Horizontal Transmission of the Symbiont Microsporidia MB in Anopheles arabiensis

Abstract

The recently discovered Anopheles symbiont, Microsporidia MB, has a strong malaria transmission-blocking phenotype in Anopheles arabiensis, the predominant Anopheles gambiae species complex member in many active transmission areas in eastern Africa. The ability of Microsporidia MB to block Plasmodium transmission together with vertical transmission and avirulence makes it a candidate for the development of a symbiont-based malaria transmission blocking strategy. We investigate the characteristics and efficiencies of Microsporidia MB transmission between An. arabiensis mosquitoes. We show that Microsporidia MB is not transmitted between larvae but is effectively transmitted horizontally between adult mosquitoes. Notably, Microsporidia MB was only found to be transmitted between male and female An. arabiensis, suggesting sexual horizontal transmission. In addition, Microsporidia MB cells were observed infecting the An. arabiensis ejaculatory duct. Female An. arabiensis that acquire Microsporidia MB horizontally are able to transmit the symbiont vertically to their offspring. We also investigate the possibility that Microsporidia MB can infect alternate hosts that live in the same habitats as their An. arabiensis hosts, but find no other non-anopheline hosts. Notably, Microsporidia MB infections were found in another primary malaria African vector, Anopheles funestus s.s. The finding that Microsporidia MB can be transmitted horizontally is relevant for the development of dissemination strategies to control malaria that are based on the targeted release of Microsporidia MB infected Anopheles mosquitoes.

Keywords: Anopheles; Microsporidia; malaria; symbiosis; vector.

FIGURE 1

Horizontal transmission of Microsporidia MB. Mosquitoes carrying Microsporidia MB are represented with blue shading in pie charts and n = number of independent experiments. (A) No transmission of Microsporidia MB was observed between An. arabiensis larvae reared in the same larval trough but separated by a screen mesh. (B) Horizontal transmission of Microsporidia MB was observed when adults were kept together in cages, and specifically when either infected males or females were housed with uninfected An. arabiensis of the opposite sex. Top row, no transmission was observed between infected and uninfected individuals of the same sex. Bottom left, transmission between Microsporidia MB infected An. arabiensis females and uninfected males was observed in 5 out of 15 cages (33%). Bottom right, out of a total of 16 experiments that had Microsporidia MB infected males and uninfected females and horizontal transmission was confirmed in 10 of these cages (56% transmission).

FIGURE 2

Distribution of Microsporidia MB across male An. arabiensis organs. (A) The screening dissected organs from 22 male An. arabiensis specimens, reveals that Microsporidia MB is detected primarily in the midguts and male gonads. (B) The intensity of Microsporidia MB infection is highest in the midgut and male gonads. (C) The screening male An. arabiensis seminal fluid revealed that Microsporidia MB was detected in 4/10 specimens. (D) The intensity of Microsporidia MB infection in An. arabiensis seminal fluid ranges from a ratio of 0.87 to 41.8 MB18S/S7. Error bars reflect SEM.

FIGURE 3

Fluorescence microscopy of Microsporidia MB in An. arabiensis male ejaculatory ducts. (A) Schematic diagram of the male Anopheles gonad shows the position of the ejaculatory duct in relation to seminal vesicle, male accessory gland and testes. (B) Fluorescence microscopy images indicate that Microsporidia MB meronts (a) are found in the male An. arabiensis ejaculatory duct. Multinucleate Microsporidia MB cells can be observed containing 4 and 8 distinct nuclei (b,c), which likely corresponds to the progression on of 4-nuclei sporogonial plasmodia into the 8-nuclei sporogonial plasmodia and ultimately into sporophorous vesicles. Scale bar = 50 μm.

FIGURE 4

Vertical transmission of horizontally acquired Microsporidia MB infections in An. arabiensis. Vertical transmission of Microsporidia MB is observed in recipient females that have become infected with Microsporidia MB after being kept with Microsporidia MB infected donor males in the first gonotrophic cycle after horizontal acquisition. Red numbers indicate Microsporidia MB infection intensity in individual An. arabiensis adults as determined by qPCR.