Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis

Background

Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness.

Results

In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission.

Conclusions

Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.

     

Interspecific transfer of bacterial endosymbionts between tsetse fly species: infection establishment and effect on host fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

Maternal effects on offspring size in a natural population of the viviparous tsetse fly

1. Theory predicts that mothers should adaptively adjust reproductive investment depending on current reserves and future reproductive opportunities. Females in better intrinsic state, or with more resources, should invest more in current reproduction than those with fewer resources. Across the lifespan, investment may increase as future reproductive opportunities decline, yet may also decline with reductions in intrinsic state. 2. Across many species, larger mothers produce larger offspring, but there is no theoretical consensus on why this is so. This pattern may be driven by variation in maternal state such as nutrition, yet few studies measure both size and nutritional state or attempt to tease apart confounding effects of size and age. 3. Viviparous tsetse flies (Glossina species) offer an excellent system to explore patterns of reproductive investment: females produce large, single offspring sequentially over the course of their relatively long life. Thus, per‐brood reproductive effort can be quantified by offspring size. 4. While most tsetse reproduction research has been conducted on laboratory colonies, maternal investment was investigated in this study using a unique field method where mothers were collected as they deposited larvae, allowing simultaneous mother‐offspring measurements under natural conditions. 5. It was found that larger mothers and those with a higher fat content produced larger offspring, and there was a trend for older mothers to produce slightly larger offspring. 6. The present results highlight the importance of measuring maternal nutritional state, rather than size alone, when considering maternal investment in offspring. Implications for understanding vector population dynamics are also discussed.     

Towards improving tsetse fly paratransgenesis: stable colonization of <Emphasis Type="Italic">Glossina morsitans morsitans</Emphasis> with genetically modified <Emphasis Type="Italic">Sodalis</Emphasis>

Background

Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness.

Results

In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission.

Conclusions

Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.

     

Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

Effect of the life-span of female Glossina palpalis gambiensis on the weight and size of its progeny

Abstract. Pupae and teneral flies of Glossina palpalis gambiensis originating from three successive reproductive cycles were compared for their size and weight. In general, pupal weight and fly weight increased, whereas fly size, measured as wing vein length, decreased with the number of reproductive cycles. The linear regression observed between weight and wing vein length of the fly demonstrated that, particularly for flies originating from the first and second larvipositions, small changes in wing vein length reflected substantial differences in weight.
The results of these laboratory experiments were compared with some field data on Glossina morsitans from Zambia and related literature. The life span of the female tsetse, affecting the size of her progeny, could clarify partially some of the field observed seasonal changes in size, whereas the correlation between fly size and weight could eventually explain the differential mortality of some size classes of tsetse flies. However, whether these laboratory observations can be extrapolated to the field has still to be confirmed.     

Reconstructing life history of hominids and humans

Aspects of life history, such as processes and timing of development, age at maturation, and life span are consistently associated with one another across the animal kingdom. Species that develop rapidly tend to mature and reproduce early, have many offspring, and exhibit shorter life spans (r-selection) than those that develop slowly, have extended periods of premature growth, mature later in life, reproduce later and less frequently, have few offspring and/or single births, and exhibit extended life spans (K-selection). In general, primates are among the most K-selected of species. A suite of highly derived life history traits characterizes humans. Among these are physically immature neonates, slowed somatic development both in utero and post-natally, late attainment of reproductive maturity and first birth, and extended post-mature survival. Exactly when, why, and through what types of evolutionary interactions this suite arose is currently the subject of much conjecture and debate. Humankind's biocultural adaptations have helped to structure human life history evolution in unique ways not seen in other animal species. Among all species, life history traits may respond rapidly to alterations in selective pressures through hormonal processes. Selective pressures on life history likely varied widely among hominids and humans over their evolutionary history. This suggests that current patterns of human growth, development, maturation, reproduction, and post-mature survival may be of recent genesis, rather then long-standing adaptations. Thus, life history patterns observed among contemporary human and chimpanzee populations may provide little insight to those that existed earlier in hominid/human evolution.     

Change across the lifespan in a psychological measure of life history strategy

Life history theory predicts that people calibrate their reproductive strategies to local levels of environmental harshness and unpredictability. While previous research has established the importance of early life cues in the development of life history strategy, the degree to which life history strategy exhibits plasticity later in life is unclear. Using longitudinal data (total N = 479) from four archival studies and a recently validated psychological measure of life history strategy, we examined mean-level trends in life history strategy at the level of psychological phenotype between the ages of 7 and 60 and found that life history strategy slowed down linearly as a function of age. Highlighting the importance of sexual selection in shaping life history strategy, we also found that men had a faster life history strategy than women at all ages and that the magnitude of this difference was constant across the lifespan. Our findings suggest that life history strategy development continues even in older adulthood. We discuss the possibility that this occurs in response to the accumulation of biological and social (e.g. offspring, relationships) capital and information about local risks and incentives.     

