**6. Conclusions**

42 Hearing Loss

Fig. 7. DPOAE suppression frequency response curves (Weibull regressions) for f2 values between 3.1 and 7.7 kHz, plotted on a normalized suppression scale (data from figs 4 and 6).

This study demonstrates that suppression of DPOAEs by contralateral pure tones can be detected in the chinchilla with real time recording. DPOAE suppression is greatest when using contralateral stimulation tones close to primary tone f2. This tonotopic response is consistent with other investigations of frequency specificity in the MOCS pathway (Chery-Croze, Moulin, & Collet, 1993; Cody & Johnstone, 1982; M. C. Liberman, 1989; Murata et al., 1980; Robertson, 1984; Robertson & Gummer, 1985; Veuillet et al., 1991; Warren III & Liberman, 1989a; Warren III & Liberman, 1989b). Unlike observations in human subjects, we did not observe any dips in fine structure DPOAEs to account for differences in the magnitude of suppression at different values of f2 or between chinchillas (Wagner,

Measurement of contralateral frequency tuning of MOCS fibers has revealed narrow band tuning equivalent in sharpness to cochlear afferent neurons (Brown, 1989; M. Liberman & Brown, 1986; Robertson, 1984). The final, divergent innervation pattern of MOCS fibers at the OHC level appears to degrade this cochleotopicity (or frequency tuning) by a factor of 4- 5 from 0.33 octaves (the approximate bandwidth of auditory afferents) to about 1.7 octaves for f2 = 3.1kHz and 1.3 octaves for f2 = 7.7kHz. The difference in tuning likely rests with the divergent OHC innervation by the MOCS fibers. Neural tracing studies in guinea pig have shown MOCS fibers innervating 15 -61 OHCs (Brown, 1989). In the cat, individual cochlear efferents contact 23 – 84 OHCs spanning 0.55-2.8mm (M. Liberman & Brown, 1986). Thus although tuning in the efferent fibers themselves appears to be as sharp as afferent tuning,

the effect of individual fibers on the organ of Corti will be much less precise.

**5.1.3 Discussion**

Heppelmann, Müller, Janssen, & Zenner, 2007).

Objective tests such as OAE and ABR are widely used in hearing screening programs and have lead to great advances in the early detection and rehabilitation of neonatal hearing

Contralateral Suppression of Otoacoustic Emissions:

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679-682.

Working Towards a Simple Objective Frequency Specific Test for Hearing Screening 45

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loss. However these tests do not provide a quick and easy means for assessing hearing threshold at different frequencies, indeed the presence of OAE does not even guarantee the presence of normal hearing. An objective frequency specific test of hearing ability would have widespread advantages, not just for neonatal testing but in many circumstances in all age groups.

In the present study we have demonstrated frequency specificity in contralateral suppression using a chinchilla model. The majority of studies shedding light onto the function of the MOCS have been derived from animal experiments. However, there is enough data in human studies to suggest that the human efferent system is qualitatively similar (Guinan Jr, 2006; James, 2006). We have shown previously that contralateral suppression of DPOAE can be assessed in real time in babies and adults (James et al., 2005) and can be used to test hearing very effectively in neonates (James, 2011). We have shown that this technique can distinguish between middle ear muscle reflexes and the OCR in an animal model (Wolter, Harrison, & James, 2011) and here show that it can be used to assess hearing threshold in a frequency specific manner. We envisage many clinical applications of this technique including the diagnosis and assessment of ANSD and more accurate hearing screening in neonatal and elderly populations.

#### **7. References**

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**1. Introduction**

**2.1 Location**

was about 21 dB(A).

**0**

**3**

*Japan*

Takahiro Tamesue

**A Prediction Method for Speech Audibility**

Securing good transmission characteristics for speech information and achieving a comfortable sound environment in buildings used by a great variety of people in public city spaces, to say nothing of spaces used for intellectual or mental work such as schools and offices, comprise two of the most important problems of environmental design. A common method for evaluating listening scores and psychological impressions for audio signal has been discussed previously (Tamesue T. et al., 2003). However, this research paid attention only to subjects in their twenties with normal hearing, and as a result the relationships between the frequency characteristics of hearing loss due to factors such as aging and the listening scores for audio signals and the psychological impressions related to speech audibility were not considered. Taking this into consideration, this chapter considers how the listening scores of audio signals and the psychological impressions for speech audibility change while taking into account the effects of hearing loss due to factors such as aging. Specifically, frequency filters for simulating hearing loss are first prepared. Next, psychological listening experiments are conducted in which both the audio signal and the noise passing through the above-mentioned filters are transmitted to subjects with normal hearing. Using the observed experimental data, the relationships between the weight-mean spectral distance (Tamesue T. et al., 2003) and the listening scores of the audio signals and psychological impressions with respect to speech audibility are investigated. Next, based on these relationships, problems associated with the prediction of listening scores and

psychological impressions with respect to speech audibility are discussed.

Psychological listening experiment I was conducted to establish the regression models of the listening scores of the audio signal and the psychological impressions related to speech

The experiment was conducted in a simple soundproof room on campus having the following dimensions: length 5.1 m, width 3.3 m, and height 2.2 m. The sound pressure level of the background noise was about 37 dB. The sound pressure level in this chapter is the value measured by a sound level meter with FLAT response. The A-weighted sound pressure level

**2. Outline of psychological listening experiment I**

audibility. The outline of the indoor experiment is as follows.

