*3.1.2. Host preferences*

The limitations discussed in terms of information required to improve the precision of PMI estimations using parasitoids can be overcome with more focused research. It is also important to note that the forensic potential of parasitic wasps is not diminished by gaps in basic knowledge of life history characteristics. In fact, parasitoids have far more potential as alternative indicator species for estimating the PMI than predatory species because of their specificity as parasites. What this means is that though some species may show variability in host preferences, progeny development is entirely tied to feeding on the host. As the fly hosts are linked directly to a corpse, the parasitoid's immature development is a second-level linkage to the conditions associated with the deceased. The same features are not true with predators that visit carrion. Another key difference is that the window of time in which a host is suitable for parasitism is more predictable and generally represents a narrower time period than that in which suitable prey are available for predation. The later is reflective of necrophilous beetles that may feed on animal remains as well as prey upon eggs and fly larvae for several days to weeks, depending on the season and ambient conditions. In comparison, the window of opportunity is quite short when pupal parasitoids (e.g., pteromalids) use puparia of calliphor‐ ids. Hosts are only of useable age for 3–5 days during warmer months, and somewhat longer as temperatures decline. Even this assessment is complicated by the observation that puparial development of *Lucilia sericata* is shorter in the presence of *N*. *vitripennis*, thereby reducing the window for parasitism by almost 1 day at temperatures near 25o C [60]. By contrast, the puparial stages suitable to serve as hosts for some sarcophagids extend over a much longer duration (1–2 weeks) [51], decreasing the precision of a PMI calculated from wasp developmental data as the age of the host at the time of parasitism is not known (**Table 2**).

## *3.1.3. Seasonality*

Seasonality of parasitoids is especially useful for the determination of whether a body has been moved. As with other aspects of parasitoid biology, seasonal occurrence of parasitic wasps is directly linked to fly hosts. For pupal parasitoids, those that enter winter dormancy in the form of diapause generally do so within the hardened puparium of a fly host. Depending on the timing of diapause onset, which is influenced by latitude, several species of calliphorids are eliminated as potential hosts as they diapause as either larvae or in an adult reproductive diapause [1]. As a consequence, sarcophagids often serve as the overwintering host for pupal parasitoids that frequent carrion. Collection of parasitized hosts with diapausing wasp larvae or parasitism of hosts by a wasp that should ordinarily be in diapause can be an indicator that the body has been moved from another region in which the seasonal conditions are substan‐ tially different from the site of discovery. Widespread use of seasonal information for pupal parasitoids is quite limited at present because diapause details are only firmly established for *N*. *vitripennis*, and even then, only for specific biogeographical locales within North America and parts of Europe [32, 61]. The situation for larval parasitoids is similarly bleak in that though the seasonal occurrence of a few species has been reported [29], diapause has only been examined in the braconid *A*. *manducator*, which synchronizes dormancy with that of its calliphorid hosts [33]. Presumably other species synchronize diapause with that of their hosts and/or enter dormancy during a time that certain fly species are not available for parasitism. It is also quite likely that in certain biogeographical regions, a true diapause does not occur and that senescence similar to that of calliphorid adults that rely on reproductive diapause occurs. Much more research needs to be devoted to examining the seasonality of parasitic Hymenoptera, as the resulting data would open new opportunities for applying parasitoid biology to medicocriminal investigations.


**Table 2.** Characteristics of idiobiont and koinobiont parasitoids associated with carrion breeding flies

#### *3.1.4. Wasp artifacts*

Further complicating the host-parasite relationship in terms of predicting wasp development times is the physiological state of the parasitoids and conditions of parasitism. Female age directly influences efficiency of foraging behavior for hosts, the length of time needed to parasitize a host, which can be especially long for parasitoids using concealed hosts, and the quality of eggs deposited on a host. Eggs from older females may fail to hatch or larvae may spend more time feeding than is typical of progeny produced by younger adults [53, 54]. A similar effect is associated with larval development on flies that have been previously parasi‐ tized by conspecifics or allospecifics [55–57]. Failure to take into account each of these influences can lead to inaccurate calculations of developmental thresholds and estimations of wasp development times, as in most instances unfavorable host conditions increases the duration of parasitoid development [51, 58, 59]. The complex interactions between parasitoids and their hosts underscore the need for standard protocols in collecting wasp development

