Carrion insects living within the bones of large mammals: insect conservation and forensic entomology implications.

Succession patterns of carrion insects on large mammal's carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone's inner cavity, was monitored with a camera to assess the insect's putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

Identification of forensically important Sarcophaga species (Diptera: Sarcophagidae) using the mitochondrial COI gene.

The identification of species of the forensically important genus Sarcophaga is very difficult and requires strong taxonomic expertise. In this study, we sequenced the mitochondrial cytochrome c oxidase subunit I (COI) gene of 126 specimens of 56 W European Sarcophaga species and added GenBank data to our database to yield a total dataset of 270 COI sequences from 99 Sarcophaga species to evaluate the COI gene as a molecular diagnostic tool for species identification in this genus. Using two simple criteria (Best Match, BM and Best Close Match, BCM), we showed that the identification success using a mini-barcode region of 127 bp was very low (80.7-82.5 %) and the use of this region is not recommended as a species identifier. In contrast, identification success was very high using the standard barcode region (658 bp) or using the entire COI region (1,535 bp) (98.2-99.3 %). Yet, there was a low interspecific sequence divergence (<2 %) in six species groups so that for 16 out of the 99 species (nine of which are of forensic importance), the use of COI barcodes as species identifier should be done with care. For these species, additional markers will be necessary to achieve a 100 % identification success. We further illustrate how such reference databases can improve local reference databases for forensic entomologists.

A new aberrant Doryctinae genus (Hymenoptera: Braconidae) from French Guiana.

A new genus, Cerritulus gen. nov., with type species, C. forticrura sp. nov., are described from French Guiana based on a single specimen that is considerably distinct morphologically from all known Neotropical genera. The new genus is only partially similar to the enigmatic, monotypic Asian genera Ceylonspathius Belokobylskij and Termitospathius Beloko-bylskij, whose species were collected inside termite nests. We provide colour pictures for the type species of the above three genera.

Preservation of iridescent colours in Phorinia Robineau-Desvoidy, 1830 (Diptera: Tachinidae).

BACKGROUND: Iridescent blue-green colours are exhibited by various organisms including several taxa in the Tachinidae (Diptera) with notable examples within the Afrotropical members of the genus Phorinia Robineau-Desvoidy, 1830. The vivid colouration observed in life quickly fades to a dull golden-yellow when a specimen is dried. Although well known, no published explanation has been given for this phenomenon. NEW INFORMATION: We illustrate the mechanism associated with this colour change. We also test and propose technical alternatives to retain the living colours in dried specimens.

Preliminary data on pupal development, lifespan and fertility of Cynomyamortuorum (L., 1761) in Belgium (Diptera: Calliphoridae).

BACKGROUND: The calliphorid Cynomyamortuorum (L., 1761) is a species of forensic interest, present mainly in the Palaearctic Region. Nearly nothing is known about its life history. NEW INFORMATION: We provide here the first data regarding pupal weight evolution during the pupal stage, female fertility and life expectancy of the species. At 22°C under a variable regime of temperatures, the egg-to-adult development time was an average of 18.05 ± 0.72 and 18.47 ± 0.67 days for females and males, respectively, in the control group. The pupal stage represented 56.7% of the total development. The development time from egg to adult and the duration of the pupal stage were significantly longer for males than for females. The measurement of pupal weight at the start of the pupal period revealed that female pupae were significantly lighter than male pupae by nearly 20%. This difference between the sexes was also observed for the dry weight of adults. An average decrease of 8.75% was observed throughout the first 8 days of the pupal stage, after which most adults started to emerge. The tested females produced an average of 176.13 ± 66.62 eggs throughout the egg-laying period. The average lifespan after emergence was 12.10 ± 4.09 days for females and 12.60 ± 2.95 days for males, with a median of 12.50 days for both sexes.

A new genus and two new species of Alysiinae (Hymenoptera: Braconidae) from Papua New Guinea.

Two new species of the genus Leptotrema van Achterberg, 1988 and one new genus of the Alysiinae from Mt Wilhelm, Madang Province, Papua New Guinea are described and illustrated. The distribution of the genus Leptotrema is extended to the Australasian region. Three new combinations are established Idiasta fulmeki (Fischer, 2010) comb. n. (=Microcrasis fulmeki Fischer, 2010), Apiasta postfurcata (Papp, 1966) comb. n. (= Microcrasis postfurcata (Papp, 1966)), and Leptotrema. bovefemora (Bhat, 1979) comb. nov. (=Aspilota bovefemora Bhat, 1979). The status of the genus Apiasta Wharton, 2002 (stat. n.) is reviewed. Keys are provided to enable their identification.

DNA barcoding and the differentiation between North American and West European Phormia regina (Diptera, Calliphoridae, Chrysomyinae).

Phormia regina (the black fly) is a common Holarctic blow fly species which serves as a primary indicator taxon to estimate minimal post mortem intervals. It is also a major research model in physiological and neurological studies on insect feeding. Previous studies have shown a sequence divergence of up to 4.3% in the mitochondrial COI gene between W European and N American P. regina populations. Here, we DNA barcoded P. regina specimens from six N American and 17 W European populations and confirmed a mean sequence divergence of ca. 4% between the populations of the two continents, while sequence divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence divergences were observed for COII (3.7%) and cyt b (5.3%), but mean divergence was lower for 16S (0.4-0.6%). Intercontinental divergence at nuclear DNA was very low (≤ 0.1% for both 28S and ITS2), and we did not detect any morphological differentiation between N American and W European specimens. Therefore, we consider the strong differentiation at COI, COII and cyt b as intraspecific mtDNA sequence divergence that should be taken into account when using P. regina in forensic casework or experimental research.

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France.

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available. We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems - BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.

Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic?

Species of the fly genus Lucilia are commonly used in forensic investigations to estimate the postmortem interval (PMI). Two close-related species Lucilia caesar and L. illustris are difficult to identify. Previous studies showed that the mitochondrial cytochrome c oxidase subunit I (COI) marker could be used to identify many Lucilia species. However, mixed results were obtained for L. caesar and L. illustris due to some European specimens showing identical haplotypes. Here, we investigated 58 new European male specimens of L. illustris and L. caesar whose morphological identifications were checked and for which COI fragments were sequenced. In addition, two other mitochondrial (cytochrome c oxidase subunit II and 16S) and two nuclear (internal transcribed spacer 2 and 28S ribosomal RNA) markers were obtained for a subset of these samples. For each marker, genetic divergence within each species was in the same range as between species, confirming the close relationship between both species. Moreover, for each of the gene fragments, both species shared at least one haplotype/genotype. Hence, none of the molecular markers tested could be used, alone or in combination, to discriminate between L. illustris and L. caesar.