Malaria is a vector-borne parasitic disease found in 91 countries with an estimated of 228 million cases occurred worldwide during 2018. The 93% (213 million) of those cases were reported in the African Region (WHO 2019). Six species of Plasmodium parasites can produce the disease but only P. falciparum and P. vivax are the predominant species globally. More than 40 species of Anopheles mosquitoes are important malaria vectors (Asley et al. 2018). Intrinsic (genetic background, parasite susceptibility) and extrinsic (feeding host preference, host diversity and availability, mosquito abundance) factors affect the capacity of mosquitoes to vector the disease (Macdonald 1952). Malaria is prevented by chemoprophylaxis, vaccination, bite-avoidance and vector-control measures. The mainstays of vector control are long-lasting insecticide (pyrethroid) treated nets and indoor residual spraying with insecticides (Asley et al. 2018). The widespread use of pyrethroid insecticides forced the emergence of insecticide resistance in malaria vectors reducing the insecticidal effect. Mosquitoes can modify their behaviour avoiding insecticide contact and so potentially reducing vector control tools efficacy. In this sense, Diop et al. (2020) investigated whether pre-exposure to an Insecticide-Treated Net (ITN) modulates the mosquito ability to take a blood meal in Anopheles gambiae. By means of video recording experiments the authors analyzed how the feeding/bitting behaviour was affected by kdr mutation genotypes (homozygous susceptible – SS-, heterozygotes -RS- and homozygous resistant -RR-) when exposed to two different insecticides (permethrin and deltamethrin). According to the results, the blood-feeding success did not differ between the three genotypes in the absence of insecticide exposure. However, authors observed differences in the feeding duration and blood meal size. In example, RR mosquitoes spent less time taking their blood meal than RS and SS. On the other hand, RS mosquitoes took higher blood volumes than RR females. These differences can affect the mosquito fitness by decreasing/increasing the likelihood to be killed by the host defensive behavior or increase the oogenesis so enhancing fecundity. Regarding the effect of exposition to insecticides authors detected a strong relationship between kdr genotype and Knock Down (KD) phenotype when mosquitoes were exposed to Permethrin. Previously, the authors have evidenced that RR mosquitoes prefer a host protected by a permethrin-treated net rather than an untreated net and that heterozygotes RS mosquitoes have a remarkable ability to find a hole into a bet net (Diop et al. 2015, Porciani et al. 2017). With data here obtained, they demonstrated that kdr homozygous resistant An. gambiae displayed enhanced feeding success when exposed to permethrin ITN. The changes observed in the feeding/biting mosquito behaviour can affect their fitness shaping the evolution of the insecticide resistance in mosquitoes’ natural populations. Moreover, this may also alter parasite transmission dynamics by modifying vector/host interactions and so vector capacity.

References

World Health Organization (2019). World malaria report 2019. Geneva: World Health Organization; 2019. ISBN 978-92-4-156572-1
Ashley EA, Pyae Phyo A, Woodrow CJ (2018). Malaria. Lancet. 391(10130):1608‐1621. doi: 10.1016/S0140-6736(18)30324-6
Macdonald G (1952). The analysis of equilibrium in malaria. Trop Dis Bull 49: 813-828.
Diop MM, Chandre F, Rossignol M, Porciani A, Château M, Moiroux N and Pennetier, C. (2020). Sub-lethal insecticide exposure affects host biting efficiency of Kdr-resistant Anopheles gambiae. bioRxiv 653980, ver. 4 peer-reviewed and recommended by PCI Zoology. doi: 10.1101/653980
Diop MM, Moiroux N, Chandre F, Martin-Herrou H, Milesi P, Boussari O, et al. (2015) Behavioral cost and overdominance in Anopheles gambiae. PLoS ONE. 10(4):e0121755. doi: 10.1371/journal.pone.0121755
Porciani A, Diop M, Moiroux N, Kadoke-Lambi T, Cohuet A, Chandre F, et al. (2017) Influence of pyrethroïd-treated bed net on host seeking behavior of Anopheles gambiae s.s. carrying the kdr allele. PLOS ONE. 12(7):e0164518. doi: 10.1371/journal.pone.0164518

Ticks are notorious vectors of diseases in humans and other vertebrates. Much effort has been expended to understand tick diversity and ecology with the aim of managing their populations to alleviate the misery they bring. Further, the fundamental question of whether ticks are usually host generalists or host specialists has been debated at length and is important both for understanding the mechanisms of their diversification as well as for focusing control of ticks [1].