**Taking Account of Hearing Loss Due to**

**Aging Under Meaningless Noise**

*Organization for Academic Information, Yamaguchi University*


**2. The dipteran life cycle**

Insect age estimation relies on their basic physiology. Insect are poikilothermic, meaning their growth and development is affected by ambient temperature [12]. Lower ambient temperatures lead to slower insect growth, while higher ambient temperatures lead to faster insect growth. Each insect species has an upper and lower threshold, above or below which the insect no longer grows [8, 13]. We can describe and predict this growth based on ambient temperature by using a mathematical formula [4, 14, 15].

Investigators are able to use the ability of insects to quickly locate and colonize carrion and the ability to mathematically estimate insect age given ambient temperature to estimate a postmortem interval, given some assumptions. If we assume that insects on an animal arrived right after death and that their growth was dictated by ambient temperature, then we can calculate how long it would take an observed insect to reach an observed developmental stage at a given ambient temperature. This calculated time is the postmortem interval [3, 4, 7]. If the exact arrival time of insects at carrion is unknown, however (i.e., before or after death, animal was blocked from insect activity, etc.) this calculated time is the time of colonization interval [4, 11].

There are two genders involved in reproduction in most species. It is the female reproductive system that produces and stores eggs, provides food for eggs, collects and stores sperm, fertilizes the eggs, and positions the eggs in the environment. The male reproductive system is responsible for sperm production and for egg fertilization presentation to the female [16].

The Diptera, or the true flies, are the main forensically significant insects. Because Diptera is typically the first insect to colonize animal remains, the most widely used in forensic cases is the dipteran life cycle. Therefore, the dipteran life cycle is considered the most important to understand [3, 7, 17]. All flies start as eggs normally laid in the environment, although some eggs remain in the female's body and are placed as larvae in the environment [18]. The adult flies enter a dead animal and lay their eggs or larvae on that animal's natural body openings and wounds. The resulting larvae feed on the body until it is ready for pupation. Flies have what is known as a complete life cycle or metamorphosis: beginning as embryos, hatching into larvae, pupating, and eclosing from the pupae into an adult. The larvae are in three stages or instars, before they finish feeding on the body and move into the dispersal or wandering stage. This dispersal stage takes the larvae 15–20 ft away from the carrion and to a protected place in which to pupate [8]. The dispersing larvae will crawl under things in the environment or dig a few centimeters down into the soil, where the outer cuticle of the larva hardens into a protective shell or pupal case. Inside this pupal casing, enzyme transforms the larvae into an adult [6].

#### **3. Calculating time of colonization**

Dipteran larvae are poikilothermic, so they do not generate their own body heat [5, 16, 19]. Rather, the ambient temperature affects them, which determines how

#### *Diptera Development: A Forensic Science Perspective DOI: http://dx.doi.org/10.5772/intechopen.90859*

position output, it can be concluded that an insect with unfettered access to a carcass arrived and colonized it within minutes of animal death. The calculation of the age of the insects colonizing the animal body (TOC estimate) can therefore be used as a measure of how long this carcass was available for insect colonization and, by logical extension, how long it was dead (postmortem interval or PMI) [4, 11].

Insect age estimation relies on their basic physiology. Insect are poikilothermic, meaning their growth and development is affected by ambient temperature [12]. Lower ambient temperatures lead to slower insect growth, while higher ambient temperatures lead to faster insect growth. Each insect species has an upper and lower threshold, above or below which the insect no longer grows [8, 13]. We can describe and predict this growth based on ambient temperature by using a mathe-

Investigators are able to use the ability of insects to quickly locate and colonize carrion and the ability to mathematically estimate insect age given ambient temperature to estimate a postmortem interval, given some assumptions. If we assume that insects on an animal arrived right after death and that their growth was dictated by ambient temperature, then we can calculate how long it would take an observed insect to reach an observed developmental stage at a given ambient temperature. This calculated time is the postmortem interval [3, 4, 7]. If the exact arrival time of insects at carrion is unknown, however (i.e., before or after death, animal was blocked from insect activity, etc.) this calculated time is the time of

There are two genders involved in reproduction in most species. It is the female reproductive system that produces and stores eggs, provides food for eggs, collects and stores sperm, fertilizes the eggs, and positions the eggs in the environment. The male reproductive system is responsible for sperm production and for egg fertiliza-

Dipteran larvae are poikilothermic, so they do not generate their own body heat [5, 16, 19]. Rather, the ambient temperature affects them, which determines how

The Diptera, or the true flies, are the main forensically significant insects. Because Diptera is typically the first insect to colonize animal remains, the most widely used in forensic cases is the dipteran life cycle. Therefore, the dipteran life cycle is considered the most important to understand [3, 7, 17]. All flies start as eggs normally laid in the environment, although some eggs remain in the female's body and are placed as larvae in the environment [18]. The adult flies enter a dead animal and lay their eggs or larvae on that animal's natural body openings and wounds. The resulting larvae feed on the body until it is ready for pupation. Flies have what is known as a complete life cycle or metamorphosis: beginning as embryos, hatching into larvae, pupating, and eclosing from the pupae into an adult. The larvae are in three stages or instars, before they finish feeding on the body and move into the dispersal or wandering stage. This dispersal stage takes the larvae 15–20 ft away from the carrion and to a protected place in which to pupate [8]. The dispersing larvae will crawl under things in the environment or dig a few centimeters down into the soil, where the outer cuticle of the larva hardens into a protective shell or pupal case. Inside this pupal casing, enzyme transforms the larvae into an adult [6].

**2. The dipteran life cycle**

*Life Cycle and Development of Diptera*

matical formula [4, 14, 15].

colonization interval [4, 11].

tion presentation to the female [16].

