Development of mixed linear models to analyze and describe the impact of malathion on the larval growth of Megaselia scalaris (Diptera: Phoridae) under various feeding media and environmental conditions.

Forensic entomology plays a crucial role in estimating the minimum postmortem interval through the study of insect larvae found at crime scenes. The precision of this estimation relies on various biotic and abiotic elements that simultaneously influence insect growth and development, encompassing factors such as temperature, humidity, photoperiod, diet, and the existence of xenobiotics in decomposing tissues. Despite numerous studies on the influence of these factors, including the impact of xenobiotics, there are currently no robust tools available for making corrections to this estimation considering concurrently all variables. In an attempt to propose an exploratory and descriptive statistical model to analyze the simultaneous effect and interaction of different variables on larval growth, this study aimed to compare the effect of malathion on the growth of Megaselia scalaris (Loew, 1866) (Diptera: Phoridae) raised in malathion-spiked porcine muscle, under controlled and uncontrolled temperature and humidity conditions (environmental conditions). Larvae were also reared using various growth media. A split-plot design that combined crossed and nested factors was employed; 2 linear mixed models were developed to assess the relationships between the variables. The model provides valuable insights into the complex interactions among xenobiotics, growth media, and environmental conditions in the size and development of M. scalaris.

Evaluation of the effect of clonazepam and its metabolites on the life cycle of Megaselia scalaris (Loew) (Diptera: Phoridae).

Fly colonization patterns and development are crucial in estimating the post-mortem interval (PMI) of decomposing corpses. Understanding the potential effects of xenobiotics on species development in cadaveric entomofauna is essential for accurate PMI estimation, given their presence in decomposing bodies. Benzodiazepines, commonly prescribed for their anxiolytic, hypnotic, and muscle relaxant effects, are of forensic interest due to their potential for abuse, dependence, intoxication, and overdose-related deaths. This study aimed to explore the effect of clonazepam and its metabolites on Megaselia scalaris, a species commonly used to estimate PMI, the alteration of which could impact the accuracy of said estimation. The S9 biotransformation fraction, an in vitro model consisting of an array of metabolic enzymes, was used to generate phase I and II metabolites for evaluating their effect on M. scalaris development, representing an innovative approach to this type of study. Megaselia scalaris larvae were reared in synthetic growth media under controlled conditions. The study compared different groups: control, clonazepam, and clonazepam with S9 fraction. Larvae were measured daily to determine growth rate, and clonazepam concentrations were analyzed using HPLC-DAD. Results showed that larvae grown in media containing clonazepam or clonazepam with S9 fraction developed faster than control larvae, reaching their pupal stage earlier. Growth rates were also altered in treated groups. In conclusion, the presence of clonazepam and its metabolites accelerated the life cycle of M. scalaris, potentially impacting the accuracy of PMI estimation. These findings underscore the importance of considering xenobiotics in forensic entomological studies for precise post-mortem interval determination.

Fannia pretiosa-group (Diptera: Fanniidae) from Mexico: six new species from the Mexico Transition Zone and an identification key.

Fannia Robineau-Desvoidy is found in all biogeographic regions. The genus contains approximately 360 known species, 18 of which have been recorded from Mexico. Herein, we describe six new species in the Fannia pretiosa-group, redescribe Fannia gilvitarsis Chillcott, and provide diagnosis, habitus images, drawings of the male terminalia, and an identification key for all species of the group that occur in Mexico. All Fannia pretiosa-group species from Mexico share a similar morphology of the mid tibia, hind femur, and male terminalia.

A miRNome analysis at the early postmortem interval.

