New approach to application of mid-infrared photoacoustic spectroscopy in forensic analysis: Study with the necrophagous blow fly Chrysomya megacephala (Diptera: Calliphoridae).

Insects can provide clues in a variety of ways to assist in criminal investigations. The FTIR-PAS technique has been successfully used to assess the cuticular chemical profiles of insect samples from different groups and for several goals. However, until now, it has never been used to evaluate samples of forensic interest, despite providing faster results, compared to the methods currently used. In this study, mid-infrared photoacoustic spectroscopy was employed to assess the cuticular chemical profiles of different stages of development of the blow fly Chrysomya megacephala sampled from two distinct populations. The results showed that this technique enabled detection of significant differences between the main vibrational modes of the chemical bonds present in the cuticles of the two populations and the different stages of development of the fly. The method enables identification of the age of individuals collected at the crime scene, as well as the distinction of different populations. Therefore, this methodology could assist in forensic investigations, in both estimating the Postmortem Interval and determining the location where the crime occurred, or whether the body had suffered some type of translocation. The technique provides high reproducibility and fast analysis, so its application for analysis of C. megacephala is a viable option in forensic crime investigations.

Proteomic analysis of the venom of the social wasp Apoica pallens (Hymenoptera: Vespidae)

ABSTRACT Wasps are a diverse group of insects that possess a sting apparatus associated with a venom gland, which is used for predation and colony defense. The biochemistry of Hymenoptera venom has been evaluated in relation to allergy and immunology, and proteomics has been shown to be a powerful tool for the identification of compounds with pharmacological potential. Data on wasps venom the of genus Apoica are scarce, so the objective of the present work was to identify the venom proteins of the eusocial wasp Apoica pallens, as a first step towards further investigation of applied uses of the venom and its protein constituents. The venom proteins were separated by two-dimensional gel electrophoresis, followed by MALDI-TOF/TOF mass spectrometry. A total of 259 spots were detected, with molecular weights from 4.9 to 141 kDa. Thirty of these proteins were identified and classified into eight functional categories: allergen, enzyme, metabolism, structural, environmental response, proteoglycan, active in DNA and RNA, and unknown function. Due to the few available proteomic data for wasp venom, many proteins could not be identified, which makes studies with proteomic analysis of Hymenoptera venom even more important.

Hydrocarbon and Fatty Acid Composition from Blowfly Eggs Represents a Potential Complementary Taxonomic Tool of Forensic Importance.

One of the most important contributions of forensic entomology is to assist criminal expertise to determine the postmortem interval, which depends on the duration of the immature stages of insects of forensic interest. On the other hand, the time of development of the different stages varies according to the species; therefore, its identification is essential. Currently, few studies have investigated the use of cuticular hydrocarbons, and none regarding fatty acids, as complementary taxonomic tools to expedite species identification. Therefore, we evaluated whether cuticular hydrocarbons together with fatty acids of eggs of flies of the family Calliphoridae, main group of forensic interest, can be used to distinguish species. The analyses were performed by chromatographic techniques. The results show that there are significant differences between the composition of cuticular hydrocarbons and fatty acids between species and, therefore, they can be used to provide a complementary taxonomic tool to expedite the forensic expertise.

Effect of temperature on the chemical profiles of nest materials of social wasps.

Social insects depend on their nests for protection against predation and abiotic threats. Accordingly, the chemical compounds present in the material wasps use to build their nests can both facilitate communication and repel predators. It is herein hypothesized that different wasp species build their nests with different structure and substrate materials and that such materials consist of chemical compounds related to unique wasp behavior and outside temperature variation. To test this hypothesis, nests were collected from three species of social wasps, the samples of which were subjected to temperature variation under laboratory conditions. The compounds present in the substrate were analyzed by gas chromatography coupled to mass spectrometry. Chemical compounds identified in the nest material of the three species responded differently to temperature variation. Chemical compounds from Polybia nests were altered significantly when subjected to temperature variation, whereas the nests of Polistes versicolor did not significantly change in relation to the control. The differences found between Polistes and Polybia nests may be related to genetic factors, but also to the type of nest they construct. It is possible that divergent evolutionary strategies for maintaining colony temperature, as a function of the chemical composition of the nests, may have appeared between wasps that have open and closed nests. In relatively small colonies, nest substrate is more resistant to temperature variation because it is composed of a greater diversity of elements and thus capable of holding heavier, longer carbon chains. Our results suggest that chemical compounds in the nest material of the three wasp species analysed responded differently to fluctuating ambient temperatures and that such variation could result from the biochemical differences of unique wasp species or from thermoregulation strategies of colonies.