**3. Calculating time of colonization**

**162**

rapidly or slowly they develop [12, 16, 19]. The warmer the temperature in the area, the faster they go through their instars and pupal stage. The higher the ambient temperature, the faster their instars and pupal stage are going through. This estate allows forensic entomologists to use information on insect growth to assess the time of colonization estimate of animal remains. This also makes temperature the most important density-independent factor for insects [4, 19].

Temperature has a profound effect on the metabolic of dipteran and the speed of development [16]. Warmer temperatures generally result in faster development, within certain temperature ranges. But, this is not accurate at extreme temperatures. Insects have both upper and lower thermal limits, below and above which the insect either dies or no longer develops. These are known as threshold temperatures [16, 19, 20]. In different species, these thermal levels are naturally different. Organisms that have evolved in tropical and warmer areas will have higher limits than those that have developed in temperate or colder areas [21]. Therefore, when it comes to temperature scales, there is a huge variation between species. The lower limits are better known than the upper limits and are significant when measuring a colonization estimate period mathematically [17, 20]. Developmental thresholds for forensically important flies are usually between 6 and 10°C and are experimentally determined. If a specific fly species developmental threshold is not available, a general rule of thumb is to use 6°C for winter species or flies in cold areas and 10°C for warm weather species or flies in warmer areas [7, 22–24].

Each stage of insect growth requires a certain amount of heat above the minimum temperature and below the maximum temperature, from egg to an adult [12, 25]. This growth rate can be represented with a linear model that represents the amount of heat necessary for a given fly to go through each of its developmental stages. These linear models are called degree day or degree hour models, because development is recorded as the temperature above the minimum developmental threshold multiplied by time (days or hours) [13, 14, 26, 27].

This model allows us to calculate the time a fly takes to develop using ambient temperature, and this formula is

(average ambient temperature minimum threshold) unit of time

This formula takes the average ambient temperature over a given time period, subtracts the minimum threshold for the insect species, and multiplies the result by unit of time. The result is called a "degree day" (if the unit of time used was a day) or a "degree hour" (if the unit of time used was an hour). These are the two most common units of time seen in this type of model, simply because weather stations often record ambient temperature at daily or hourly intervals.

The formula may be used to calculate how many degree days or degree hours are necessary for an insect to get through various stages in its life cycle, as well as how many degree days have been accumulated over time. The DD/DH necessary for an insect to get through its lifecycle is determined by experiment. The data are recorded as hours needed for a species to develop at a given temperature, and these data may be converted into DD or DH simply by using the formula.

**Table 1** shows several forensically important insects and the time it takes each insect to develop at various temperatures. Take, for example, the hours necessary for the black blow fly, *Phormia regina* (Meigen)*,* to develop at 27°C. According to research, it takes *P. regina* eggs 16 h to go from freshly laid to hatching, 18 h to go through the first larval instar, 11 h to go through the second larval instar, and 36 h to go through the third larval instar. To turn this information into degree hours, simply plug the data into the equation

Egg DH:

$$\begin{aligned} &(27^{\circ}\text{C} - 10^{\circ}\text{C} \times 16\text{ h}) \\ &(17^{\circ}\text{C}) \times 16\text{ h} \\ &272\text{ DH} \end{aligned}$$

This calculation shows that 5 degree days accumulated over that 1 day. Each passing day may be calculated in the same way, and the results are added up to

Average temperature (°C) on 4 separate days: 15°C, 20°C, 21°C, and 18°C

Once all this data is determined, the DD required for the growth of an insect species is added to the total DD accumulated over a period of time to decide how long it would take for an insect to develop into an observed stage of life given at the

Note that the dates are described in descending order; this method allows an entomologist to begin with the date on which evidence of insects was collected and to work back in time to determine the minimum period of insect colonization. In **Table 1**, 11 DD were accumulated on May 15, 15 DD on May 14, 12 DD on May 13, etc. In total, it takes 57.14 DD to develop from egg through the end of the third

*Example of average ambient temperatures and calculated DD/ADD from a weather station.*

If maggots at the very end of the third instar were found on an animal at the end of the day on May 15, the ADD necessary for those maggots to develop through the third instar may be used to determine when the eggs were laid. In this case, *P. regina* needs 57.14 DD to develop through the third instar. There were not enough DD accumulated on May 15 to reach that number, so the eggs could not have been laid on May 15 and have had time to reach the observed life stage. Since only 25 DD accumulated between May 14 and May 15, there was still not enough time for the fly to reach its third instar. Enough DD is accumulated between May 11 and May 15, however, for the insect to reach its observed life stage. In this instance, we can say the eggs had to have been laid sometime on May 11 or earlier to reach the third instar by May 15. This gives a minimum time of colonization estimation, based on

The feasibility of this approach of the colonization estimation time degree day

model depends on the availability of growth rate information for different necrophagous species. Such data are collected experimentally and appear to be reported as a life table or the rate of development of different insect species. Arthropods associated with carrion are associated with ecological groups: necrophagous species, which feed upon the animal remains; omnivorous species, which feed upon both the animal remains and other organisms colonizing those remains; predators and parasites of the necrophagous or omnivorous species;

produce the accumulated degree days over any given period of time:

ADD over the 4 days: 5 DD + 10 DD + 11 DD + 8 DD = 34 DD

reported ambient temperature. It is best to display this data in table form:

**Date Average temp (°C) DD ADD** May 15 21 11 11 May 14 25 15 26 May 13 22 12 38 May 12 18 8 46 May 11 22 12 58 May 10 20 10 68 May 9 15 5 73

DD for each day: 5 DD, 10 DD, 11 DD, 8 DD

*Diptera Development: A Forensic Science Perspective DOI: http://dx.doi.org/10.5772/intechopen.90859*

the life history of the flies colonizing an animal.

instar.

