A possible malaria control approach involves the dissemination in mosquitoes of inherited symbiotic microbes to block Plasmodium transmission. However, in the Anopheles gambiae complex, the primary African vectors of malaria, there are limited reports of inherited symbionts that impair transmission. We show that a vertically transmitted microsporidian symbiont (Microsporidia MB) in the An. gambiae complex can impair Plasmodium transmission. Microsporidia MB is present at moderate prevalence in geographically dispersed populations of An. arabiensis in Kenya, localized to the mosquito midgut and ovaries, and is not associated with significant reductions in adult host fecundity or survival. Field-collected Microsporidia MB infected An. arabiensis tested negative for P. falciparum gametocytes and, on experimental infection with P. falciparum, sporozoites aren’t detected in Microsporidia MB infected mosquitoes. As a microbe that impairs Plasmodium transmission that is non-virulent and vertically transmitted, Microsporidia MB could be investigated as a strategy to limit malaria transmission.

The root knot nematode, Meloidogyne is one of the most economically damaging plant parasitic nematode groups, and are widely distributed in Kenyan agro-ecosystems. The aim of this study was to determine the diversity of Meloidogyne species in Kenyan sweet potato fields and identify sweet potato varieties that exhibit resistance to M. incognita. Meloidogyne species were collected from Nyanza, Western, Eastern and Central Provinces of Kenya. Mitochondrial DNA was used to differentiate Meloidogyne species. The most common species in all sampled regions was M. incognita. Meloidogyne hapla was recorded for the first time in Kenyan sweet potato growing areas (Mosocho, Matayos, Teso South, Manyatta, and Nzaui sub-counties), while M. enterolobii was observed in Kiharu, Matayos and Mosocho sub-counties and a novel Meloidogyne sp. was identified in Kiharu sub-county. Seventy-two sweet potato varieties collected from both agricultural fields and research stations in Kenya were evaluated for resistance to M. incognita under greenhouse conditions in two separate trials. Known susceptible (Beauregard) and resistant (Tanzania) sweet potato varieties were included as controls. Responses of sweet potato varieties to M. incognita infection was assessed by the number of eggs present and level of galling on a scale of 1–5, where 0 = 0 galls and 5 ≥ 100 galls. The reproduction index (RI) was used to classify the varieties as resistant or susceptible. There was a significant difference (P < 0.001) in the number of eggs, GI and RI among the varieties tested. Forty nine sweet potato varieties were considered very resistant and may be used in breeding programs to incorporate resistance against M. incognita into commercial cultivars of sweet potato or to use them in crop rotation programmes for management of RKN. The results on Meloidogyne species diversity in Kenyan sweet potato fields will also be useful in nematode management programs

Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b (cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 (COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera (Aedes (Ae. vittatus from Ae. metallicus), Culex (Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles (An. gambiae s.s. from An. arabiensis) and Mansonia (Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia (Ad. africana and Ad. furfurea), Mimomyia (Mi. hispida and Mi. splendens) and Coquillettidia (Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes.

Background: Insect symbionts have the potential to block the transmission of vector-borne diseases by their hosts. The advancement of a symbiont-based transmission blocking strategy for malaria requires the identification and study of Anopheles symbionts. Methods: High throughput 16S amplicon sequencing was used to profile the bacteria associated with Anopheles gambiae sensu lato and identify potential symbionts. The polymerase chain reaction (PCR) with specific primers were subsequently used to monitor symbiont prevalence in field populations, as well as symbiont transmission patterns. Results: We report the discovery of the bacterial symbiont, Spiroplasma, in Anopheles gambiae in Kenya. We determine that geographically dispersed Anopheles gambiae populations in Kenya are infected with Spiroplasma at low prevalence levels. Molecular phylogenetics indicates that this Anopheles gambiae associated Spiroplasma is a member of the insolitum clade. We demonstrate that this symbiont is stably maternally transmitted across at least two generations and does not significantly affect the fecundity or egg to adult survival of its host. Conclusions: In diverse insect species, Spiroplasma has been found to render their host resistant to infection by pathogens. The identification of a maternally transmitted strain of Spiroplasma in Anopheles gambiae may therefore open new lines of investigation for the development of symbiont-based strategies for blocking malaria transmission.

Anopheles mosquitoes are colonized by diverse microorganisms that may impact on host biology and vectorial capacity. Eukaryotic symbionts such as fungi have been isolated from Anopheles, but whether they are stably associated with mosquitoes and transmitted transstadially across mosquito life stages or to subsequent generations remains largely unexplored. Here, we show that a Leptosphaerulina sp. fungus isolated from the midgut of An. gambiae can be stably associated with An. gambiae host and that it imposes low fitness cost when re-introduced through co-feeding. This fungus is transstadially transmitted across An. gambiae developmental stages and to their progeny. It is present in field-caught larvae and adult mosquitoes at moderate levels across geographical regions. We observed that Leptosphaerulina sp. induces a distinctive melanotic phenotype across the developmental stages of mosquito. As a eukaryotic symbiont that is stably associated with An. gambiae Leptosphaerulina sp. can be explored for paratransgenesis.

Environmental factors play a crucial role in the population dynamics of arthropod endosymbionts, and therefore in the deployment of Wolbachia symbionts for the control of dengue arboviruses. The potential of Wolbachia to invade, persist and block virus transmission depends in part on its intracellular density. Several recent studies have highlighted the importance of larval rearing temperature in modulating Wolbachia densities in adults, suggesting that elevated temperatures can severely impact some strains, while having little effect on others. The effect of a replicated tropical heat cycle on Wolbachia density and levels of virus blocking was assessed using Aedes aegypti lines carrying strains w Mel and w AlbB, two Wolbachia strains currently used for dengue control. Impacts on intracellular density, maternal transmission fidelity and dengue inhibition capacity were observed for w Mel. In contrast w AlbB-carrying Ae. aegypti maintained a relatively constant intracellular density at high temperatures and conserved its capacity to inhibit dengue. Following larval heat treatment, w Mel showed a degree of density recovery in aging adults, although this was compromised by elevated air temperatures. When choosing the Wolbachia strain to be used in a dengue control programme it is important to consider the effects of environmental temperatures on invasiveness and virus inhibition. Author summary: In the past decades, dengue incidence has dramatically increased all over the world. An emerging dengue control strategy utilizes Ae. aegypti mosquitoes artificially transinfected with the bacterial symbiont Wolbachia , with the ultimate aim of replacing wild mosquito populations. Wolbachia is transmitted from mother to offspring and is able to interfere with virus transmission within the mosquito vector. However, the rearing temperature of mosquito larvae is known to impact on some Wolbachia strains. In this study, we compared the effects of a temperature cycle mimicking natural breeding sites in tropical climates on two Wolbachia strains, currently used for open field trials. We observed that the strain w Mel was susceptible to high larval rearing temperatures, while the strain w AlbB resulted to be more stable. These results underlines the importance of understanding the impact of environmental factors on released mosquitoes, in order to ensure the most efficient strategy for dengue control.