Background

Mosquitoes are insects with high medical importance. They not only disturb people, but also transmit serious diseases, such as malaria, dengue, encephalitis B, etc. In recent years, dengue epidemics in provinces Guangdong, Yunnan, Guangxi, and Taiwan of China, make mosquito control a very urgent battle (Meng et al., 2015). But at present, the weapon used against mosquito is mainly chemical insecticides, which caused environmental polution, and ecosystem disturbance, and in the same time, induced resistance of mosquitoes to chemicals (Zhao et al., 2014; Meng et al., 2015; Cai et al., 2015). Mosquito biological control is a kind of methods that can protect environment and ecosystem. But, at present, mosquito biological control is not popular yet. One of the main problems is the lack of ideal agent for use, and now only Bacillus thuringiensis var. israelensis (Bti), and Bacillus sphaericus (Bs) are practially in application. But they have different shortages. For example, the effective duration of Bti is only about 7-14 days, which is too short, and the ability of Bs to kill mosquitoes is relatively weak (private communication). In comparison, mosquito-killing fungus, Pythium guiyangense (Pg) can keep controlling mosquito larvae for an obviously longer time, and it has advantages such as fast reproduction, simpleness of production, wide spectrum of mosquito host (3 gena, 14 species), and safety to non-target organisms etc. (Wang, 2008; Su, 2010). But, it also has shortages shuch as unstable virulence. Rejuvenation is an useful method to overcome it, that isolating mycelia with higher infectivity from mosquito larvae and infecting mosquito larvae again. Through the repeating isolating-infection process, fungal ability to infect mosquito larvae can be rebuild up. Here, we report our work to rejuvenate the fungus during July 22^th, through December 12^th in 2013.

1 Materials and Methods

1.1 Materials

Pg used in the experiment was isolated from garden soil of Guizhou Medical University (formerly Guiyang Medical College), and kept in our lab. The media used to culture fungus were SFE and KPYG2, and that for isolating fungus from larval corpse was water agar medium (1 000 ml water add 15 g agar). The test targets were Culex quinquefaciatus larvae from insectarium with temperature of 25℃±1℃, light cycle L:D=14:10, and RH 70%.

1.2 Methods

1.2.1 Bioassay of Pg

Pg mycelium was planted on fresh SFE agar plates, and cultured for 5-7 days in 25℃. In each desinfected plastic container were added with 200 ml of tap water, 25 larvae and 8 drops of 4% chicken liver powder suspension. In each cup of experimental groups, 3 pecies (each with area about 113 mm²) of agar with fungal mycelia were added, while in control groups 3 pecies of blank agar were added. Observations were made twice a day, dead larvae were picked up and checked with microscope for fungal infection, mortality and infection rates were calculated. The mortality or infection rate=number of dead larvae or infected larvae/number of tested larvae × 100%.

1.2.2 Isolation of Pg

Infected larva was washed 3 times with double desinfected water after immersed in 75% alcohole for 10 s, soaked away residual water and then planted onto water plates, SFE plates, or KPYG2 plates according to the need of experiments. Plates were cultured in 25℃ for 5-7 days before use.

1.2.3 Rejuvenation of the fungus

Newly isolated fungus was used to infect larvae, and then reisolated from infected larvae (dead or alive) with solid body and exuberant fungal mycelia on it in test cups that had higher infection rates.

1.2.4 Observation indexes

The main index was the level of the highest group average infectivity (GHI) among the test groups in each experiment, and the supplementary indexes were the highest and lowest infectivity (CHI and CLI) in individual cups, and the number of alive infected larvae (NAL).

2 Results

2.1 The GHI

The virulence of Pg to mosquitoes dropped significantly by subculturing on artificial media for long time, and the mosquito infection rates were commonly around 10%. After rejuvenation, the GHI increased obviously (Figure 1). In the beginning of rejuvenation, the GHI was low. Along with the progress of rejuvenation, the GHI grew up markedly, and exceeded 40%. 80% was one of the peaks which was achieved in about 2 monthes. There was a low ebb between the 84^th and 119^th days, because the media used for fungus were not fresh. When the problem was resolved, the GHI tended to higher and stabler.

