Transinfection of buffalo flies (Haematobia exigua) with Wolbachia and effect on host biology

A widespread insect endosymbiont Wolbachia is currently of much interest for use in novel strategies for the control of insect pests and blocking transmission of insect-vectored diseases. Wolbachia-induced effects can vary from beneficial to detrimental depending on host biology and the genetic background of the infecting strains. As a first step towards investigating the potential of Wolbachia for use in the biocontrol of buffalo flies (BF), embryos, pupae, and adult female BF were injected with three different Wolbachia strains (wAlbB, wMel and wMelPop). BF eggs were not easily injected because of their tough outer chorion and embryos were frequently damaged resulting in less than 1% hatch rate of microinjected eggs. No Wolbachia infection was recorded in flies successfully reared from injected eggs. Adult and pupal injection gave a much higher survival rate and resulted in somatic infection and germinal tissue infection in surviving flies with transmission to the succeeding generations on a number of occasions. Investigations of infection dynamics in flies from injected pupae confirmed that Wolbachia were increasing in numbers in BF somatic tissues and ovarian infections were confirmed with wMel and wMelPop in some instances, though not with wAlbB. Measurement of fitness traits indicated reduced longevity, decreased and delayed adult emergence, and reduced fecundity in Wolbachia-infected flies in comparison to mock-injected flies. Furthermore, fitness effects varied according to the Wolbachia strain injected with most marked reductions seen in the wMelPop-injected flies and least severe effects seen with the wAlbB strain.

50 Wolbachia, are maternally inherited endosymbionts of insects, that are of much interest for use in the 51 biological control of pests, most particularly as a basis for area-wide integrated control strategies for a 52 range of insect species [9][10][11]. Wolbachia has been used in insect control programs in two main ways.

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First, it has been used as a means to achieve population replacement, where Wolbachia-infected 54 insects impart unique characteristics such as pathogen blocking or fitness deficits, and second, by the 55 incompatible insect technique (IIT) in which Wolbachia-infected males released into the population 56 cause the production of non-viable eggs, similar to the sterile male technique [11][12][13][14]. Both of these 57 strategies are based on cytoplasmic incompatibility (CI) and the resultant ability of Wolbachia to spread 58 though uninfected or differentially infected populations [14]. Some of the novel fitness costs induced by 59 Wolbachia include decreased fecundity and male competitiveness, seen in Anopheles stephensi 60 infected with wAlbB, lifespan reduction, egg mortality, delayed larval development and altered feeding 61 behaviour seen in Aedes aegypti infected with wMelPop [15][16][17][18][19][20]

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The first step towards developing Wolbachia based control programs is the establishment of Wolbachia 71 transinfected lines of the target pest. The most common method used to transinfect new hosts with 72 Wolbachia has been embryonic microinjection, although injection into other stages, such as adults and 73 pupae have also given some success [14]. Of the available transinfection procedures, embryonic 74 microinjection is mostly preferred as Wolbachia are directly introduced to the pole cells of pre-75 blastoderm embryos using a fine needle inserted at the posterior end of the egg, desirably resulting in 76 germline and somatic cell infection. In contrast, adult injection is usually carried out into the thoracic or 77 abdominal regions of adults where Wolbachia must successfully evade or overcome a number of 78 membrane barriers and the host immune response to become established in the germinal tissues for 79 next-generation transmission [14].

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Nevertheless, using cell lines of the intended host artificially infected with Wolbachia as the donor 89 source has been suggested as advantageous for obtaining a high density and host context adapted      wMelPop were found positive with infection percentages of 5%, 22%, and 10% respectively, suggesting 260 transmission via the germline tissues in these instances (see Table 2).

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Effect of Wolbachia on egg production 303 Difference between infected females and non-infected females in egg production was also analysed 304 following pupal injection with the three different strains of Wolbachia. Over 14 days there was a 305 significant reduction in the total eggs laid by females infected with wAlbB (p=0.012), wMel (p=0.0052), 306 and wMelPop (p=0.0051) in comparison with the mock-injected flies (Fig. 9). 307 308

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Embryonic microinjection is by far the most frequently used technique to develop Wolbachia-310 transinfected insect lines, mainly because Wolbachia injected into the germ cells of the developing 311 embryo provides a direct route for infection of the germ tissues in the early stage of differentiation [14].

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However, this technique is also the most challenging step because the invasive procedure of egg 313 microinjection can result in high mortality of eggs and optimal methods differ for different insect species 314 [14,45,46]. Another disadvantage of this technique is that inability to determine the sex of an embryo 315 prior to injection means that approximately half of the injected flies will be males that do not transmit 316 Wolbachia to the next generation [14]. This means that many thousands of eggs must often be 317 microinjected using specialised equipment before successful Wolbachia transinfection is achieved [14] 318 and as male embryos cannot be identified, half of this effort is functionally wasted. With BF, less than

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Although embryonic microinjection has been the primary method used to develop transinfected insects, 328 adult microinjection can be advantageous in that females can be selected for injection [14]. Further, 329 adult microinjection can be performed using a simple syringe and small-bore needles delivering higher 330 volumes of Wolbachia to overcome the host immunological response [14].