One elegant resolution of this question is to consider most tick species to be global generalists but local specialists [1]. This is well illustrated in a series of studies of the seabird tick, Ixodes uriae, which is comprised of host-specific races that show genetic [2], morphological [3] and host performance [4] differences associated with the seabirds they feed on. Such a pattern has clear ramifications for sympatric speciation; however, the factors that potentially act to drive these differences have remained elusive.

Dupraz et al. [5] have now made intriguing and important steps toward bridging the gap between demonstrating local patterns of tick host association and understanding the physiological mechanisms that may facilitate such divergences. They collected I. uriae ticks from the nests of two seabirds – Atlantic puffins and common guillemots – on the north side of Iceland. Four populations of ticks were sampled, with one island providing both puffin ticks and guillemot ticks, to give two tick populations from each of the two seabird host species. They then washed the ticks in solvent and analyzed the dissolved cuticular hydrocarbons (CHCs) using GC mass spectrometry, revealing 22 different hydrocarbon compounds common to most of these samples. CHCs are known to be important across arthropods for a variety of functions ranging from reducing water loss to facilitating communication and recognition between individuals with species.

Dupraz et al. [5] found three hydrocarbons that distinguished puffin ticks most consistently from guillemot ticks. A cross-validation test for host type also assigned 75% of the tick pools to the seabird host of origin. However, with these limited sample sizes, statistical analysis revealed no significant difference in CHC profiles between the host types, although a tendency was evident. Nonetheless, this study revealed a number of potentially diagnostic CHCs for tick host type, as well as some that may be more diagnostic of locations. This provides a fascinating and actionable foundation for further work using additional sites and host types, as well as an entry point into discerning the mechanisms at play in producing the diversity, complexity and adaptability that make ticks such medical menaces.

References

[1]  McCoy, K.D., Léger, E., Dietrich, M., 2013. Host specialization in ticks and transmission of tick-borne diseases: a review. Front. Cell. Infect. Microbiol. 3. https://doi.org/10.3389/fcimb.2013.00057

[2]  McCoy, K.D., Chapuis, E., Tirard, C., Boulinier, T., Michalakis, Y., Bohec, C.L., Maho, Y.L., Gauthier-Clerc, M., 2005. Recurrent evolution of host-specialized races in a globally distributed parasite. Proc. R. Soc. B Biol. Sci. 272, 2389–2395. https://doi.org/10.1098/rspb.2005.3230

[3]  Dietrich, M., Beati, L., Elguero, E., Boulinier, T., McCoy, K.D., 2013. Body size and shape evolution in host races of the tick Ixodes uriae. Biol. J. Linn. Soc. 108, 323–334. https://doi.org/10.1111/j.1095-8312.2012.02021.x

[4]  Dietrich, M., Lobato, E., Boulinier, T., McCoy, K.D., 2014. An experimental test of host specialization in a ubiquitous polar ectoparasite: a role for adaptation? J. Anim. Ecol. 83, 576–587. https://doi.org/10.1111/1365-2656.12170

[5] Dupraz, M., Leroy, C., Thórarinsson, T. L., d’Ettorre, P. and McCoy, K. D. (2022) Within and among population differences in cuticular hydrocarbons in the seabird tick Ixodes uriae. bioRxiv, 2022.01.21.477272, ver. 5 peer-reviewed and recommended by Peer Community in Zoology. https://doi.org/10.1101/2022.01.21.477272

The critical role that gut microbiota play in many aspects of an animal’s life, including pathogen resistance, detoxification, digestion, and nutritional physiology, is becoming more and more apparent (Engel and Moran 2013; Lindsay et al., 2020). Gut microbiota recruitment and maintenance can be largely affected by the surrounding environment (Chandler et al., 2011; Callens et al., 2020). The environment may thus dictate gut microbiota composition and diversity, which in turn can affect organismal responses to stress. Only few studies have, however, taken the gut microbiota into account to estimate life histories in response to multiple stressors in aquatic systems (Macke et al., 2016). 

