IT202000004381A1 - Artificial diet for the breeding of Diptera larvae of forensic interest. - Google Patents

Description

Artificial diet for the breeding of Diptera larvae of forensic interest

Artificial diet for rearing Diptera larvae of forensic interest

Technical field

The present invention refers to an artificial diet for the breeding of diptera larvae of forensic interest.

Known art

Forensic entomology? a branch of entomology that studies the life cycles of those insects which, developing on decaying human remains, can be used for determining the dating and causes of death.

The insects that play an important role in this area are mainly Diptera and Coleoptera.

The families of insects belonging to the order of Diptera that have a forensic importance are very numerous and different from each other, so how diverse are their ecological characteristics. They complete their life cycles on perishable, dirty, unhygienic and foul-smelling natural diets, which hinder the breeding work in an internal laboratory environment. To overcome this unsanitary condition,? felt the need? to have a simple, hygienic and non-smelly artificial diet available for the breeding of these insects in a controlled environment.

The use of artificial diets is necessary for the development of all those studies that require large populations of insects (eg breeding of sterile males, breeding of parasitoids and predators in which the need to provide live prey has been eliminated). Artificial diets allow insects to be raised year round regardless of natural food sources which may be seasonal or not available in quantity? sufficient.

The speed? development of insect larvae? mainly determined by environmental conditions, mainly by temperature and secondarily by humidity? and photoperiod. Equally important, on a par? environmental conditions, appears to be the substrate on which the larvae develop, which can? significantly alter the development times and the possibility? of the larvae to continue the life cycle and complete the metamorphosis, transforming into adult forms.

In the field of forensic entomology? the estimation of the long-term post-mortem interval (PMI) is important. A reliable estimate of the PMI requires a proven assessment of insect development rates under controlled laboratory conditions. Breeding conditions can have a considerable influence on the growth and development of larvae (Niederegger S. 2010. Forensic Science International 199 (1? 3): 72? 78). If these influences are not adequately considered, there? can lead to a miscalculation of the PMI.

Most studies on larval growth rates do? focused on factors such as temperature (Grassberger M, 2002. Forensic Entomology International 128: 177? 182 / Ames C.2003. Medical Veterinary Entomology 17: 178? 186 / Boatright SA. 2010. Journal of Medical Entomology 47 (5): 917 ? 923), larval aggregation and crowding or drug effects (Introna F. 2001 Forensic Science International 120: 42? 47 / Gunn J.A. 2006. Journal of Analytical Toxicology 30 (8): 519? 523). A noticeable variation of food substrates? been used in these studies, such as eg. mammalian muscle, ground beef (Gunn et al 2006), mouse / rat carcasses (Putman R.J. 1977. Journal of Animal Ecology 46: 853? 866 / Tarone A.M. 2006. Journal of Medical Entomology 43 (5): 1023? 1033) , fish (D'Almeida J.M. 1996. Brazil. Mem I Oswaldo Cruz 91 (1): 131? 136), or even artificial diets containing components such as whole milk powder, baking powder and wheat germ (Calabres E.J., 1974. Jounal of insect physiology 20 (2): 383? 393) but these prove to be efficient for the breeding of only some dipterans of forensic interest and therefore not very functional to a possible standardization of breeding in the laboratory

However, for practical reasons? and Low Cost Many forensic entomologists continue to use animal organs such as the liver of sheep, cattle, or pigs to determine development rates. Natural diets, however, have a series of disadvantages that cause severe limitations to breeding in the laboratory such as easy perishability, non-hygiene. and the generation of bad smells (Sherman R.A. 1995. Medicine. Veterinary Entomology. 9: 393-398.).

We must also not forget that the type of organ used can? influence the rate of larval development. For example, nearby Calliphora larvae grow longer. quickly if raised on heart, lung, brain and kidney compared to porcine liver (Kaneshrajah G.2004. Internation Journal of Legal Medicine 118 (4): 242? 244), while the larvae of Lucilia sericata grow with equal speed? on different organs of porcine origin (Clark K.

