients with G3I had significantly lower mean left ventricular ejection fraction than those in G2I (44.58 ± 7.23 vs. 48.44 ± 7.61, p = 0.001). As expected, in-hospital mortality rate increased proportionally with the increase in ischemia grade (p = 0.036). There were significant differences in percentage of lymphocytes (p = 0.010) and percentage of neutrophils (p = 0.004), and therefore, NLR was significantly different between G2I and G3I patients (p < 0.001). Multivariate logistic regression analysis revealed that only NLR was the independent variable with a significant effect on ECG ischemia grade (odds ratio = 1.254, 95% confidence interval 1.120–1.403, p < 0.001). Conclusion: We found an association between G3I and elevated NLR in patients with STEMI. We believe that such an association might provide an additional prognostic value for risk stratification in patients with STEMI when combined with standardized risk scores. .

Fundamento: A relação neutrófilos/linfócitos (N/L) tem sido descrita como boa preditora de eventos cardiovasculares adversos futuros em pacientes com infarto agudo do miocárdio com elevação do segmento ST (IAMEST). Mudanças na porção terminal do complexo QRS também têm sido associadas a eventos adversos após IAMEST. Objetivo: Investigar a associação entre o grau de isquemia no ECG e a relação N/L em pacientes com IAMEST para determinar fatores de risco convencionais adicionais na estratificação precoce de risco. Métodos: Pacientes com IAMEST foram investigados. O grau de isquemia foi analisado a partir do ECG obtido à admissão. A contagem de leucócitos e seus subtipos foi realizada a partir de hemograma automatizado. De acordo com o grau de isquemia presente no ECG de admissão, os pacientes foram classificados em dois grupos, isquemia grau 2 (IG2) e isquemia grau 3 (IG3). Resultados: Pacientes com IG3 apresentaram valores médios significativamente menores de fração de ejeção do ventrículo esquerdo do que os pacientes com IG2 (44,58 ± 7,23 versus 48,44 ± 7,61; p = 0,001). Como esperado, a taxa de mortalidade intra-hospitalar aumentou proporcionalmente com o aumento no grau de isquemia (p = 0,036). Houve diferenças significativas nas porcentagens de linfócitos (p = 0,010) e de neutrófilos (p = 0,004) e, portanto, a relação N/L diferiu significativamente entre pacientes com IG2 e IG3 (p < 0,001). À análise de regressão logística multivariada, apenas a relação N/L emergiu como variável independente com efeito significativo sobre o grau de isquemia no ECG (odds ratio = 1,254; intervalo de confiança de 95% 1,120-1,403; p < 0,001). Conclusão: Nós encontramos uma associação entre IG3 e relação N/L aumentada em pacientes com IAMEST. Acreditamos que esta associação possa oferecer um valor prognóstico adicional para estratificação de risco em pacientes com IAMEST quando usado em combinação com escores de risco padronizados. .

Adenotrophic viviparity in tsetse flies: potential for population control and as an insect model for lactation.

Tsetse flies (Glossina spp.), vectors of African trypanosomes, are distinguished by their specialized reproductive biology, defined by adenotrophic viviparity (maternal nourishment of progeny by glandular secretions followed by live birth). This trait has evolved infrequently among insects and requires unique reproductive mechanisms. A key event in Glossina reproduction involves the transition between periods of lactation and nonlactation (dry periods). Increased lipolysis, nutrient transfer to the milk gland, and milk-specific protein production characterize lactation, which terminates at the birth of the progeny and is followed by a period of involution. The dry stage coincides with embryogenesis of the progeny, during which lipid reserves accumulate in preparation for the next round of lactation. The obligate bacterial symbiont Wigglesworthia glossinidia is critical to tsetse reproduction and likely provides B vitamins required for metabolic processes underlying lactation and/or progeny development. Here we describe findings that utilized transcriptomics, physiological assays, and RNA interference-based functional analysis to understand different components of adenotrophic viviparity in tsetse flies.

[Glossina feeding habits and diversity of species of trypanosomes in an active focus of human African trypanosomiasis in Gabon]. / Régime alimentaire des glossines et diversité des espèces de trypanosomes dans un foyer actif de trypanosomose humaine africaine au Gabon.

