Mosquito Parasitic Nematode Aggregation Behavior And Their Infection Strategies

Mosquitoes are the most important pests not only because their nuisance biting but also because they transmit more than 80 pathogens shared by human and animals such as arboviruses,malaria and filarial nematodes.According to WHO incompletely collected data,there are 300 to 500 million people infected by malaria every year and about one million people dead among these infected people.Currently,mosquito control approach mainly relies on chemical pesticide spray.However,those synthetic pesticides such as Malathion and Permethrin are not only highly toxic to mammalians but also cause environmental hazards.Particularly,the potential damage to pregnant women and infants are inestimable.Mermithid mosquito parasitic nematodes are natural enemies of mosquito larvae that possess great potential as biological control agents.These nematodes infect lower instars of mosquito larvae in water and kill their hosts right after they emerge from the larvae.These developed adults will aggregate,mate and lay eggs in the mosquito larval habitat.The eggs will hatch into infective pre-parasites under suitable condition and continually attack mosquitoes.The great advantages of these nematodes are that they can be used as inundative release(short term control like using pesticide)as well as inoculative release(long term control).These parasites are very host specific and infect no species other than mosquitoes and therefore extremely safe to human and other animals.Therefore,to study the biological aspects and infection strategies of these parasites will provide further fundamental knowledge to guide us in mass production and field application of these beneficial organisms.Using mosquito parasitic mermithid nematodes Strelkovimermis spiculatus and Romanomermis iyengari as materials,we have studied their aggregation behavior,dialpaused eggs hatching and foraging behavior of their pre-parasites under laboratory conditions.First,we have investigated the mating aggregation behavior in Strelkovimermis spiculatus.Female post-parasites,through their attraction of males and,remarkably,other females,drive the formation of mating clusters.During our three-week sand plate study,post-parasites joined mating clusters increased from 32.10%of week 1 to 76.5%of week 3.Sand columns that contain cluster reduced from week one's 25.00%to week three's 10.4%indicating clusters may grow in size by merging with other individual or clusters.Female molting to the adult stage and reproductive success are enhanced in larger clusters.As the cluster size increased from 2 to 20 nematodes,the post-parasites molting rate increased from 40.4%to 99.1%and the egg production increased from 1145.2 to 4115.6 per female.Our data also showed that both male and female nematodes can mate multiple times.Formation of cluster may also provide the opportunity for mating competition to ensure the quality of their next generation.Our results indicated that although forming mating cluster risks being attacked by natural enemies,the benefits of increasing their fitness must overweighed the risks for the behavior to be evolved.Due to the nematode cluster formation,their eggs were distributed in highly aggregated pattern in their habitat.Therefore,it is important to maximize their infectivity to their available hosts under high competition of their own species.Through laboratory experiments,we investigated the following related topics:1.How diapaused eggs respond to their host density and stages.2.How their infective pre-parasites search their host habitat and host effectively after hatching.There are two situations to the eggs laid by Strelkovimermis spiculatus.The first situation is in the temporary flooded water body such as riverbank.When the water dries up,the eggs become dehydrated in the sand or soil.When the next flood comes,all dehydrated eggs hatch into pre-parasites to look for hosts come with the flood.The second situation is in permanent ponds where nematode eggs become diapaused under normal temperature.We investigated how these diapaused eggs hatch in responding to host availability and population density using diapaused eggs from Strelkovimermis spiculatus and their host Culex pipiens pipiens under laboratory conditions.The results indicated that the presence of the host induced the egg hatching.The hatching rate increased when larger numbers of host larvae were present.Second instar mosquito larvae induced significantly higher hatching rates(69.1%)than any other stages.These findings explain how S.spiculatus synchronizes its life cycle with its host life cycle and population dynamics to increase its fitness when the natural habitat is constantly covered by water.Direct exposure of the nematode eggs to host larvae resulted in consumption of as many as 20 eggs per host.The eggs consumed caused 0-70%host mortality depending on the number consumed,which indicated an infection path through gut penetration although it may represent a rare situation in nature.The result of host cue assays showed that the combination of chemical cues and physical vibration induced the highest egg hatching(56.9%),which may increase the sensitivity of detecting host availability and population density.These findings suggest that eggs hatch synchronously with the most susceptible mosquito stage and with peak mosquito larval density.To understand the infection mechanisms of the mosquito parasitic mermithid nematodes Romanomermis iyengari and Strelkovimermis spiculatus,we have investigated the foraging strategy of the pre-parasites of both species.Romanomermis iyengari with body length of 0.99 mm(twice as S.spiculatus 0.55 mm)swims faster(2.76cm/min)than S.spiculatus(1.61cm/min).The nematode pre-parasites of R.iyengari negatively responded to natural sunlight toward shade area,where mosquito larvae tend to aggregate.This behavior helped the nematode to gain significantly higher parasitism when shade was present(37.7%)than no shade present(11.3%).In contrast,S.spiculatus pre-parasites had no response to host aggregation habitat(shade area)and therefore,shade contributed no parasitism gain to this species.The host searching pattern data indicated that the two species used different strategy for hot searching.When no host present,R.iyengari tended to do long range search and responded to host aggregation habitat but slowed down and do more localized search when host present.However,S.spiculatus tended to do localized movement when hosts were absent regardless the shade area.Once host larvae present in nearby location,the nematodes responded to the host cue and moved faster toward their hosts.Study also revealed that host chemical cues are responsible for the nematodes to respond for host finding but not the vibration generated by host movement like the case in egg hatch.Our data indicated that R.iyengari was more suitable against mosquito species in larger water environment like rice field.On the other hand,S.spiculatus was more suitable against mosquito species in small pond like Culex pipiens.In conclusion,benefits overweighed the risks for forming clusters in mosquito parasitic mermithid nematode adults.The nematodes have evolved strategies to maximize the infection against their hosts,which includes response to host density and host stage for egg hatch and to use host habitat and host chemical sues for host location.Our finding will provide further understanding of the nematode biology,behavior and relationship with their hosts and therefore better guide the nematode massive production and mosquito management practices.