The limitations discussed in terms of information required to improve the precision of PMI estimations using parasitoids can be overcome with more focused research. It is also important to note that the forensic potential of parasitic wasps is not diminished by gaps in basic knowledge of life history characteristics. In fact, parasitoids have far more potential as alternative indicator species for estimating the PMI than predatory species because of their specificity as parasites. What this means is that though some species may show variability in host preferences, progeny development is entirely tied to feeding on the host. As the fly hosts are linked directly to a corpse, the parasitoid's immature development is a second-level linkage to the conditions associated with the deceased. The same features are not true with predators that visit carrion. Another key difference is that the window of time in which a host is suitable for parasitism is more predictable and generally represents a narrower time period than that in which suitable prey are available for predation. The later is reflective of necrophilous beetles that may feed on animal remains as well as prey upon eggs and fly larvae for several days to weeks, depending on the season and ambient conditions. In comparison, the window of opportunity is quite short when pupal parasitoids (e.g., pteromalids) use puparia of calliphor‐ ids. Hosts are only of useable age for 3–5 days during warmer months, and somewhat longer as temperatures decline. Even this assessment is complicated by the observation that puparial development of *Lucilia sericata* is shorter in the presence of *N*. *vitripennis*, thereby reducing the

stages suitable to serve as hosts for some sarcophagids extend over a much longer duration (1–2 weeks) [51], decreasing the precision of a PMI calculated from wasp developmental data

Seasonality of parasitoids is especially useful for the determination of whether a body has been moved. As with other aspects of parasitoid biology, seasonal occurrence of parasitic wasps is directly linked to fly hosts. For pupal parasitoids, those that enter winter dormancy in the form

C [60]. By contrast, the puparial

data for use in forensic entomology [31].

window for parasitism by almost 1 day at temperatures near 25o

as the age of the host at the time of parasitism is not known (**Table 2**).

*3.1.2. Host preferences*

74 Forensic Analysis - From Death to Justice

*3.1.3. Seasonality*

The vast majority of parasitoids utilizing carrion-inhabiting flies as hosts emerge as adults from host puparia, regardless of whether they are larval or pupal parasitoids. Exit holes chewed in puparia reflect species preferences for emergence location and the size of the hole often typifies species [1]. Pupal exuvia also remain within the puparium, along with any unemerged larvae or pupae, providing clues regarding species identity and possibly devel‐ opmental duration, which are useful in establishing a PMI based on a particular wasp species and host. Such information has also proven useful to the specialized discipline of forensic archaeoentomology, in which parasitized puparia provided insight into the burial practices of pre-Columbian civilizations in Peru [42].

Molecular artifacts may also be associated with parasitic wasps, potentially revealing infor‐ mation regarding developmental conditions for the wasps and/or host species. The artifacts are in the form of heat shock proteins (hsps), produced in response to various stresses experienced during development, most frequently while progeny are feeding on fly hosts. For example, larvae of *N*. *vitripennis* demonstrate up-regulation of hsp 23, 60, and 70 when developing on hosts that have experienced overcrowded conditions in larval aggregations [52]. Hsp expression levels correspond with species and size of maggot masses experienced by the host. Similarly, fly hosts synthesize specific hsps in response to maggot mass dynamics, and the expression continues during pupal and early pharate adult development [62], a window of time in which pupal parasitoids oviposit on discovered flies. These observations suggest the possibility that molecular markers associated with hsp expression or associated with altered gene expression of other proteins may be useful in deciphering the developmental conditions experienced by parasitic wasps prior to discovery, and may also reveal limited but useful information concerning ambient temperatures realized by their fly hosts [52]. Much more research is needed to determine if such possibilities are feasible.