Feeding host is an important factor upon which depend the Glossina infection rate and the proportion of different species of trypanosome. Glossina feed both upon animals and humans. In order to identify species of trypanosomes present in the Komo-Mondah focus and to verify whether there is any relationship between the prevalence of sleeping sickness and the feeding habits of Glossina, we have carried out an entomological survey in this focus of Gabon. Flies were dissected and organs were analysed by PCR, while the origin of blood meals was determined by ELISA. Three species of trypanosomes were found: Trypanosoma congolense "forest type" (14/104; 13.46%), Trypanosoma vivax (11/104; 10.58%) and Trypanosoma brucei s.l. (65/104; 62.5%) with 13.46% (14/104) of mixed infections of T. brucei s.l. and T. congolense Glossina palpalis palpalis was caught in all biotopes investigated (91.85%) and was infected by all these species of trypanosomes. Glossina caliginea was not infected and Glossina fuscipes fuscipes was infected by T. brucei s.l. Tsetse flies feed more on animals than on humans in almost all villages, but there was no significant difference between the number of blood meals taken upon these two groups of vertebrates (Chi 2 = 7.43; p > 0.05). A negative correlation was found between the zoophylic/anthropophylic index and the prevalence of HAT. This result is insufficient to conclude that this index can be used as an indicator of the degree of prevalence of HAT. In contrary, the trypanosomian risk seems to be an appropriate indicator of the prevalence of HAT in an area. The identification of the reservoir hosts in this focus would be useful for a good understanding of the HAT epidemiology.

Effects of flow rate and temperature on cyclic gas exchange in tsetse flies (Diptera, Glossinidae).

Air flow rates may confound the investigation and classification of insect gas exchange patterns. Here we report the effects of flow rates (50, 100, 200, 400 ml min(-1)) on gas exchange patterns in wild-caught Glossina morsitans morsitans from Zambia. At rest, G. m. morsitans generally showed continuous or cyclic gas exchange (CGE) but no evidence of discontinuous gas exchange (DGE). Flow rates had little influence on the ability to detect CGE in tsetse, at least in the present experimental setup and under these laboratory conditions. Importantly, faster flow rates resulted in similar gas exchange patterns to those identified at lower flower rates suggesting that G. m. morsitans did not show DGE which had been incorrectly identified as CGE at lower flow rates. While CGE cycle frequency was significantly different among the four flow rates (p<0.05), the direction of effects was inconsistent. Indeed, inter-individual variation in CGE cycle frequency exceeded flow rate treatment variation. Using a laboratory colony of closely related, similar-sized G. morsitans centralis we subsequently investigated the effects of temperature, gender and feeding status on CGE pattern variation since these factors can influence insect metabolic rates. At 100 ml min(-1) CGE was typical of G. m. centralis at rest, although it was significantly more common in females than in males (57% vs. 43% of 14 individuals tested per gender). In either sex, temperature (20, 24, 28 and 32 degrees C) had little influence on the number of individuals showing CGE. However, increases in metabolic rate with temperature were modulated largely by increases in burst volume and cycle frequency. This is unusual among insects showing CGE or DGE patterns because increases in metabolic rate are usually modulated by increases in frequency, but either no change or a decline in burst volume.

Factors affecting trypanosome maturation in tsetse flies.

Trypanosoma brucei brucei infections which establish successfully in the tsetse fly midgut may subsequently mature into mammalian infective trypanosomes in the salivary glands. This maturation is not automatic and the control of these events is complex. Utilising direct in vivo feeding experiments, we report maturation of T. b. brucei infections in tsetse is regulated by antioxidants as well as environmental stimuli. Dissection of the maturation process provides opportunities to develop transmission blocking vaccines for trypanosomiasis. The present work suggests L-cysteine and/or nitric oxide are necessary for the differentiation of trypanosome midgut infections in tsetse.

The effect of intersubspecific hybridization and gamma radiation on the reproductive biology of Glossina palpalis palpalis (Robineau-Desvoidy) and Glossina palpalis gambiensis Vanderplank.