**165**

**Table 1.**

In this case, it takes *P. regina* 272 DH to go through its entire egg stage. This same calculation may be used to determine DD:

Egg DD:

$$\begin{aligned} &(27^{\circ}\text{C} - 10^{\circ}\text{C} \times 0.67 \text{ days}) \\ &(17^{\circ}\text{C}) \times 0.67 \text{ days} \\ &\mathbf{11.39} \text{ DD} \end{aligned}$$

The number of DD or DH necessary for each stage of *P. regina* development may be calculated in this same way:

First instar DH/DD:

$$\begin{aligned} &(27^{\circ}\text{C} - 10^{\circ}\text{C} \times 18\text{ h}) \\ &(17^{\circ}\text{C}) \times 18\text{ h} \\ &306\text{ DH} \quad \text{or} \quad 12.75\text{ DD} \end{aligned}$$

Second instar DH/DD:

```
ð27°C � 10°C � 11 h
ð Þ� 17°C 11 h
180 DH or 7:5 DD
```
Third instar DH/DD:

```
ð27°C � 10°C � 36 h
ð Þ� 17°C 36 h
612 DH or 25:5 DD
```
The results may be summed up to determine the accumulated degree days (ADD) or accumulated degree hours (ADH) necessary for *P. regina* to develop from freshly oviposited egg through the end of the third instar:

11*:*39 DD þ 12*:*75 DD þ 7*:*5 DD þ 25*:*5 DD ¼ 57*:*14 DD

This may be calculated for any range of developmental stages in an insect's life cycle.

Once the ADD or ADH necessary for insect development is known, it is possible to calculate the time necessary for the DD to accumulate at recorded ambient temperatures. The species threshold temperature is used in the same DD formula, but the average temperature is a weather station recording over a given period of time.

If, for example, a weather station recorded an average temperature of 150 C over 1 day, and then the DD accumulated over that 1 day may be calculated as

$$\begin{aligned} &(\mathbf{15^{\circ}C} - \mathbf{10^{\circ}C}) \times \mathbf{1} \text{ day} \\ &(\mathbf{5^{\circ}C}) \times \mathbf{1} \text{ day} \\ &\mathbf{5 DD} \end{aligned}$$

*Diptera Development: A Forensic Science Perspective DOI: http://dx.doi.org/10.5772/intechopen.90859*

This calculation shows that 5 degree days accumulated over that 1 day. Each passing day may be calculated in the same way, and the results are added up to produce the accumulated degree days over any given period of time:

Average temperature (°C) on 4 separate days: 15°C, 20°C, 21°C, and 18°C DD for each day: 5 DD, 10 DD, 11 DD, 8 DD ADD over the 4 days: 5 DD + 10 DD + 11 DD + 8 DD = 34 DD

Once all this data is determined, the DD required for the growth of an insect species is added to the total DD accumulated over a period of time to decide how long it would take for an insect to develop into an observed stage of life given at the reported ambient temperature. It is best to display this data in table form:

#### **Table 1.**

Egg DH:

Egg DD:

ð27°C � 10°C � 16 h ð Þ� 17°C 16 h

In this case, it takes *P. regina* 272 DH to go through its entire egg stage. This same

ð27°C � 10°C � 0*:*67 days ð Þ� 17°C 0*:*67 days

The number of DD or DH necessary for each stage of *P. regina* development may

ð27°C � 10°C � 18 h ð Þ� 17°C 18 h

306 DH or 12*:*75 DD

ð27°C � 10°C � 11 h ð Þ� 17°C 11 h 180 DH or 7*:*5 DD

ð27°C � 10°C � 36 h ð Þ� 17°C 36 h

612 DH or 25*:*5 DD

11*:*39 DD þ 12*:*75 DD þ 7*:*5 DD þ 25*:*5 DD ¼ 57*:*14 DD

Once the ADD or ADH necessary for insect development is known, it is possible to calculate the time necessary for the DD to accumulate at recorded ambient temperatures. The species threshold temperature is used in the same DD formula, but the average temperature is a weather station recording over a given period of time. If, for example, a weather station recorded an average temperature of 150

ð15°C � 10°CÞ � 1 day

C over

This may be calculated for any range of developmental stages in an insect's

1 day, and then the DD accumulated over that 1 day may be calculated as

5 DD

ð Þ� 5°C 1 day

The results may be summed up to determine the accumulated degree days (ADD) or accumulated degree hours (ADH) necessary for *P. regina* to develop from

freshly oviposited egg through the end of the third instar:

272 DH

11*:*39 DD

calculation may be used to determine DD:

*Life Cycle and Development of Diptera*

be calculated in this same way: First instar DH/DD:

Second instar DH/DD:

Third instar DH/DD:

life cycle.

**164**

*Example of average ambient temperatures and calculated DD/ADD from a weather station.*

Note that the dates are described in descending order; this method allows an entomologist to begin with the date on which evidence of insects was collected and to work back in time to determine the minimum period of insect colonization. In **Table 1**, 11 DD were accumulated on May 15, 15 DD on May 14, 12 DD on May 13, etc. In total, it takes 57.14 DD to develop from egg through the end of the third instar.