2006. Forensic Science International 156: 145? 149).

This natural variability? in the development times of the Calliphoridae larvae of forensic interest involves difficulties? objective with respect to the standardization of methods and procedures at an international level and often exposes entomological dating to procedural criticisms in the trial stage.

From what was written previously, you can? note how numerous studies on arthropods of forensic interest have been performed all over the world, in Italy these studies are very limited and scarce, perhaps due to the lack of standardization of laboratory diets, feeding protocols, and breeding of insects, which makes research on behavioral, medical and forensic entomology problematic.

The development of a standardized artificial diet for the larvae of the main species of diptera that make up the cadaverous entomofauna would therefore meet the need? to overcome the limitations previously described, offering a substrate that allows the breeding of the larvae collected at the crime scene in an internationally standardized way, allowing to fill the existing gap. Furthermore, the use of an artificial diet would improve the conditions of conservation. and transport of the sampled larvae, which are otherwise now placed on a fresh animal organ, usually bovine liver, with obvious difficulties. maintenance in conditions of fitness? of the substrate.

The production of a standard substrate, reproducible everywhere and produced in a uniform way is the possibility? to subject the food substrate to rigorous quality controls? during production, it would allow to obtain repeatable results within the single laboratory and reproducible also between laboratories located anywhere in the world, allowing to provide the basis for accreditation or in any case the recognition by certification bodies, of the procedures used and of the achieved results.

A composition suitable as an artificial diet for a Diptera of forensic interest such as Lucilia sericata? described in Tachibana, 2001 (Tachibana S. 2001.Applied Entomology and Zoology 36: 521-523). The diet ? composed of wheat germ, milk powder, yeast and agar.

This diet causes larval growth retardation of more than 2 days compared to liver-reared larvae. For this reason, although apparently very similar to ours, this formulation does not respond to what? one of the main objectives, that is, the production of a standard larval substrate, capable of making homogeneous the performance in terms of development times, of the larvae reared for more than one year. forensic laboratories and therefore guarantee repeatability? of the entomological test.

Some diets tested for diptera of forensic interest (eg Rikta Khatun 2018. Asian Australas. Journal Bioscience and Biotechnology. 2018, 3 (2), 86-92) involve the use of powdered milk in association with egg yolk . The diet ? been tested in our laboratories with unsatisfactory results. The farmed insects have shown not only a lengthening of development times but a high percentage of mortality. (? 80%). Such a result? due to the nutritional characteristics of the formulation. In particular, the excessive presence of lipids and the absence of essential amino acids (eg tryptophan) prevent the activation of a series of mechanisms necessary for the production of hormones involved in the growth and development of insects.

Document CN 101978823A describes an artificial diet for diptera belonging to the Hermetia illucens species (family: Stratiomyidae) containing soy flour, peanuts, wheat bran and water. The diet described? suitable for the breeding of this species but can not? be adapted to other insects of forensic interest due to the absence of proteins of animal origin, necessary to guarantee an adequate growth rate.

Summary of the invention

AND? an artificial diet which overcomes the drawbacks of the known art has now been developed and constitutes the object of the present invention.

The artificial diet of the invention does not show significant differences in terms of development duration, palatability. and sustainability? cheaper than traditional natural diets such as eg. muscles, heart, liver or minced meat or fish and has the undeniable merit of standardization.

Taken together, the tests reported below have shown that the artificial diet of the invention facilitates the breeding of insects of interest in the field of forensic entomology not only from a hygienic point of view, but also in terms of cost ratio. effectiveness.