Effect of temperature on survival and cuticular composition of three different ant species.

Climatic factors, such as temperature variation, interfere with the survival of insects. To respond to these variations, insects have some specific characteristics. These include water content of the body, thickness of the lipid layer, as well as the qualitative and quantitative characteristics of cuticular chemical components. This study hypothesizes that different ant species respond to temperature changes in different ways and that such differences may be associated with cuticle hydrocarbons (CHCs) and fatty acids. As model ant species, Atta sexdens, Odontomachus bauri and Ectatomma brunneum were used for experimental analyses. Ants were submitted to a water bath for 5 h at different temperatures, and their CHCs and fatty acids were identified and quantified, followed by correlating these chemical compounds with temperature variations and the survival. Temperatures below 30 °C did not affect the survival of the three species. E. brunneum had a higher percentage of survival at temperatures above 30 °C. O. bauri was the most sensitive species with 100% mortality at 40 °C. Survival was found to be unrelated to any of the identified fatty acids. However, CHCs underwent significant quantitative and qualitative variation, as shown by an increased percentage of CHCs with longer chain length of linear alkanes at temperatures above 30 °C. These increase enables these ants to maintain the integrity of their cuticle and survive at temperatures above 30 °C. It can be concluded that the forager ants studied respond differently to temperature variation and that changes in the conformation of CHCs are in line with the ecological characteristics of the different studied species because, they vary in terms of diurnal/nocturnal foraging and types of environments foraged. Among the three species, E. brunneum foragers were found to be more active under adverse conditions and more tolerant to temperature variation with the correspondingly appropriate changes in CHCs composition.

New method for estimating the post-mortem interval using the chemical composition of different generations of empty puparia: Indoor cases.

Most flies of forensic importance are in two superfamilies, the Muscoidea and the Oestroidea, with similar life stages including the puparium. Upon completion of metamorphosis the adult fly emerges from the puparium, leaving behind an exuvia that is of potential significance in forensic investigation. The empty puparium is a durable piece of entomological evidence lasting several years. Through the study of chemical compounds, specifically the hydrocarbons of these puparia, it is possible to identify the species, in addition to how long they have been exposed to weathering and for this reason, these parameters can assist forensic entomologists in estimating long-term postmortem interval (minPMI). In corpses that take a relatively longer time to decompose, insects may use the same corpses for several oviposition cycles. Therefore, the aim of this study was to develop a new method to determine the PMI based on chemical compounds of the puparia from different oviposition cycles of the fly Chrysomya megacephala. The chemical composition of 50 puparia from different cycles of oviposition were evaluated by Gas Chromatography-Mass Spectrometry (GC-MS). In total, 60 compounds were identified ranging from C18 to C34, 38 of those were common to all generations. Our results demonstrate that chemical profiles can be used to differentiate puparia collected from successive cycles, and therefore valuable in the estimation of minPMI.

Observation of intra- and interspecific differences in the nest chemical profiles of social wasps (Hymenoptera: Polistinae) using infrared photoacoustic spectroscopy.

The aim of this study was to explore whether the nest chemical profile (NCP) can be used to determine intra- and interspecific differences in social wasps of the subfamily Polistinae. For this purpose, Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) was used to directly analyze small pieces of nest as well as the gasters of females. An advantage of the methodology was that no sample preparation was required. FTIR-PAS combined with multivariate discriminant analyzes was used, to the best of our knowledge for the first time, to evaluate the NCPs of six species of polistine wasps, observing the influence of the nesting environment and investigating the relationships among the nest and female cuticular chemical profiles. The results revealed significant inter-species differences among the NCPs, as well as strong correlations with the environments in which the nests were located. The Mischocyttarus and Polistes species belong to the same tribe (Polistini) and therefore exhibited similar NCPs. These species were separated from the Polybia species in the NCP dendrogram, mainly because the Polybia belong to another tribe (Epiponini). Correlation was observed between the NCPs and the cuticular chemical profiles of females. The findings of the study demonstrated the importance of the NCP for differentiation of species and environments, and the utility of FTIR-PAS for identification of correlations between individuals and nests. The results confirmed the hypothesis that the NCP can be used as an additional tool for intra- and interspecific differentiation in social wasps of the subfamily Polistinae.