If maggots at the very end of the third instar were found on an animal at the end of the day on May 15, the ADD necessary for those maggots to develop through the third instar may be used to determine when the eggs were laid. In this case, *P. regina* needs 57.14 DD to develop through the third instar. There were not enough DD accumulated on May 15 to reach that number, so the eggs could not have been laid on May 15 and have had time to reach the observed life stage. Since only 25 DD accumulated between May 14 and May 15, there was still not enough time for the fly to reach its third instar. Enough DD is accumulated between May 11 and May 15, however, for the insect to reach its observed life stage. In this instance, we can say the eggs had to have been laid sometime on May 11 or earlier to reach the third instar by May 15. This gives a minimum time of colonization estimation, based on the life history of the flies colonizing an animal.

The feasibility of this approach of the colonization estimation time degree day model depends on the availability of growth rate information for different necrophagous species. Such data are collected experimentally and appear to be reported as a life table or the rate of development of different insect species.

Arthropods associated with carrion are associated with ecological groups: necrophagous species, which feed upon the animal remains; omnivorous species, which feed upon both the animal remains and other organisms colonizing those remains; predators and parasites of the necrophagous or omnivorous species;

adventive species, which use the remains as an extension of the environment; and incidental species which are associated with animal remains due to happenstance [3, 28].

Necrophagous insects arrive on and in a corpse in a somewhat predictable sequence and are arguably the most useful for forensic entomology. This is the ecological succession of insects and is influenced by the environment, season, and the decomposition state of the carrion. The insects arrive in blending waves, called seres, of organisms, each comprised of different species attracted to a particular state of decay [3, 29].

the third instar. The oldest maggots will begin to disperse from the carrion to find pupation sites. These maggots will crawl under objects in the environment or burrow into the soil to become pupae. The early seres or pioneer flies cease to be attracted to the corpse, making way for those insects that prefer later stages of decay. Predatory maggots are much more abundant at this stage and may be feeding upon other species in the maggot mass [34]. Predatory beetles lay their eggs in the corpse and their larvae then hatch to feed on the dead animal and other insects colonizing that animal. Parasitic wasps are even more common, attacking the huge maggot masses and developing pupae. Active decay attracts a new sere characterized by beetles in Dermestidae family and grease moths. As active decay moves into butyric fermentation, two additional seres are attracted: first, a sere consisting of cheese skippers, *Fannia* sp.; Silphid beetles; and beetles in the family Cleridae arrives at the body. The second sere consists of dump flies, flies in the family

As the carrion is stripped of most of its soft tissue, the remaining tissue begins to

Once the carrion has completely dried out, the body attracts insects that can feed on hair and dried skin. This dry stage of decomposition is highly attractive to a new sere consisting of mites, followed by a sere of beetles in Dermestidae and Tingidae, and finally a sere of primarily Ptinidae and Tenebrionidae. This stage tends to last for a long while, since the business of feeding on dried out tissue takes a great deal of time and digestive enzymes. The beetles will remain on the bones as long as the

After the dried tissue has been cleaned by the beetles, moths, and mites, only the bones remain along with empty pupal casings. There is no longer any major insect activity, and any further decomposition is accomplished by bacteria and physical

By way of comparison, a buried animal has fewer seres with lower species diversity [37]. Fresh buried animals tend to attract flies in Calliphoridae, Muscidae, Sarcophagidae, and Phoridae. Buried animals in active decay may attract root-

eating beetles, and those in dry decay are populated by rove beetles.

*General arrival of forensically important insect families by phase of decomposition.*

dry out. The reduction in soft food makes the body less palatable to the mouth hooks of maggots and more suitable for the chewing mouthparts of beetles. Beetle adults and larvae feed on skin and ligaments, while certain late stage flies, such as the cheese fly, arrive to feed on whatever moist flesh remains. Predators and parasites are still prevalent at this stage, feeding on the straggling maggots or other

Phoridae, beetles in the family Silphidae, and clown beetles [3].

soft-bodied insects in the vicinity [3, 35].

*Diptera Development: A Forensic Science Perspective DOI: http://dx.doi.org/10.5772/intechopen.90859*

carrion is undisturbed and has dried tissue [36].

factors [31].

**Figure 1.**

**167**

Early research in entomology indicated that the number of seres varies according to the placement of the carrion. Mègnin [30] showed that animals exposed to the environment yielded eight distinct insect seres, while those that were buried attracted only three distinct insect seres. This difference speaks to the availability of the tissue for colonizing insects, along with the ability of those insects to reach tissue that is blocked from easy access. Different insects are attracted to different stages of generalized decomposition. While stages of decomposition are not discrete and can sometimes be difficult to characterize with precision, historically scientists have broken up decomposition of animal remains into five major stages: fresh, putrefaction, active decay, butyric fermentation, and dry decay [31]. Each of these stages attracts one or more seres of insects and other arthropods in a predictable sequence.

From the moment of death, the insect fauna of an animal body begins to change. Any ectoparasites associated with the body leave relatively quickly as the body cools and the blood ceases to circulate [18]. Myiasis-causing flies may or may not die—it is dependent upon if they are obligate parasites of living tissue or facultative parasites that can feed on both living and dead animals. Botflies, for example, are dependent upon a living host, and if the host dies, the botfly dies. On the other hand, *Cochliomyia* sp. can feed on living or dead hosts and may just continue to feed after the animal dies [4, 18].