The composition of the artificial diet of the invention includes:

- Soybean flour, characterized by a protein content of 35-40g / 100 and carbohydrates of 15-20g / 100 (50-55% of the total dry weight);

- Casein, particularly suitable? the casein from bovine milk (7-10% of the total dry weight) which supplements that already? present in powdered milk as it contains a series of essential amino acids for the development of insects such as: tryptophan, methionine, leucine, isoleucine and lysine;

- Yeast (Saccharomyces cerevisiae) in water soluble powder (10-15% of the total dry weight);

- Powdered milk for babies, particularly suitable? powdered milk? Nipiol 1? which guarantees the part in fats 20-30g (26.7g / 100) both saturated and unsaturated, as well as? an adequate supply of linoleic acid 4000-6000mg / 100gr. This product ? source of many vitamins including vitamin C 100-110 (104mg / 100g) and thiamine 0.7-1 (0.9mg / 100g) which promotes digestibility? (10-15% of the total dry weight);

- 10gr agar (8-10% by weight).

Another object of the invention? the process of making the diet to feed the dipterans.

Still another object? a product in the form of agar gel, portionable and comprising: 65-70% of water, 17-18% of soy flour, 2.5-3% of casein, 3-5% of yeast, 3-5% of milk powder and 2.5-3% of agar.

Further objects and advantages will become apparent from the detailed description of the invention.

Detailed description

The present invention aims to provide a nutritional substrate for the breeding of insects of interest in the field of forensic entomology, specifically diptera.

Such a nutrient substrate? suitable for larvae belonging not only to different species but also to different families, such as:

Hermetiaillucens: Family Stratiomidae

Calliphora Vicino and Lucilia sericata: Family Calliphoridae

Musca domestica: Family Muscidae

Sarcophaga argyrostoma: Sarcophagidae family

The composition (in terms of dryness, without the addition of water) of the artificial diet includes:

- Soybean meal, particularly suitable? soy flour? Molino lipstick? produced by, characterized by a protein content equal to 35-40g / 100g and carbohydrates equal to 15-20g / 100g (50-60% total by weight);

- Casein, particularly suitable? casein from bovine milk? Sigma ?.

Manufactured from Other caseins can be used (5-10% by weight);

- Water soluble baking powder? Biolife? produced from an excellent source of vitamins of group B (2-3%) / (10-15% by weight);

- Powdered milk, with a casein content of 10-11% / 100g, particularly suitable? powdered milk? Nipiol 1? which also guarantees the part in fats (25-28g / 100) both saturated and unsaturated (mono- and poly-unsaturated) as well as? an adequate supply of linoleic acid (800-900mg / 100g), which is important in regulating insect stage changes. This product ? source of many vitamins including vitamin C (100-110mg / 100g) which favors the development of wings in adult insects and thiamine (0.8-1mg / 100g) which favors their digestibility. (10-15% by weight);

- Agar, (8-10g / 100g) particularly suitable? la highly purified biolife gelidium seaweeds (Rodophiceae)? biolife? produced by

Other types of agar can be used

All components are readily available on the market and at low cost.

In total, the artificial diet produced by us has a protein part of about 30-40g / 100g (60% of vegetable origin and 40% of animal origin) a quantity of carbohydrates of about 3-5g / 100g and fats equal to 10-15g / 100g (20% of animal origin and 80% of plant origin)

The diet ? prepared following the following main steps:

Step 1: Dissolve the agar in water at a temperature of 90-100 ° C until the solution is clarified. AND? Shaking is preferred to prevent the agar powder from settling on the bottom. Typical concentrations at which agar is used are between 0.5% and 2% with respect to water.

Step 2: Dry mix the components of the diet consisting of: soy flour, casein, yeast, milk powder, with the following ratios: 17-18% soy flour, 2.5-3% casein, 3-5% yeast, 3-5% milk powder. Step 3: Add the agar solution to the mixture of the components of the diet at a temperature of about 45-50 ° C and keep the pH in the range 6.8 - 7.5;

Step 4: Stir for 1-2 minutes and quickly pour onto a horizontal stand, possibly into 90mm Petri dishes

Step 5: Spray on the surface of the agar mixture like this obtained a fungicide (optional step)

Step 6: Allow to solidify and keep cool, preferably at 4 ° C.