Chemotaxonomic Profile and Intraspecific Variation in the Blow Fly of Forensic Interest Chrysomya megacephala (Diptera: Calliphoridae).

Necrophagous insects such as blow flies (Diptera: Calliphoridae) are considered crucial in forensic entomology. Identification at species level and determination of larval stage are the basis for estimation of postmortem interval (PMI). Insect evidence can also be used in the determination of crime scenes, since body displacement is common. The aim of this study was to determine the chemotaxonomic profile and intraspecific variability of the forensically important blow fly Chrysomya megacephala (F. 1794). Adults were collected in the municipalities of Dourados-MS (Brazil) and Rio Claro-SP (Brazil), and then transferred to the laboratory for oviposition and development of the immature stages. Chemical analysis of cuticular compounds was performed by gas chromatography. Cuticular chemical profiles varied significantly between the two populations, as well as between developmental stages, supporting the use of these compounds as a complementary tool to help identify the species and its stages, along with geographical variability. This could greatly accelerate forensic investigations, eliminating the need to allow the fly larvae to develop until adult stage in order to confirm the species identity and sample origin.

Action of Ants on Vertebrate Carcasses and Blow Flies (Calliphoridae).

Forensic entomology is a science that uses insect fauna as a tool to assist in criminal investigations and civil proceedings. Although the most researched insects are the Diptera and Coleoptera, ants may be present in all stages of decomposition. The aim of this study was to evaluate the role of ants and their action on blow flies during the decomposition process. Experiments were performed in which four pig carcasses were exposed in the cold and dry season (November/2012 and March/2013) and four in the hot and wet season (May/2013 and August/2013). Flies were the first insects to detect and interact with the carcasses, and six species of the Calliphoridae family were identified. Ants (Hymenoptera: Formicidae) were the second group, with six subfamilies identified. Myrmycinae represented 42% of the species, followed by Formicinae (28%), Ectatominae and Ponerinae (both 10%), and Ecitoninae and Dolichoderinae (both 5%). The ants acted on the carcasses as predators of visiting species, omnivores, and necrophagous, in all cases significantly affecting the decomposition time, slowing it down when the ants preyed on adult and immature insects consuming the carcass, or accelerating it by consuming the carcass and creating holes that could serve as gateways for the action of other organisms. The ants also generated artifacts that could lead to forensic misinterpretation.

Polydomy in the ant Ectatomma opaciventre.

Tropical ants commonly exhibit a hyper-dispersed pattern of spatial distribution of nests. In polydomous species, nests may be satellites, that is, secondary structures of the main nest, where the queen is found. In order to evaluate whether the ant Ectatomma opaciventre Roger (Formicidae: Ectatomminae) uses the strategy of building polydomous nests, the spatial distribution pattern of 33 nests in a 1,800 m(2) degraded area located in Rio Claro, SP, Brazil, were investigated using the nearest neighbor method. To complement the results of this investigation, the cuticular chemical profile of eight colonies was analyzed using Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). The nests of E. opaciventre presented a hyper-dispersed or regular distribution, which is the most common in ants. The analysis of the cuticular hydrocarbons apparently con-firmed the hypothesis that this species is polydomous, since the chemical profiles of all studied colonies with nests at different sites were very similar to the chemical signature of the single found queen and were also different from those of colonies used as control.