2.2 The CHI

CHI was another index to show the infectivity of the fungus, which was significantly higher after rejuvenation than before (Figure 2), even reached the peak of 100% (in other data).

2.3 The CLI

Before rejuvenation, there was always 0% infection rate in some test cups. Along with the increase of Pg virulence, 0% infection rate reduced, and the CLI rised (Figure 3), even reached 80% (in other data).

2.4 NAL

In the beginning of rejuvenation, generally only dead larvae were found with fungal mycelia covering the body. Along with the process of rejuvenaion, more and more alive infected larvae were found (Figure 4).

3 Discussion

Degeneration is a common problem in culture and production of microbals, which appears in different patterns. The degeneration of Pg mainly manifests as decrease of virulence to mosquito. Acording to researchers, the reasons for the degeneration may be diversified, which involves physiological and biochemistry mechanisms, as well as disturbence of virus and effects of cultural conditions. Among which, the influences from heredity might be the most important part, or maybe, heredity causes the degeneration through other factors mentioned above (Tang et al., 1996; Liu et al., 2001).

The genetic factors that causes the degeneration including gene mutation, gene recombination, chromosome mutation, genetic equilibrium changes etc. For Pg, a species of multicellular fungus, the explain of degeneration might be the theory of heterocaryon (Tang et al., 1996). In the process of fungal growing and reproduction, different nuclei with different gene combination are easily formed by repeated miosis and mitosis. These nuclei may have different expression characteristics because of the different gene combination. Changes of the ratio of various types of nuclei lead to a big variation range of fungal virulence to mosquitoes. When fungus be cultured on artificial media for a long time, its nuclei that express genes fitting to saprophytic life become prepotent, which cause the dramatical decrease of the virulence. In the process of continuous rejuvenation passing its hosts, strains with higher virulence were selected, by which, the ratio of nuclei that express genes fitting to parasitic life increases, and thus the fungal virulence be enhanced. As another proof for the virulence increase is the number of alive infected larvae getting bigger. When the fungal virulence is low, the infected larvae are found dead and covered by mycelia. It could be speculated that the invasion and infection of the fungus into the larval body was slow, and the larval death and fungal growth are generally not obvious until they die. After rejuvenation, the process of invasion and infection become faster, and the fungal mycelia appears on larval body even before the death of larvae. Pg strains reisolated from alive infected larvae have higher virulence than those from dead ones, of which, the reason might be that they can express more genes fitting parasitic life.

One of the bottle necks for the application of Pg in real battle against mosquitoes is unstability of its virulence, and to raise and stabilize its virulence is a key to its utilization. Although genetic engineering and protoplast breeding are very powerful tools for modifying strains of microbial, traditional rejuvenation passing host is more convenient, economic, and simple, thus is more practicable. Our results showed that, after rejuvenation, all of the Pg average infection rates in the highest group, and the highest and lowest individual cup infection rates raised to 40% and above, and, even, the highest individual cup infection rate once reached up to 100%, which means we have successfully made the virulence of once degenerated Pg strain recovered. Although there was a setback in the process because of the degeneration of media material, the virulence increased again when the problem was resolved, which proved again culturing condition, especially culture media influenced the physiological status strongly. These findings provide very useful reference for industry practice. Rejuvenation passing hosts is an effective method to prevent Pg strain from degeneration caused by repeating subculture in artificial media, which is a very important part of the Pg manufacture business.

In order to overcome the defect of Pg, one new formula has been designed, that Pg mycelia combine with Bti to work together. When apply in mosquito breeding water, Bti kills larvae quickly, and fade away in short time, thus Pg grow up in water environment consuming organic material and larval corpses. The stronger Pg take the job to control mosquito larvae for a relative longer period which depends on conditions. The longest time of Pg to control mosquito larvae observed was 273 days (Yang et al., 2016).

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