Prospects for using pyroproxyfen-treated targets for tsetse control

Using¹⁴C cholesterol as a marker a positive correlation was established between the amount of oil (a chlorinated n-alkane containing 43–46% chlorine, ‘cereclor S45’) picked up by an adult tsetse fly exposed by tarsal contact to a treated surface and the duration of such exposure. Only a poor uptake was achieved from netting surfaces treated with less than 50% oil in acetone. Terylene netting treated with radioactive pyriproxyfen, [1-methyl-2-(4-phenoxyphenoxy)ethoxy] pyridine, dissolved in cereclor, was exposed in the field for a year. After 9 months 20% of the original radioactivity remained and was shown to be 95% authentic pyriproxyfen. Brief tarsal contact (up to 5 seconds) with such netting, by adult females ofGlossina morsitans morsitans Westwood, reduced the viability of their offspring to 28–43% of untreated control values. The effect was greatest in the reproductive cycle immediately following contact. Between 10 and 12 months after treatment of the fabric the radioactivity fell to only 7% of the original level but was associated mainly (>80%) with intact pyriproxyfen. Exposure of female flies to this netting resulted in a positive correlation between the duration of exposure and the extent of suppression of offspring viability, such that 2 min was sufficient to reduce offspring viability to zero for the life of the female. Traps or targets impregnated with conventional formulations of pyrethroids to kill tsetse would have lost all their activity by this time. Results are discussed in terms of the prospects for using pyriproxyfen-treated targets to sterilize female tsetse directly and also indirectly through the contamination of males prior to mating through contact with such targets.     

Reproductive senescence and terminal investment in female Barbary macaques (Macaca sylvanus) at Salem

The reproductive history of 207 female Barbary macaques, living in a large outdoor enclosure in Southwest Germany, was studied during an 11-year period. The results yielded a significant relationship between female age and fecundity, with fertility rates lower than expected among young and old females. Analysis of the reproductive history of individual females revealed a significant decline in fertility from prime age (7–12 years) to mid age (13–19 years), and from mid age to old age (20–25 years). The proportion of long interbirth intervals increased steadily among aging females. Infant survival was not significantly related to maternal age, but offspring of old females showed the highest survivorship. Behavioral observations revealed that old mothers weaned their offspring significantly later than younger mothers, suggesting that prolongation of interbirth intervals is due not only to deteriorating physical condition but also to increased maternal investment, as life history theory predicts. Reproduction ceased during the middle of the third decade of life. Final cessation of estrous cycling invariably occurred 3 or 4 years after the birth of the last offspring, but a postreproductive life span of 5 years appears to be common in this population. Available data suggest that reproductive senescence and menopause are more common among nonhuman primates than widely believed and that both traits are part of an adaptive life history strategy.     

Differences in Patterns of Reproductive Allocation between the Sexes in Nicrophorus orbicollis

Organisms are selected to maximize lifetime reproductive success by balancing the costs of current reproduction with costs to future survival and fecundity. Males and females typically face different reproductive costs, which makes comparisons of their reproductive strategies difficult. Burying beetles provide a unique system that allows us to compare the costs of reproduction between the sexes because males and females are capable of raising offspring together or alone and carcass preparation and offspring care represent the majority of reproductive costs for both sexes. Because both sexes perform the same functions of carcass preparation and offspring care, we predict that they would experience similar costs and have similar life history patterns. In this study we assess the cost of reproduction in male Nicrophorus orbicollis and compare to patterns observed in females. We compare the reproductive strategies of single males and females that provided pre- and post-hatching parental care. There is a cost to reproduction for both males and females, but the sexes respond to these costs differently. Females match brood size with carcass size, and thus maximize the lifetime number of offspring on a given size carcass. Males cull proportionately more offspring on all carcass sizes, and thus have a lower lifetime number of offspring compared to females. Females exhibit an adaptive reproductive strategy based on resource availability, but male reproductive strategies are not adaptive in relation to resource availability.     

Predicting trait values and measuring selection in complex life histories: reproductive allocation decisions in Soay sheep

Accurate prediction of life history phenomena and characterisation of selection in free-living animal populations are fundamental goals in evolutionary ecology. In density regulated, structured populations, where individual state influences fate, simple and widely used approaches based on individual lifetime measures of fitness are difficult to justify. We combine recently developed structured population modelling tools with ideas from modern evolutionary game theory (adaptive dynamics) to understand selection on allocation of female reproductive effort to singletons or twins in a size-structured population of feral sheep. In marked contrast to the classical selection analyses, our model-based approach predicts that the female allocation strategy is under negligible directional selection. These differences arise because classical selection analysis ignores components of offspring fitness and fails to consider selection over the complete life cycle.     