Necrophagous insects or those attracted to dead tissue are attracted to the body within minutes of death and are associated with the fresh stage of decay. In general, the first adults are observed on a newly dead animal within an hour after death, as long as there is an adequate access to the body [4]. Some investigators report female flies arriving within 15 s of death [7]. Eggs and early instar maggots tend to appear with the onset of autolysis. In general, the animal will be characterized in this early sere by the presence of adult flies and fly eggs. Eggs will be found near natural bodily opening, on wounds, and sometimes in protected folds of skin or coverings. Those eggs will hatch to first instars quickly in warm environments, but there will not be a large maggot mass on the body; the tissue will still look fresh. The most common groups of insects found during this fresh stage include species from the fly families of Calliphoridae, Muscidae, and Sarcophagidae [32, 33].

As the carrion begins to putrefy, the young maggots will move throughout the body, spreading bacteria, secreting digestive enzymes, and feeding on tissue. They move as a maggot mass, benefitting from communal heat and shared digestive secretions. Larvae first feed between muscles then on the muscle fibers themselves as the maggots grow and the digestive juices get to work. The rate of decay increases, and the odors emitted from the body attract more blow flies, flesh flies, beetles, and mites. They are joined by parasitic wasps that lay their eggs inside maggots and later inside pupae. The most common insects associated with this putrefaction stage, however, are species in the fly families of Calliphoridae and Sarcophagidae [3].

Once the carrion enters in active decay, there will be several generations of maggots present on the body. Some of the maggots will be large well and enter into

#### *Diptera Development: A Forensic Science Perspective DOI: http://dx.doi.org/10.5772/intechopen.90859*

adventive species, which use the remains as an extension of the environment; and incidental species which are associated with animal remains due to

Necrophagous insects arrive on and in a corpse in a somewhat predictable sequence and are arguably the most useful for forensic entomology. This is the ecological succession of insects and is influenced by the environment, season, and the decomposition state of the carrion. The insects arrive in blending waves, called seres, of organisms, each comprised of different species attracted to a particular

Early research in entomology indicated that the number of seres varies according to the placement of the carrion. Mègnin [30] showed that animals exposed to the environment yielded eight distinct insect seres, while those that were buried attracted only three distinct insect seres. This difference speaks to the availability of the tissue for colonizing insects, along with the ability of those insects to reach tissue that is blocked from easy access. Different insects are attracted to different stages of generalized decomposition. While stages of decomposition are not discrete and can sometimes be difficult to characterize with precision, historically scientists have broken up decomposition of animal remains into five major stages: fresh, putrefaction, active decay, butyric fermentation, and dry decay [31]. Each of these stages attracts one or more seres of insects and other arthropods in a

From the moment of death, the insect fauna of an animal body begins to change. Any ectoparasites associated with the body leave relatively quickly as the body cools and the blood ceases to circulate [18]. Myiasis-causing flies may or may not die—it is dependent upon if they are obligate parasites of living tissue or facultative parasites that can feed on both living and dead animals. Botflies, for example, are dependent upon a living host, and if the host dies, the botfly dies. On the other hand, *Cochliomyia* sp. can feed on living or dead hosts and may just continue to feed

Necrophagous insects or those attracted to dead tissue are attracted to the body within minutes of death and are associated with the fresh stage of decay. In general, the first adults are observed on a newly dead animal within an hour after death, as long as there is an adequate access to the body [4]. Some investigators report female flies arriving within 15 s of death [7]. Eggs and early instar maggots tend to appear with the onset of autolysis. In general, the animal will be characterized in this early sere by the presence of adult flies and fly eggs. Eggs will be found near natural bodily opening, on wounds, and sometimes in protected folds of skin or coverings. Those eggs will hatch to first instars quickly in warm environments, but there will not be a large maggot mass on the body; the tissue will still look fresh. The most common groups of insects found during this fresh stage include species from the fly

As the carrion begins to putrefy, the young maggots will move throughout the body, spreading bacteria, secreting digestive enzymes, and feeding on tissue. They move as a maggot mass, benefitting from communal heat and shared digestive secretions. Larvae first feed between muscles then on the muscle fibers themselves

as the maggots grow and the digestive juices get to work. The rate of decay increases, and the odors emitted from the body attract more blow flies, flesh flies, beetles, and mites. They are joined by parasitic wasps that lay their eggs inside maggots and later inside pupae. The most common insects associated with this putrefaction stage, however, are species in the fly families of Calliphoridae and

Once the carrion enters in active decay, there will be several generations of maggots present on the body. Some of the maggots will be large well and enter into

families of Calliphoridae, Muscidae, and Sarcophagidae [32, 33].

happenstance [3, 28].

*Life Cycle and Development of Diptera*

state of decay [3, 29].

predictable sequence.

after the animal dies [4, 18].

Sarcophagidae [3].

**166**

the third instar. The oldest maggots will begin to disperse from the carrion to find pupation sites. These maggots will crawl under objects in the environment or burrow into the soil to become pupae. The early seres or pioneer flies cease to be attracted to the corpse, making way for those insects that prefer later stages of decay. Predatory maggots are much more abundant at this stage and may be feeding upon other species in the maggot mass [34]. Predatory beetles lay their eggs in the corpse and their larvae then hatch to feed on the dead animal and other insects colonizing that animal. Parasitic wasps are even more common, attacking the huge maggot masses and developing pupae. Active decay attracts a new sere characterized by beetles in Dermestidae family and grease moths. As active decay moves into butyric fermentation, two additional seres are attracted: first, a sere consisting of cheese skippers, *Fannia* sp.; Silphid beetles; and beetles in the family Cleridae arrives at the body. The second sere consists of dump flies, flies in the family Phoridae, beetles in the family Silphidae, and clown beetles [3].