Foraging strategies of the ant Ectatomma vizottoi (Hymenoptera, Formicidae)

Foraging strategies of the ant Ectatomma vizottoi (Hymenoptera, Formicidae). Foraging activity may be limited by temperature, humidity, radiation, wind, and other abiotic factors, all of which can affect energy costs during foraging. Ectatomma vizottoi's biology has only recently been studied, and no detailed information is available on its foraging patterns or diet in the field. For this reason, and because foraging activity is an important part of the ecological success of social insects, the present study aimed to investigate E. vizottoi's foraging strategies and dietary habits. First, we determined how abiotic factors constrained E. vizottoi's foraging patterns in the field by monitoring the foraging activity of 16 colonies on eight different days across two seasons. Second, we characterized E. vizottoi's diet by monitoring another set of 26 colonies during peak foraging activity. Our results show that E. vizottoi has foraging strategies that are similar to those of congeneric species. In spite of having a low efficiency index, colonies adopted strategies that allowed them to successfully obtain food resources while avoiding adverse conditions. These strategies included preying on other ant species, a foraging tactic that could arise if a wide variety of food items are not available in the environment or if E. vizottoi simply prefers, regardless of resource availability, to prey on other invertebrates and especially on other ant species.

Division of labor in colonies of the eusocial wasp, Mischocyttarus consimilis.

The division of labor between castes and the division of labor in workers according to age (temporal polyethism) in social wasps are crucial for maintaining social organization. This study evaluated the division of labor between castes, and the temporal polyethism in workers of Mischocyttarus consimilis Zikán (Hymenoptera: Vespidae). To describe the behavioral repertory of this species, observations were made of 21 colonies, with 100 hours of observations. In order to observe temporal polyethism, each newly emerged wasp was marked with colored dots on the upper area of the thorax. This allowed the observation of behavioral acts performed by each worker from the time of emergence to its death. Through hybrid multidimensional scaling, a clear division between queens and workers could be identified, in which the behaviors of physical dominance and food solicitation characterized the queen caste; while behaviors such as adult-adult trophallaxis, destruction of cells, alarm, foraging for prey, foraging for nectar, and unsuccessful foraging characterized the worker caste. Hybrid multidimensional scaling characterized two groups, with intra-nest activities preferentially accomplished by younger workers, while extra-nest activities such as foraging were executed more frequently by older workers.

First report of interspecific facultative social parasitism in the paper wasp genus Mischocyttarus Saussure (Hymenoptera, Vespidae) / Primeiro registro de parasitismo social facultativo interespecífico em vespas do gênero Mischocyttarus Saussure (Hymenoptera, Vespidae)

First report of interspecific facultative social parasitism in the paper wasp genus Mischocyttarus Saussure (Hymenoptera, Vespidae). Parasitism of colonies of the social wasp Mischocyttarus cerberus Ducke, 1918 by females of Mischocyttarus consimilis Zikán, 1949 was observed in a rural area of Dourados, state of Mato Grosso do Sul, Brazil. In all monitored cases, the invasion occurred in the pre-emergence colony stage, generally by a single female of M. consimilis. The period of establishment of the foreign female in the host colony was marked by antagonistic behaviors between the host female and the invasive. In general, the architecture of the parasitized nest was modified from the typical architecture of the host species nest.

Primeiro registro de parasitismo social facultativo interespecífico em vespas do gênero Mischocyttarus Saussure (Hymenoptera, Vespidae). Parasitismo de colônias da vespa social Mischocyttarus cerberus Ducke, 1918 por fêmeas de Mischocyttarus consimilis Zikán, 1949 foram registrados em uma área rural no município de Dourados estado de Mato Grosso do Sul no Brasil. Em todos os casos monitorados a invasão ocorreu na fase colonial de pré-emergência, e em geral foi executado por uma única fêmea de M. consimilis. O período de estabelecimento da fêmea estrangeira na colônia hospedeira foi marcado por comportamentos antagônicos entre as fêmeas interespecíficas. Em geral, a arquitetura do ninho parasitado foi modificada em relação à arquitetura típica do ninho da espécie hospedeira.

Colony cycle of the social wasp Mischocyttarus consimilis Zikán (Hymenoptera, Vespidae)

Colony cycle of the social wasp Mischocyttarus consimilis Zikán (Hymenoptera, Vespidae). This study describes some aspects of the colony cycle of the Neotropical social wasp Mischocyttarus consimilis, from data obtained under field conditions. Our results showed that the colony cycle in M. consimilis is annual and asynchronous in relation to the months of the year. The colonies remained active for approximately eight months. Most of the abandonments were associated with natural causes, and were most frequent in the pre-emergence stage. The nests were constructed preferentially in man-made structures, especially in sites protected from direct sunlight and rain. Colony foundation was either by haplometrosis or pleometrosis, being the first form predominant.