The microbiota influences the Drosophila melanogaster life history strategy

Organisms are locally adapted when members of a population have a fitness advantage in one location relative to conspecifics in other geographies. For example, across latitudinal gradients, some organisms may trade off between traits that maximize fitness components in one, but not both, of somatic maintenance or reproductive output. Latitudinal gradients in life history strategies are traditionally attributed to environmental selection on an animal's genotype, without any consideration of the possible impact of associated microorganisms (“microbiota”) on life history traits. Here, we show in Drosophila melanogaster, a key model for studying local adaptation and life history strategy, that excluding the microbiota from definitions of local adaptation is a major shortfall. First, we reveal that an isogenic fly line reared with different bacteria varies the investment in early reproduction versus somatic maintenance. Next, we show that in wild fruit flies, the abundance of these same bacteria was correlated with the latitude and life history strategy of the flies, suggesting geographic specificity of the microbiota composition. Variation in microbiota composition of locally adapted D. melanogaster could be attributed to both the wild environment and host genetic selection. Finally, by eliminating or manipulating the microbiota of fly lines collected across a latitudinal gradient, we reveal that host genotype contributes to latitude‐specific life history traits independent of the microbiota and that variation in the microbiota can suppress or reverse the differences between locally adapted fly lines. Together, these findings establish the microbiota composition of a model animal as an essential consideration in local adaptation.     

Effects of reproductive strategies on pollutant concentrations in pinnipeds: a meta‐analysis

In marine mammals such as pinnipeds (seals, sea lions and walruses), reproductive strategies reveal how species acquire, store and allocate energy to offspring. During lactation, females can allocate energy acquired from concurrent resources (income breeding); or utilize energy stored prior to reproduction (capital breeding). Mothers transfer a large proportion of energy to their pups via lipid rich milk, meaning that pollutants such as polychlorinated biphenyls (PCBs) and mercury (Hg) will also transfer, raising concern for negative health effects. To quantify the effect of reproductive strategy on maternal pollutant transfer in pinnipeds, we developed a proxy for income and capital breeding by focusing on the lactational component of reproduction, and arranged species along this gradient. We found a strong positive relationship between lipid content in milk and degree of capital breeding. We tested this gradient against maternal pollutant transfer expressed as a concentration ratio by meta‐analysis. In mother‐pup pairs, the concentration ratio of PCBs was one order of magnitude higher than for mercury. PCB concentrations in pups and juveniles were similar to adult females, but mercury was always lower in young offspring than in females. We found no effect of reproductive strategy between studies investigating mother‐pup pairs and non‐related females and juveniles (< 1 year old), however data were strongly biased towards capital breeders. Our results suggest that either: 1) reproductive strategy does not affect pollutant bioaccumulation; or 2) a lack of income breeder data prevents us from testing the overall effect of reproductive strategy on maternal pollutant transfer. The finding that PCB concentrations in juveniles are similar to females is of concern due to early life stage exposure. We recommend data collection from income breeding species such as the sea lions to elucidate whether reproductive strategy, and potentially other life history traits, has an overall effect on maternal pollutant transfer.     

Flexible responses to stage‐specific offspring threats

When caring for their young, parents must compensate for threats to offspring survival in a manner that maximizes their lifetime reproductive success. In birds, parents respond to offspring threats by altering reproductive strategies throughout the breeding attempt. Because altered reproductive strategies are costly, when threats to offspring are limited, parents should exhibit a limited response. However, it is unclear if response to offspring threat is the result of an integrated set of correlated changes throughout the breeding attempt or if responses are a flexible set of dissociable changes that are stage‐specific. We test these hypotheses in a system where house wrens (Troglodytes aedon) compete for nesting cavities with Carolina chickadees (Poecile carolinensis) by usurping and destroying their nests during the early stage of the breeding attempt (the egg stage). Due to the specificity of the house wren threat, we can test whether parental responses to an offspring threat show flexibility and stage specificity or if parental strategies are an integrated and persistent response. We monitored nests in a natural population to compare life history traits of chickadees nesting in boxes that were in the presence of house wrens to chickadees nesting in boxes that did not overlap with house wrens. Carolina chickadees that nested near house wrens laid significantly smaller clutch sizes (early change in reproductive strategy) but did not alter nestling provisioning or nestling stage length (late change in reproductive strategy), suggesting that chickadees respond in a flexible and stage‐specific manner to the threat of house wrens. By responding only when a threat is highest, parents minimize the cost of antithreat responses. Our study suggests that parents can respond in subtle and nuanced ways to offspring threats in the environment and specifically alter reproductive behaviors at the appropriate stage.     