Houwenhuyse et al., investigate how the microbiome affects life histories in response to ecologically relevant single and multiple biotic stressors (an oomycete-like parasite, and a toxic cyanobacterium) in Daphnia magna (Houwenhuyse et al., 2023). Daphnia is an excellent model, because this aquatic system lends itself extremely well for gut microbiota transplantation and manipulation. This is due to the possibility to sterilize eggs (making them free of bacteria), horizontal transmission of bacteria from the environment, and the relative ease of culturing genetically similar Daphnia clones in large numbers. 

The authors use an elegant experimental design to show that the Daphnia gut microbial community differs when derived from a laboratory versus natural inoculum, the latter being more diverse. The authors subsequently show that key life history traits (survival, fecundity, and body size) depend on the stressors (and combination thereof), the microbiota (structure and diversity), and Daphnia genotype. A key finding is that Daphnia exposed to both biotic stressors show an antagonistic interaction effect on survival (being higher), but only in individuals containing laboratory gut microbiota. The exact mechanism remains to be determined, but the authors propose several interesting hypotheses as to why Daphnia with more diverse gut microbiota do less well. This could be due, for example, to increased inter-microbe competition or an increased chance of contracting opportunistic, parasitic bacteria. For Daphnia with less diverse laboratory gut microbiota, a monopolizing species may be particularly beneficial for stress tolerance. Alongside these interesting findings, the paper also provides extensive information about the gut microbiota composition (available in the supplementary files), which is a very useful resource for other researchers. 

Overall, this study reveals that multiple, interacting factors affect the performance of Daphnia under stressful conditions. Of importance is that laboratory studies may be based on simpler microbiota systems, meaning that stress responses measured in the laboratory may not accurately reflect what is happening in nature. 

REFERENCES

Callens M, De Meester L, Muylaert K, Mukherjee S, Decaestecker E. The bacterioplankton community composition and a host genotype dependent occurrence of taxa shape the Daphnia magna gut bacterial community. FEMS Microbiology Ecology. 2020;96(8):fiaa128. https://doi.org/10.1093/femsec/fiaa128

Chandler JA, Lang JM, Bhatnagar S, Eisen JA, Kopp A. Bacterial communities of diverse Drosophila species: ecological context of a host-microbe model system. PLOS Genetics. 2011;7(9):e1002272. https://doi.org/10.1371/journal.pgen.1002272

Engel P, Moran NA. The gut microbiota of insects - diversity in structure and function. FEMS Microbiology Reviews. 2013;37(5):699-735. https://doi.org/10.1111/1574-6976.12025

Houwenhuyse S, Bulteel L, Vanoverberghe I, Krzynowek A, Goel N et al. Microbiome mediated tolerance to biotic stressors: a case study of the interaction between a toxic cyanobacterium and an oomycete-like infection in Daphnia magna. 2023. OSF, ver. 2 peer-reviewed and recommended by Peer Community in Zoology. https://doi.org/10.31219/osf.io/9n4mg

Lindsay EC, Metcalfe NB, Llewellyn MS. The potential role of the gut microbiota in shaping host energetics and metabolic rate. Journal of Animal Ecology. 2020;89(11):2415-2426. https://doi.org/10.1111/1365-2656.13327

Macke E, Tasiemski A, Massol F, Callens M, Decaestecker E. Life history and eco-evolutionary dynamics in light of the gut microbiota. Oikos. 2017;126(4):508-531. https://doi.org/10.1111/oik.03900

Human African Trypanosomiasis (HAT), or sleeping sickness, is caused by trypanosome parasites. In sub-Saharan Africa, two forms are present, Trypanosoma brucei gambiense and T. b. rhodesiense, the former responsible for 95% of reported cases. The parasites are transmitted through a vector, Genus Glossina, also known as tsetse, which means fly in Tswana, a language from southern Africa. Through a blood meal, tsetse picks up the parasite from infected humans or animals (in animals, the parasite causes Animal African Trypanosomiasis or nagana disease). Through medical interventions and vector control programs, the burden of the disease has drastically reduced over the past two decades, so the WHO neglected tropical diseases road map targets the interruption of transmission (zero cases) for 2030 (WHO 2022).