The pH of the final composition? between 6.8 and 7.5, this range? optimal for larval growth since it positively influences the palatability? and the activation of enzymes involved in digestion.

The support cos? obtained ? an artifact consisting of an agar matrix, portionable to be used in the quantities? pi? suitable according to the number of diptera to be fed. Said agar matrix includes the following components: 65-70% of water, 17-18% of soy flour, 2.5-3% of casein, 3-5% of yeast, 3-5% of milk powder and 2.5-3 % of agar.

The diet is suitable for breeding in the laboratory of Diptera of forensic interest, therefore mainly necrophagous and scavenger species that feed in their larval stage on substrates, of animal or vegetable origin, in an advanced state of decomposition. This can? be used both in the typical farms set up with the aim of dating the ages? larval specimens sampled during the inspection and both to set up mass farms for research purposes. In fact, the elements that compose it are particularly balanced so as to guarantee not only the optimal growth up to the adult stage, avoiding anomalies in the physiology of the insect (eg alterations of the growth hormone) but also a good capacity? mating and egg production.

The diet of the invention? preferably suitable for the breeding of diptera larvae of forensic interest and therefore for insects that feed on decaying organic material of animal origin.

The diet ? been tested to verify its suitability for the species Calliphora Vicino and Lucilia sericata (Family: Calliphoridae) and for other Diptera belonging to different families such as Musca domestica (Family: Muscidae), Hermetia illucens (Family: Stratiomyidae) and Sarcophaga argyrostoma (Family: Sarcophagidae) For all species the data obtained confirm that the larval, pupal and flicker development times do not differ significantly with respect to rearing on bovine liver, while the Adequacy Index for larvae developed on an artificial diet? been more? high compared to liver-reared larvae.

The process foresees that in Step 1 a first homogeneous mixture is prepared, subjected to high temperatures (about 100? C) for 25-30?, Of water (200ml) and agar (10gr), then added to the solution to the various components of the diet in Step 3.

In Step 2 the diet component is prepared by mixing the individual components dry: from 50-55% soy flour, 5-10% casein, 10-15% baking powder 10-15% milk powder, as indicated in table 1. A fungicide can? be distributed on the surface of the diet so? prepared in order to inhibit the formation of mold. The diet ? stored in the refrigerator (? 4? C) until use.

The studies described in the Examples part were performed in parallel between two laboratories, IZSLER in Reggio Emilia and Rutgers University (USA), demonstrating the robustness and reproducibility? of the formulation.

The natural diets currently used for diptera generate bad smells and unsanitary conditions that do not adapt well to laboratory environments, the artificial diet formulated by us therefore proposes to be more? hygienic, economical and nutritious for the breeding of diptera of forensic interest and above all? a standardized diet which? suitable for all Diptera thanks to the presence of essential amino acids (tryptophan, methionine, leucine, isoleucine and lysine) in quantity? high and well balanced, such as to make the substrate particularly digestible and easily absorbed by the larvae of many diptera of forensic interest.

The cost of starting materials for the artificial diet? very low, given that it is based on ingredients available on the market all year round, with a very low procurement cost. The processes for production and storage are simple and fast and this has a favorable impact on the cost / benefit ratio linked to the operator.

The following examples are to be considered exemplary and not limitative of the scope of the invention.