Nutritional levels of pregnant and postpartum tsetse Glossina pallidipes Austen captured in artificial warthog burrows in the Zambezi Valley of Zimbabwe

In Zimbabwe, female tsetse Glossina pallidipes Austen, collected from artificial warthog burrows and subjected to ovarian dissection and nutritional analysis, provide the first field estimates of resource allocation from mother to offspring across all of pregnancy. Approximately 45% of 1833 females captured are full‐term pregnant on entering the burrow. The remainder presumably use burrows as refuges at temperatures >32 °C. Maternal residual dry weight (RDW) increases by 1.5 mg after the first feed post‐larviposition but, thereafter, only by 1.4 mg by 90% of pregnancy completion. Uterine RDW changes little by 60% of pregnancy completion but increases by >6 mg by parturition. Between the times of 5% and 90% pregnancy completion, maternal RDW is approximately constant: it then declines 2.8 mg by parturition, balancing larval gains of 2.6 mg. Mothers accumulate 6.3 mg of fat in the first 80% of pregnancy, while uterine fat increases by only 0.8 mg. Thereafter, by parturition, larval fat increases by 4.1 mg, whereas maternal fat declines by 3.3 mg. The larva deposited is 5% heavier than its mother and has 52% more fat. RDW and fat levels, corrected for fly size and haematin, are 1 and 2 mg, respectively, lower in females from traps than those from burrows. Burrow catches provide an improved picture of tsetse pregnancy dynamics, highlighting a reproductive strategy involving resource commitment to the larva being delayed until late pregnancy when sufficient stores guarantee viable pupal production. Larval development in tsetse starts significantly later than the analogous changes during pregnancy in two mammals and two viviparous fish.     

Early is better: seasonal egg fitness and timing of reproduction in a zooplankton life-history model

Timing of reproduction influences future prospects of offspring and therefore the reproductive value of parents. Early offspring are often more valuable than later ones when food availability and predation risk fluctuate seasonally. Marine zooplankton have evolved a diversity of life history strategies in response to seasonality. We present a state-dependent life history model for the annual and herbivorous high-latitude copepod Calanoides acutus . Individuals are characterised by four states; developmental stage, structural size, energy reserves and vertical location. There are two habitats, a surface habitat with seasonal predation risk and food availability, and a safer deep habitat with no food and low metabolism (diapause). Optimal life histories (diapause and energy allocation strategies) are found by dynamic programming. Seasonal egg fitness (reproductive value) emerges from the model and peak values are typically before the feeding season. Disentangling the fitness components, we conclude that seasonality in egg fitness is caused both by environmental seasonality in food and predation risk and by time-constraints on development and diapause preparation. Realised egg production, as predicted from population simulations, does not match the seasonal peak in offspring fitness but is delayed relative to peak egg fitness. We term this an 'internal life history mismatch' as constraints and tradeoffs cause sub-optimal birth dates for most eggs whereas mothers maximise their reproductive value by high number of eggs rather than few and optimally timed eggs. The earliest eggs have a disproportionately high contribution to population recruitment, emphasising the importance of early eggs and the need to understand seasonal patterns in offspring fitness.     

Transgenerational effects of nutrition are different for sons and daughters

Food shortage is an important selective factor shaping animal life‐history trajectories. Yet, despite its role, many aspects of the interaction between parental and offspring food environments remain unclear. In this study, we measured developmental plasticity in response to food availability over two generations and tested the relative contribution of paternal and maternal food availability to the performance of offspring reared under matched and mismatched food environments. We applied a cross‐generational split‐brood design using the springtail Orchesella cincta, which is found in the litter layer of temperate forests. The results show adverse effects of food limitation on several life‐history traits and reproductive performance of both parental sexes. Food conditions of both parents contributed to the offspring phenotypic variation, providing evidence for transgenerational effects of diet. Parental diet influenced sons’ age at maturity and daughters’ weight at maturity. Specifically, being born to food‐restricted parents allowed offspring to alleviate the adverse effects of food limitation, without reducing their performance under well‐fed conditions. Thus, parents raised on a poor diet primed their offspring for a more efficient resource use. However, a mismatch between maternal and offspring food environments generated sex‐specific adverse effects: female offspring born to well‐fed mothers showed a decreased flexibility to deal with low‐food conditions. Notably, these maternal effects of food availability were not observed in the sons. Finally, we found that the relationship between age and size at maturity differed between males and females and showed that offspring life‐history strategies in O. cincta are primed differently by the parents.     

Tsetse-trypanosome interactions: rites of passage.

Trypanosomes that cause sleeping sickness (Trypanosoma brucei rhodesiense and T. b. gambiense) are entirely dependent on tsetse for their transmission between hosts, but the flies are not easily infected. This situation has not arisen by chance - the tsetse has evolved an efficient defence system against trypanosome invasion. In this review, Susan Welburn and Ian Maudlin chart the progress of trypanosomes through the fly and identify some of the hazards faced by both parasite and fly that affect vector competence of tsetse.