The closely related tsetse fly subspecies Glossina palpalis palpalis (Nigeria origin) and Glossina palpalis gambiensis (Burkina Faso origin) hybridize readily in the laboratory. Hybridized G.p.palpalis females produced less offspring than the parental intrasubspecific crosses. Adult emergence was below 70% with at least 78% being females. Most female hybrids were fertile whereas most of the male hybrids were sterile when backcrossed to the G.p.palpalis parental line. All F1 males were capable of transferring a spermatophore but their mates rarely had sperm-impregnated spermathecae. Their testes rarely contained mature sperm; moreover, sperm, when present had low or no motility. During laboratory cage tests with virgin females of both subspecies and either sexually mature male G.p.palpalis or G.p.gambiensis, there was no indication for selective mating. The same was true when gamma irradiated males (120 Gy treatment in air) were used. In the latter case complete sterility was induced causing embryonic arrest in all inseminated female mates. Consequently, in ratio tests with untreated virgin G.p.palpalis females, untreated G.p.palpalis males and an increasing number of irradiated G.p.gambiensis males, there was a gradual decrease in production of viable offspring. The results of the present study are discussed with a view of using a combined hybridization and induced sterility in distinct geographical zones where the two subspecies are present.

The use of freeze-dried blood in the membrane feeding of tsetse flies (Glossina p. palpalis, Diptera: Glossinidae).

Rearing of tsetse flies using membrane feeding became a method of routine during recent years. HOwever, the use of blood does require donor animals. Now, for the first time, a technique has been developed using freeze-dried blood as nutrient for Glossina p. palpalis. This report contains an account of the experiments carried out in obtaining a freeze-dried blood product. Initially, fresh bovine blood was processed prior to freeze-drying. It was later reconstituted with distilled water and the osmolarity adjusted to that of fresh blood. ATP was necessary for optimal blood uptake and the addition of albumin increased the weights of puparia. A colony of flies was reared for more than two years by feeding the flies with this reconstituted blood formulation. Later, in a modified freeze-drying programme the processing of the blood prior to drying was eliminated. The addition of ATP was still required, but the quality of the product was not improved anymore by enrichment with albumin, plasma or serum. In comparisons between freeze-dried blood, fresh blood and in vivo feeding of tsetse flies, no differences were found, except the puparial weights were considerably higher in the colony fed on freeze-dried blood than those of the fresh blood colony. This membrane feeding technique eliminates the dependence on blood donor animals for rearing of tsetse flies and can be recommended for breeding colonies in Africa.

Cyclic AMP is a likely mediator of ovulation in the tsetse fly.

Ovulation in tsetse flies is normally induced by mating, but virgins can be stimulated to ovulate with an injection of dibutyryl cyclic AMP, cholera toxin (a cyclic AMP generator), or aminophylline (a phosphodiesterase inhibitor). Thus, elevation of cyclic AMP is a likely link in the events leading to ovulation.

Physiology of an ATP receptor in labellar sensilla of the tsetse fly Glossina morsitans morsitans Westw. (Diptera: Glossinidae).

Electrophysiological recordings have been made from cells in the eight large, labellar sensilla of g. morsitans. One of these cells in each sensillum was shown to respond to ATP over a concentration range of 10(-6)-10(-3) M. It was also sensitive to several other adenosine phophates, but much less sensitive to CTP, GTP and ITP. The activity of the receptor was depressed below pH 7, and sometimes considerably increased above pH 9. These aspects the receptor's physiology support the results of behavioural studies. It is concluded that the eight receptors mediate the flies' behavioural response to ATP.

Structural and functional dynamics of oogenesis in Glossina austeni: general features, previtellogenesis and nurse cells.

Glossina austeni oogenesis throughout its nine-day pregnancy cycle is described with the focus on previtellogenic stages. The ultrastructural details of the oocyte-nurse cell relationship and cyst formation is presented. The oocyte develops in a syncytial association with 15 nurse cells with the entire unit surrounded by a follicular epithelium. The nurse cells have large elaborate nucleoli. Evidence of nuclear emissions and the presence of an unusual cytoplasmic membrane association were found. A variety of nuclear inclusions are seen in the oocyte. Glycogen, lipid, ribosomes and membrane organelles accumulate in the oocyte during previtellogenesis.