Ciclo colonial da vespa social Mischocyttarus consimilis Zikán (Hymenoptera, Vespidae). Este estudo descreve alguns aspectos do ciclo colonial da vespa social Neotropical Mischocyttarus consimilis, sendo realizado em condições de campo. Os resultados desse estudo mostraram que o ciclo colonial em M. consimilis é anual, bem como assincrônico em relação aos meses do ano. Em geral, as colônias permanecem em atividade por aproximadamente oito meses. Os abandonos de colônia estão associados principalmente a causas naturais, e são mais freqüentes na fase colonial de pré-emergência. Os ninhos são estabelecidos preferencialmente em construções humanas, especialmente em locais protegidos da incidência direta de luz solar e água da chuva. Fundações de colônia ocorrem por uma ou outra forma haplometrose ou pleometrose, sendo a primeira predominante.

Nest architecture, colony productivity, and duration of immature stages in a social wasp, Mischocyttarus consimilis.

Tis study examined the nest architecture, colony productivity, and duration of the immature stages of the social wasp Mischocyttarus consimilis Zikán (Hymenoptera: Vespidae). The study was carried out under field conditions. Nests of M. consimilis consist of a single uncovered comb, which is attached to the substratum by a single petiole. The data for the nest architecture showed a positive and significant correlation between the size of the comb and the diameter of the petiole, and also between the height and diameter of the cells. The nests were constructed on horizontal, vertical, and sloping substrata with no apparent preference for a specific orientation. The colonies produced 72.9 cells and 40.7 adults on average. The mean frequency of productive cells was 33.3%, and 19.4% of the cells were reused. The mean duration of the immature stages combined was 69.7 days and the egg, larval, and pupal stages had mean durations of 14.9, 36.0, and 18.8 days, respectively. The duration of each immature stage was significantly shorter in the warm-humid season, and the larval and pupal stages were shorter during the colony pre-emergence stage.

Nest structure and occurrence of three species of Azteca (Hymenoptera, Formicidae) in Cecropia pachystachya (Urticaceae) in non-floodable and floodable pantanal areas / Arquitetura de ninho e ocorrência de três espécies de Azteca (Hymenoptera, Formicidae) em Cecropia pachystachya (Urticaceae) em ambiente alagável e não alagável no Pantanal

Thirty Cecropia pachystachya trees were examined in non-floodable and floodable areas to investigate the association between C. pachystachya and Azteca ants in the Pantanal of Mato Grosso do Sul, Brazil. The species Azteca ovaticeps, Azteca isthmica, and Azteca alfari were found nesting inside domatia of C. pachystachya. A. ovaticeps was the most frequent species in the trees in the floodable area, while A. isthmica and A. alfari, in the non-floodable area. A. ovaticeps and A. isthmica maintained more entrance/exit holes in comparison to A. alfari. All Azteca species maintained entrance/exit holes in the closest domatia to the apical area of the branch, due to proximity to Müllerian and pearl bodies, suggesting that these species of Azteca were influenced by their environment during evolution and became specialized. All internodal septa of each examined branch were perforated by ants, indicating the branches were inhabited by a single colony.

Foram analisadas 30 plantas de Cecropia pachystachya em cada ambiente alagável e não alagável no Pantanal sul-mato-grossense, Brasil, com o objetivo de investigar a associação entre formigas Azteca e C. pachystachya. Foram encontradas as espécies Azteca ovaticeps, Azteca isthmica e Azteca alfari nidificando nas domáceas da planta. A. ovaticeps foi mais frequente em plantas de área alagável, enquanto A. isthmica e A. alfari em plantas em área não alagável. A. ovaticeps e A. isthmica apresentaram maior quantidade de orifícios de entrada/saída em relação à A. alfari e todas as espécies mantêm próximo da região apical do ramo, orifícios de entrada/saída nas domáceas, devido à proximidade com os corpúsculos müellerianos e pérola. Isto é, essas espécies de Azteca especializaram-se ao longo da evolução influenciadas pelo ambientes. Todos os septos internodais de cada ramo analisados apresentaram-se perfurados pelas formigas, sugerindo que os mesmos são habitados por uma única colônia.