As the carrion is stripped of most of its soft tissue, the remaining tissue begins to dry out. The reduction in soft food makes the body less palatable to the mouth hooks of maggots and more suitable for the chewing mouthparts of beetles. Beetle adults and larvae feed on skin and ligaments, while certain late stage flies, such as the cheese fly, arrive to feed on whatever moist flesh remains. Predators and parasites are still prevalent at this stage, feeding on the straggling maggots or other soft-bodied insects in the vicinity [3, 35].

Once the carrion has completely dried out, the body attracts insects that can feed on hair and dried skin. This dry stage of decomposition is highly attractive to a new sere consisting of mites, followed by a sere of beetles in Dermestidae and Tingidae, and finally a sere of primarily Ptinidae and Tenebrionidae. This stage tends to last for a long while, since the business of feeding on dried out tissue takes a great deal of time and digestive enzymes. The beetles will remain on the bones as long as the carrion is undisturbed and has dried tissue [36].

After the dried tissue has been cleaned by the beetles, moths, and mites, only the bones remain along with empty pupal casings. There is no longer any major insect activity, and any further decomposition is accomplished by bacteria and physical factors [31].

By way of comparison, a buried animal has fewer seres with lower species diversity [37]. Fresh buried animals tend to attract flies in Calliphoridae, Muscidae, Sarcophagidae, and Phoridae. Buried animals in active decay may attract rooteating beetles, and those in dry decay are populated by rove beetles.

*General arrival of forensically important insect families by phase of decomposition.*

Since Meginin's original experiments, there have been many, many attempts to characterize the number of seres that show up on animals of different sizes. The consensus is that these successional waves range from 8 to 10, and any attempts to discreetly define the arthropod community associated with the seres is confounded by the continual nature of decomposition. There is a broad general agreement of orders and families that show up on a decomposing body [3, 4, 7]. There is also a general agreement of sequence and the idea that the first sere is primarily composed of Dipteran species. Succession is affected by the location of the carrion, any covering, animal type, local insect species, time of year, and a myriad of other factors. At the family level, the succession looks like as shown in **Figure 1**. Knowing the general succession of insects on a corpse in a particular area enables the entomologist to extend the time of colonization estimation even if the primary colonizers of the first sere have come and gone.

**References**

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*3.1. Feeding Phases*

Based on the outcomes of the experiment, it was divided into phases. The main characteristics of the feeding phases, except for the start-up phase, are reported in Table 5. Indeed, the start-up phase was characterized by high instability of the main process parameters as well as by a rapid variation of admixture feeding rates over time. Thus, the remaining feeding phases were labeled from 1 to 7.

**Table 5.** Main parameters (with their standard deviations) of anaerobic digestion trials on hemp residues.

1 OLR means "organic loading rate"; 2 HRT means "hydraulic retention time"; 3 C means "percentage of new hemp in the admixture"; 4 R means "digestate recirculation ratio".

Feeding phases were classified according to enzymatic treatment, OLR, HRT, C, and R (Table 5). The two C reference values applied in this study are close to the organic loadings used in the industrial plants of anaerobic digestion.

#### *3.2. Management of the Reactor and Process Stability*

Management of the reactor -TS and VSd.b. trends of the material in the feeding hopper, in the reactor sludge, and the digestate tank are reported in Figures 2 and 3.

**Figure 2.** TS (total solids) (**a**) and VSd.b. (volatile solids on a dry basis); (**b**) trends of the material in the feeding hopper and the digestate tank, concerning consecutive sampling.

**Figure 3.** TS (total solids) (**a**) and VSd.b. (volatile solids on a dry basis); (**b**) trends of the reactor sludge, concerning consecutive sampling.

By comparing the TS and VSd.b. trends in the feeding hopper and digestate, it can be seen that from the 70th sampling, these parameters were notably lower in the discharged sludge than in the corresponding feeding mixtures. The 70th sampling corresponds to the beginning of the 5th feeding phase and this occurrence emerged from the observations related to the 6th and 7th phases as well. In the previous feeding phases, a distinction between the TS and VSd.b. evolution in the fed slurry and the corresponding digestate cannot be seen. This result is related to the higher reference value of C (C2), which was better than the other one (C1).

With regard to Figure 3, the reactor sludge did not show relevant differences in terms of TS and VSd.b. by comparing the loading side to the discharging side, mainly due to a certain mixing of the sludge along the longitudinal section of the reactor. TS and VSd.b. seemed to increase from phase "1" to phase "4" and to decrease in the subsequent phases (characterized by the reference value, C2).

Management of process stability: Concerning the main design and operation process parameters of the reactor, the trends of HRT and OLR are shown in Figures 4 and 5.

**Figure 4.** Hydraulic retention time (HRT) trend.

**Figure 5.** Organic loading rate (OLR) trend.

HRT did not show significant variations during the entire experiment (Figure 4) and it was excluded from the statistical analysis.

OLR (Figure 5) was monitored through the daily determination of TS and VSd.b. in the feeding admixture and adjusted by setting the hemp share in the feeding admixture and the loading flow rate (Figure 2).

Regarding the process stability parameters, the trends of FOS/TAC and pH, biogas production, and biogas composition (CH4, CO2, NH3, H2S) are reported in Figures 6–9.

**Figure 6.** FOS/TAC (volatile fatty acids/buffer capacity) and pH trends.

**Figure 7.** Biogas production trends (GPR) (via biogas metering).

**Figure 8.** Biogas composition (CH4 and CO2, [%wt]) detected by the portable gas analyzer during the experimental period.

