Effetti dell’insetticida diofenolan su Musca domestica Linnaeus, 1758

Musca domestica Linnaeus, 1758, a relevant synanthropous pest, is the most frequent dipteran species in animal farms and waste landfills. Fly infestations cause heavy economic losses and often lawsuits against cattle or poultry farms near residential areas. For control of pest arthropods, including M. domestica, since 1940 neurotoxic adulticides with very low selectivity and high environmental impact were employed regardless of their toxicity to vertebrates, bioaccumulation and development of resistance. A more recent insecticide class, the Insect Growth Regulators (IGR), has species-specific toxicity and low health and environmental impact. The IGR activity is mostly exerted on larval stages but some of them also induce sterility in adults. The aim of this doctoral thesis is to investigate on a sensitive strain of M. domestica the effects of diofenolan, an IGR analogue of juvenile hormone, unregistered for control of this species in Italy. The toxicity of diofenolan on immature stages was evaluated by topical treatments on 3rd instar larvae at different concentrations, verifying the main toxicological parameters and monitoring morphological effects of the active ingredient by optical and scanning electron (SEM) microscopy. The effects of diofenolan were also investigated on female reproductive ability (egg laying and hatching) by suitable tests along the gonotrophic cycles, monitoring ovarian and ovariole alterations by optical and transmission electron (TEM) microscopy. The interactions between diofenolan and ecdysteroids, produced by ovariole follicle cells in adult females and involved in the ovarian cycle, were also investigated by enzymatic immunoassay (EIA). The interactions between diofenolan and the activity of corpora allata, endocrine gland producing juvenile hormone required for ovarian development, were also tested by morphological and ultrastructural investigations. The results showed a dose-dependent toxicity of diofenolan against M. domestica, mainly affecting pupae by inducing larviform puparia and apparently normal ones with altered respiratory plates, together with puparium-trapped adults. Tests on reproductive ability according to the gonotrophic cycle showed that treated females were able to lay eggs only in the first cycle and that hatching ability reduced to 24% along the gonotrophic cycles. In the second gonotrophic cycle the reproductive ability was 1/6 of the initial one and in the third cycle lowered to 1/16 of the initial one. The results show that diofenolan is very efficient in affecting the reproductive ability (egg laying and hatching) in M. domestica. The morphological investigations on ovarioles at different time intervals since emerging from puparium showed nine morphotypes which could be arranged into three “transformation paths”: a “precocious degenerative” path involving the primary follicle and associated to secondary follicle hypertrophy, an “intermediate degenerative” path involving the secondary follicle, and a “degenerative/hypertrophic” path in which the secondary follicle may degenerate or increase its size by hypertrophy. The most frequent path is the “intermediate degenerative” one and these data agree with the previous ones obtained on M. domestica reproductive ability. The ecdysteroid content measured by EIA in M. domestica females treated at different time intervals shows a modified profile in comparison to controls, closely related to the degenerative events detected in ovariole follicles. In adult insects corpora allata are known to exhibit periodical activation/inactivation phases related to the ovarian cycle. The effects of diofenolan on corpora allata, investigated and confirmed for the first time at the ultrastructural level in M. domestica adults, together with original data on cell number and volume of the gland, show in treated females widespread alterations of nucleus, cytoplasm and mitochondria which strongly suggest that the gland undergoes a rapid transition from an inactive stage to a degenerative one. This study also shows the presence within the gland of light and dark cells, never previously identified in morphological and ultrastructural investigations of corpora allata in M. domestica. In conclusion, the main effects of the juvenile hormone analogue diofenolan in this species include inhibition of emerging from puparium and abatement of the reproductive ability in terms of egg laying and hatching, probably related to anomalous ecdysteroid profiles and marked alterations in corpora allata. Further investigations may involve the effects of diofenolan on other stages of the biological cycle and on male reproductive ability. The biocide and sterilizing effects of diofenolan in M. domestica are very interesting for integrated pest management programs since the multiple activity of this active ingredient may significantly reduce the need for adulticides with higher environmental and health impact. It is therefore recommended to extend studies on this IGR in M. domestica by appropriate field trials.