Meaningful vector control programs utilize traps for the removal of animals and for surveillance, along with different methods of spraying insecticides. However, in existing HAT risk areas, it will be essential to understand the ecology of the vector species to implement control programs in a way that areas cleared from the vector will not be reinvaded from other populations. Thus, it will be crucial to understand basic population genetics parameters related to population structure and subdivision, migration frequency and distances, population sizes, and the potential for sex-biased dispersal. The authors utilize genotyping using nine highly polymorphic microsatellite markers of samples from Chad collected in differently affected regions and at different time points (Ravel et al., 2023). Two major HAT zones exist that are targeted by vector control programs, namely Madoul and Maro, while two other areas, Timbéri and Dokoutou, are free of trypanosomes. Samples were taken before vector control programs started.

The sex ratio was female-biased, most strongly in Mandoul and Maro, the zones with the lowest population density. This could be explained by resource limitation, which could be the hosts for a blood meal or the sites for larviposition. Limited resources mean that females must fly further, increasing the chance that more females are caught in traps. 

The effective population densities of Mandoul and Maro were low. However, there was a convergence of population density and trapping density, which might be explained by the higher preservation of flies in the high-density areas of Timbéri and Dokoutou after the first round of sampling, which can only be tested using a second sampling. 

The dispersal distances are the highest recorded so far, especially in Mandoul and Maro, with 20-30 km per generation. However, in Timbéri and Dokoutou, which are 50 km apart, very little exchange occurs (approx. 1-2 individuals every six months). A major contributor to this is the massive destruction of habitat that started in the early 1990s and left patchily distributed and fragmented habitats. The Mandoul zone might be safe from reinvasion after eradication, as for a successful re-establishment, either a pair of a female and male or a pregnant female are required. As the trypanosome prevalence amongst humans was 0.02 and of tsetse 0.06 (Ibrahim et al., 2021) before interventions began, medical interventions and vector control might have further reduced these levels, making a reinvasion and subsequent re-establishment of HAT very unlikely. Maro is close to the border of the Central African Republic, and the area has not been well investigated concerning refugee populations of tsetse, which could contribute to a reinvasion of the Maro zone. The higher level of genetic heterogeneity of the Maro population indicates that invasions from neighboring populations are already ongoing. This immigration could also be the reason for not detecting the bottleneck signature in the Maro population. 

The two HAT areas need different levels of attention while implementing vector eradication programs. While Madoul is relatively safe against reinvasion, Maro needs another type of attention, as frequent and persistent immigration might counteract eradication efforts. Thus, it is recommended that continuous tsetse suppression needs to be implemented in Maro.  

This study shows nicely that an in-depth knowledge of the processes within and between populations is needed to understand how these populations behave. This can be used to extrapolate, make predictions, and inform the organisations implementing vector control programs to include valuable adjustments, as in the case of Maro. Such integrative approaches can help prevent the failure of programs, potentially saving costs and preventing infections of humans and animals who might die if not treated.

References

Ibrahim MAM, Weber JS, Ngomtcho SCH, Signaboubo D, Berger P, Hassane HM, Kelm S (2021) Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro, Southern Chad- Southern Chad-A matter of concern for zoonotic potential? PLoS Neglected Tropical Diseases, 15, e000 323. https://doi.org/10.1371/journal.pntd.0009323

Ravel S, Mahamat MH, Ségard A, Argiles-Herrero R, Bouyer J, Rayaisse JB, Solano P, Mollo BG, Pèka M, Darnas J, Belem AMG, Yoni W, Noûs C, de Meeûs T (2023) Population genetics of Glossina fuscipes fuscipes from southern Chad. Zenodo, ver. 9 peer-reviewed and recommended by PCI Zoology. https://doi.org/10.5281/zenodo.7763870

WHO (2022) Trypanosomiasis, human African (sleeping sickness). https://www.who.int/news-room/fact-sheets/detail/trypanosomiasis-human-african-(sleeping-sickness), retrieved 17. March 2023

Molecular markers have drastically changed and improved our understanding of biological processes. In combination with PCR, markers revolutionized the study of all organisms, even tiny insects, and eukaryotic pathogens amongst others. Microsatellite markers were the most prominent and successful ones. Their success started in the early 1990s. They were used for population genetic studies, mapping of genes and genomes, and paternity testing and inference of relatedness. Their popularity is based on some of their characteristics as codominance, the high polymorphism information content, and their ease of isolation (Schlötterer 2004). Still, microsatellites are the marker of choice for a range of non-model organisms as next-generation sequencing technologies produce a huge amount of single nucleotide polymorphisms (SNPs), but often at expense of sample size and higher costs.
 