**Figure 9.** Gas production rate (GPR) trends of biogas and methane.

By considering the entire experimental campaign, pH varied between 7.2 and 8.0, accompanied by higher values during the first phase.

The FOS/TAC ratio increased over time, from about 0.170 to values close to 0.270. These values are very similar to the typical ones of the industrial plants treating lignocellulosic biomass, such as corn silage.

Overall, the FOS/TAC trend is consistent with the increase in the percentage of new hemp in the feeding admixtures (C2 treatment) (see Table 5) from the fifth feeding phase to the seventh one. It can be supposed that the introduction of higher amounts of hemp provoked a shift of the anaerobic digestion microbial dynamics towards the predominance of acidogenic reactions.

Daily biogas production (Figure 7) was characterized by high variability during the experimental campaign. It showed a significant increase of the biogas produced during the experimental phases from "5" to "7" (C2), accompanied by a rapid increase during the fifth phase and a decrease over the sixth one (without enzymes). The last feeding phase showed rising values for most of the days (to reach about 1.1 m3 biogas per day), corresponding to the coupling of higher C and enzymatic treatment. It can be considered as the most suitable among all the treatments applied. On average, the CH4 content [%wt] in the biogas produced during the entire experimental campaign was 53.8 ± 2.2. CO2 content [%wt] was 45.6 ± 2.4. CH4 concentration showed a slow reduction from the phase "1" to the phase "4". From the phase "5", a gradual increase of its concentration was detected. CH4 and CO2 trends (Figure 8) did not show any peak attributed to organic overload.

Performance parameters: The performance parameters, SGP and GPR, of the anaerobic digestion trials are shown in Figures 9 and 10.

**Figure 10.** Specific gas production (SGP) trends of biogas and methane.

SGP of biogas/methane is the energy yield of an anaerobic digestion system regardless of OLR, thus it plays an important role when assessing the energy yield of the considered process.

Both trends showed an increase starting in the fifth feeding phase. It was more evident during the seventh period.

By comparing these outcomes with those obtained on a laboratory scale [30,32], it can be pointed out that the present work provided higher SGP values both for biogas and methane than the previous literature on anaerobic digestion trials of hemp straw. Overall, the management of the reactor and the process stability was enhanced with respect to biogas production, especially in the last part of the experiment, characterized by the combination of higher values of C and the application of enzymes.

SGPs of the present work are lower compared to those obtained in the same pilot plant, using vegetable feedstock characterized by high degradability (lower lignin contents and higher starch content). Due to the lignocellulosic nature of this crop, the specific biogas/methane production of hemp straw was lower than those related to raw potatoes (0.68 Nm3 of biogas ·kgVS−1 and 0.37 Nm3 of methane ·kgVS−1) [53], potato chips (0.81 Nm3 of biogas ·kgVS−1 and 0.47 Nm3 of methane ·kgVS−1) [54], and fruit and vegetable wastes (0.78 Nm3 of biogas ·kgVS−1 and 0.43 Nm3 of methane ·kgVS−1) [55].

Experiments carried out on the same pilot digester using admixtures made of different kinds of feedstock (admixture composition: 30%wt of shredded corn, the remaining part consisting in whey, vegetable water, pomace pitted, and manure to maintain the OLR between 2.5 and 3.5 kgVS·m3reactor·d−1) reported SGPbiogas from 0.623 ± 0.212 Nm3·kgVS−1 to 0.768 ± 0.227 Nm3·kgVS−1 and SGPmethane from 0.281 ± 0.160 Nm3·kgVS−1 to 0.438 ± 0.096 Nm3·kgVS−1 [56]. Also, hemp straw residues' performance in anaerobic digestion was found to be similar to other lignocellulosic crops [57], the specific methane production of which is between 0.17 and 0.39 Nm3·kgVS−1. Results are comparable to those reported by [58] for other agricultural crops, such as oats, flax, and sorghum, but lower than the hemp energy yields considered there.

Thus, hemp straw residues used in the same pilot plant showed lower SGP of biogas and methane probably because of the lignocellulosic composition. The results obtained by the residues considered in this work could be a ffected by the harvesting time, which is the reproductive stage when the lignin content is higher than in the vegetative stage. By considering that the reproductive stage is the core of the supply chain scenario assumed in this study, related to the extraction of oil from seeds, the results already discussed about the potential of biogas production from straw residues suggest the recovery of this low-value by-product to energy conversion. By considering the ultimate analysis, the carbon:nitrogen ratio is useful to define the biomass suitability for biochemical (ratio <30) or thermochemical processes (ratio >30). The ratio of hemp straw residues is 28.9 (Table 3), thus it can be considered for both, but with slightly higher suitability for biochemical conversion.


2.2.2. Tensile Strength (TS)

In this study, the splitting tensile strength (TS) of cylindrical specimens is accepted as the main tensile TS measurement for a concrete (and HPC) material (sample). Its test(s) can be performed in accordance with ASTM C496 [30].

#### 2.2.3. Flexural Strength (FS)

The primary property for a concrete material (sample) to demonstrate is CS, but its tensile behaviours are also important. When reinforced concrete (RC) is subjected to tensile force, cracks and expansion will occur, depending largely on the TS of the concrete material. Direct tension tests of concrete are seldom performed, mainly because the device that holds

the specimen may introduce secondary stresses that introduce error in the measurement. Another test for estimating the TS of a concrete material (sample) is the ASTM C78 (third point) flexural loading test, the result of which is referred to as flexural strength (FS) in this study [31].