EXAMPLE 1

The breeding tests of Calliphora vicinal larvae (Diptera: Calliphoridae) on artificial diet object of the present invention are illustrated below. The larvae of C. nearby are particularly important in forensic entomology because? manage to identify and colonize a carcass already? in the first hours after death thanks to their typically necrophagous larvae (which feed on animal carcasses). The preparation of the diet involved mixing the following ingredients:

- 50g of soy flour with a protein part equal to 38g per 100g of product and carbohydrates equal to 16g per 100g of product

-10g of Casein from bovine milk

- 15g of water soluble yeast extract

- 15g of powdered milk for babies (Nipiol 1)

- 10g of Agar highly purified biolife gelidium seaweeds (Rodophiceae)

- 200ml of distilled water

- 1-1.5ml of pure propiconazole-based fungicide

The diet ? been prepared following the following steps:

Step 1: Dissolve 10g of agar in 200ml of distilled water

Step 2: Mix the other ingredients in a separate Becker

Step 3: Add the agar solution to the other ingredients

Step 4: Mix and pour immediately into 90mm Petri dishes Step 5: Bring under a chemical hood and spray the fungicide on the surface of the diet

Step 6: Leave to solidify, uncovered, for about 10 minutes Step 7: Store at 4 ° C

The development of larval, pupal and adult stages of C. neighbors raised with the artificial diet we have devised? was compared with those raised with bovine liver and pork muscle. The adults used in the experimentation derive from field sampling during the summer period, using attractive traps triggered with pieces of pork muscle. In the laboratory, the adults were kept in special cages (dimensions 30x30x30cm) at a constant temperature of 25? C (? 1? C), Humidity? Relative of 70% (? 5%) and a photoperiod with a light-to-dark ratio of 16-8h. The effectiveness and sustainability? of our diet, for each species raised,? was compared with 2 different natural diets (calf liver and pork muscle). Groups of 100-150 eggs were placed on a petri dish with 100 g of artificial diet or natural diet in special plastic boxes (width 10.5 cm x height 25 cm). The larvae were fed ad libitum until reaching the pupal stage. A pool of 10 larvae, taken randomly,? was measured at 24h intervals. upon reaching the pupal stage these were transferred to boxes with a bottom covered with sawdust and a pool of 10 specimens? been weighed at 24h intervals. The flicker of adults? been recorded daily and in the end? the sex ratio was assessed (n = 3).

_Data analysis

The mean lengths of larvae are analyzed for the two artificial diets and liver or meat (n = 3) (Table 2).

RESULTS

Larval and pupal life span, pupal weight, sex ratio and mortality? for Lab1 (IZSLER seat of Reggio Emilia) and Lab2 (Rutgers University? USA) are shown respectively in Table 1 and Table 2.

Table 1_duration of the larval and pupal period, pupal weight, sex-ratio and mortality? of neighboring C. raised on different diets in Lab1 a = Average? SD (n = 3).

Table 2_duration of the larval and pupal period, pupal weight, sex-ratio and mortality? of neighboring C. raised on different diets in Lab2 a = Average? SD (n = 3).

The suitability? of two diets? was determined using an index that incorporates growth, development, fertility? (indicated in terms of pupal weight) and survival in an empirical factor, described by Seth and Sharmanel 1998 (Seth R.H., Sharma V.P. In Proceedings of a final Research Co-ordination Meeting: Evaluation of Lepidoptera population suppression by radiation induced sterility of 1998) . (Table 3 and Table 4).

, Table 4_Growth and development of C. neighbor on different diets: index of adequacy calculated by Lab2.

The data show that the artificial diet? suitable for breeding in the laboratory of C. close as? causes a low mortality? in both larvae and pupae. The larval period is comparable with other diets tested. The adequacy index for larvae developed on an artificial diet? pi? high compared to the larvae developed on the liver. The cost of the starting materials? been around ~ 1.5? ($ 1.66) / 100 g, needed for the growth of 200 larvae.

EXAMPLE 2

The artificial diet object of our invention? been tested for the development of Hermetia illucens (Diptera: Stratiomyidae) whose larvae have a typically detritivorous diet. The artificial diet? was produced in the same way? of example 1. Larval growth and development of Hermetia illucens reared with our artificial diet were compared with development on 3 other different substrates: adult pork muscle, bovine liver and a standard diet normally used for mass farming of this insect (Gainesville diet) based on wheat bran (50%), maize flour (20%) and alfalfa (30%) not suitable for breeding other diptera of forensic interest. Are the second generation of H. illucens raised on artificial diets? was used in the experiment. The masses of eggs laid on artificial diets or natural diet (liver, meat

and standard diet) were placed in plastic containers (width 10.5 cm x height 25 cm). The diet was renewed every two days. Pupae (10 pupae every 24 hours) were weighed (n = 3) every day.