The high level of polymorphism of microsatellite markers, which consist of one to six base-pair nucleotide motifs replicated up to 10 or 20 times, results from slippage events during DNA replication. Short hairpin loops might shorten the template strand or extend the new strand. However, such slippage events might occur during PCR amplification resulting in additional bands or peaks. Such stutter alleles often appear to differ by one repeat unit and might be hard to interpret but definitively reduce automated scoring of microsatellite results.
 
A standalone software package available to handle stuttering is Microchecker (van Oosterhout et al., 2004, which nowadays faces incompatibilities with updated versions of different operating systems. Thus, de Meeûs and Noûs (2022), in their manuscript, tackled the stuttering issue by developing an OS-independent analysis pipeline based on standard spreadsheet software such as Microsoft Office (Excel) or Apache Open Office (Calc). The authors use simulated populations differing in the mating system (pangamic, selfing (30%), clonal) and a different number of subpopulations and individuals per subpopulation to test for differences among the null model (no stuttering), a test population with 2 out of 20 loci (10%) with stuttering, and the latter with stuttering cured. Further to this, the authors also re-analyse data from previous studies utilising organisms differing in the mating system to understand whether control of stuttering changes major parameter estimates and conclusions of those studies.
 
Stuttering of microsatellite loci might result in increased heterozygote deficits. The authors utilise the FIS (inbreeding coefficient) as a tool to compare the different treatments of the simulated populations. Their method detected stuttering in pangamic and selfing populations, while the detection of stuttering in clonal organisms is more difficult. The cure for stuttering resulted in FIS values similar to those populations lacking stuttering. The re-analysis of four previously published studies indicated that the new method presented here is more accurate than Microchecker (van Oosterhout et al., 2004) in a direct comparison. For the Lyme disease-transmitting tick Ixodes scapularis (De Meeûs et al., 2021), three loci showed stuttering and curing these resulted in data that are in good agreement with pangamic reproduction. In the tsetse fly Glossina palpalis palpalis (Berté et al., 2019), two out of seven loci were detected as stuttering. Curing them resulted in decreased FIS for one locus, while the other showed an increased FIS, an indication of other problems such as the occurrence of null alleles. Overall, in dioecious pangamic populations, the method works well, and the cure of stuttering improves population genetic parameter estimates, although FST and FIS might be slightly overestimated. In monoecious selfers, the detection and cure work well, if other factors such as null alleles do not interfere. In clonal organisms, only loci with extremely high FIS might need a cure to improve parameter estimates.
 
This spreadsheet-based method helps to automate microsatellite analysis at very low costs and thus improves the accuracy of parameter estimates. This might certainly be very useful for a range of non-model organisms, parasites, and their vectors, for which microsatellites are still the marker of choice. 
 
References

Berté D, De Meeus T, Kaba D, Séré M, Djohan V, Courtin F, N'Djetchi KM, Koffi M, Jamonneau V, Ta BTD, Solano P, N’Goran EK, Ravel S (2019) Population genetics of Glossina palpalis palpalis in sleeping sickness foci of Côte d'Ivoire before and after vector control. Infection Genetics and Evolution 75, 103963. https://doi.org/0.1016/j.meegid.2019.103963

de Meeûs T, Chan CT, Ludwig JM, Tsao JI, Patel J, Bhagatwala J, Beati L (2021) Deceptive combined effects of short allele dominance and stuttering: an example with Ixodes scapularis, the main vector of Lyme disease in the U.S.A. Peer Community Journal 1, e40. https://doi.org/10.24072/pcjournal.34

de Meeûs T, Noûs C (2022) A simple procedure to detect, test for the presence of stuttering, and cure stuttered data with spreadsheet programs. Zenodo, v5, peer-reviewed and recommended by PCI Zoology. https://doi.org/10.5281/zenodo.7029324

Schlötterer C (2004) The evolution of molecular markers - just a matter of fashion? Nature Reviews Genetics 5, 63-69. https://doi.org/10.1038/nrg1249

van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4, 535-538. https://doi.org/10.1111/j.1471-8286.2004.00684.x