_Data analysis

The mean lengths of larvae are analyzed for the two artificial diets and liver and meat (n = 3)

RESULTS

All the parameters measured to evaluate the normal larval growth and development of the insect are shown in table 5

Table 5 duration of the larval period, pupal, pupal weight, sex-ratio and mortality? of H. illucens raised on different diets (n = 3).

The suitability of the 4 diets? was determined using an index that incorporates growth, development, fertility? (indicated in terms of pupal weight) and survival in an empirical factor, described by Seth and Sharma in 1998 (Seth R.H., Sharma V.P. In Proceedings Meeting: Evaluation of Lepidoptera population suppression by radiation induced sterility of 1998). (see table 6)

Table 6 Growth of Hermetia illucens on 4 diets tested. (a) Growth index =% adult excretions / total development time. (b) Index of Adequacy = (pupal weight / larval duration) x% Adult flickers

From table 2 it is clear that our diet can? be compared with a typical standard diet already? used for H. illucens both in terms of life cycle duration and in terms of adequacy index. However, our diet guarantees a survival percentage and a higher growth index than the 4 tested larval substrates. So even in this case, the data collected show that our artificial diet can? be used not only for species with a specifically necrophagous diet but also for species whose larvae essentially feed on debris of both animal and vegetable origin.

Claims (1)

CLAIMS 1. Composition for an artificial diet of diptera comprising: - soy flour with a protein content of 35-40g / 100 and carbohydrates of 15-20g / 100 (50-55% by weight); - Caseins (7-10% by weight); - Saccharomyces cerevisiae yeast powder (10-15% by weight); - Powdered milk, (10-15% by weight); - Agar 8-10g for 200ml of water 2. Composition according to claim 1 wherein the caseins are bovine milk caseins. 3. Composition according to claims 1-2 wherein the yeast? in the form of water-soluble powder. 4. Composition according to any one of claims 1-3 wherein the powdered milk has a fat quantity of 20-30g / 100g, linoleic acid in quantity? 4-6g / 100g, Vitamin C 100-110g / 100g, Thiamine 0.7-1mg / 100g. 5. Composition according to any one of claims 1-4 wherein the protein part? about 35-40g / 100g, the amount in carbohydrates? of about 15-20g / 100g and the fats are equal to 20-30g / 100g. 6. Process for preparing the composition according to any one of claims 1-5 which includes the following steps: Step 1: Dissolve the agar in water at a temperature of 90-100 ° C until the solution is clarified, the agar concentrations being between 0.5% and 2% with respect to water; Step 2: Dry mix the other components of the diet Step 3: Add the agar solution to the mixture of diet components and keep the pH of the mixture in the range 6.8 - 7.5 Step 4: Mix and quickly pour onto a horizontal support 7. Process according to claim 6 which includes Step 5: to spray a fungicide on the surface of the agar mixture obtained Process according to any one of claims 6-7 which further comprises Step 6 of allowing to solidify and keep cool 9. Agar product comprising the following components: 65-70% of water, 17-18% of soy flour, 2.5-3% of casein, 3-5% of yeast, 3-5% of milk powder and 2.5- 3% agar. 10. Use of a composition comprising: soy flour with a protein content of 35-40g / 100 and carbohydrates of 15-20g / 100 (50-55% by weight); Caseins (7-10% by weight); Saccharomyces cerevisiae yeast powder (10-15% by weight); Powdered milk, (10-15% by weight); 8-10g agar for 200ml of water to feed Diptera