The postmortem interval (PMI) is the time elapsing since the death of an individual until the body is examined. Different molecules have been analyzed to better estimate the PMI with variable results. The miRNAs draw attention in the forensic field to estimate the PMI as they can better support degradation. In the present work, we analyzed the miRNome at early PMI in rats' skeletal muscle using the Affymetrix GeneChip™ miRNA 4.0 microarrays. We found 156 dysregulated miRNAs in rats' skeletal muscle at 24 h of PMI, out of which 84 were downregulated, and 72 upregulated. The miRNA most significantly downregulated was miR-139-5p (FC = -160, p = 9.97 × 10-11), while the most upregulated was rno-miR-92b-5p (FC = 241.18, p = 2.39 × 10-6). Regarding the targets of these dysregulated miRNAs, the rno-miR-125b-5p and rno-miR-138-5p were the miRNAs with more mRNA targets. The mRNA targets that we found in the present study participate in several biological processes such as interleukin secretion regulation, translation regulation, cell growth, or low oxygen response. In addition, we found a downregulation of SIRT1 mRNA and an upregulation of TGFBR2 mRNA at 24 h of PMI. These results suggest there is an active participation of miRNAs at early PMI which could be further explored to identify potential biomarkers for PMI estimation.

Multi-locus phylogeny of the catfish genus Ictalurus Rafinesque, 1820 (Actinopterygii, Siluriformes) and its systematic and evolutionary implications.

BACKGROUND: Ictalurus is one of the most representative groups of North American freshwater fishes. Although this group has a well-studied fossil record and has been the subject of several morphological and molecular phylogenetic studies, incomplete taxonomic sampling and insufficient taxonomic studies have produced a rather complex classification, along with intricate patterns of evolutionary history in the genus that are considered unresolved and remain under debate. RESULTS: Based on four loci and the most comprehensive taxonomic sampling analyzed to date, including currently recognized species, previously synonymized species, undescribed taxa, and poorly studied populations, this study produced a resolved phylogenetic framework that provided plausible species delimitation and an evolutionary time framework for the genus Ictalurus. CONCLUSIONS: Our phylogenetic hypothesis revealed that Ictalurus comprises at least 13 evolutionary units, partially corroborating the current classification and identifying populations that emerge as putative undescribed taxa. The divergence times of the species indicate that the diversification of Ictalurus dates to the early Oligocene, confirming its status as one of the oldest genera within the family Ictaluridae.

Neriidae (Diptera: Schizophora) from Mexico: new species, new records and an identification key.

We conducted a study of material from the Colección de Artrópodos de Referencia Forense, UNAM (CARF), Colección Nacional de Insectos, UNAM (CNIN) and Colección de Insectos del Bosque Seco, Estación de Biología Chamela, UNAM (EBCH), which revealed three new distributional records of cactus flies species in Mexico as follows: Cerantichir enderleini (Hennig, 1937), Eoneria blanchardi Aczél, 1951, and Glyphidops durus (Cresson, 1926). This study also described two new species: Cerantichir mexicana Gomes & Sepúlveda and Eoneria ramirezi Gomes & Sepúlveda. We update the distribution records of the cactus flies (Neriidae) distributed in Mexico. The list of species includes: Glyphidops filosus (Fabricius, 1805), Glyphidops flavifrons (Bigot, 1886), Glyphidops filosus (Fabricius, 1805), Glyphidops xanthopus (Schiner, 1868), Nerius pilifer Fabricius, 1805, Nerius plurivittatus Bigot, 1886, Nerius purpusianus Enderlein, 1922, Odontoloxozus longicornis (Coquillett, 1904), and Odontoloxozus pachycericola Mangan & Baldwin, 1986. This study increases the number of species known to occur in Mexico from eight to 13.

Effect of Malathion on the Development of Megaselia scalaris (Loew, 1866) (Diptera: Phoridae), a Forensically Important Fly.

Malathion is a widely used organophosphorus pesticide; it is also a molecule of forensic interest due to its moderate to high toxicity in nontarget organisms, humans included. This compound is present in some fatal intoxications, accidental or intentional; its presence in the tissues on which the cadaveric entomofauna feeds may affect its growth rate and life cycle duration leading to an error in the estimation of the minimum postmortem interval (PMImin). Since the toxic effect of malathion on the cadaveric entomofauna could affect the estimation of the PMImin, the aim of this work was to study the toxic effect of malathion on the growth and development of the scuttle fly, Megaselia scalaris, a fly of forensic interest which plays an important role in forensics cases related to human remains found indoors or in concealed environments. The study was complemented with some morphological observations; no morphological changes were observed in the larvae, nor the adult flies exposed to malathion. Malathion affects the viability of the egg and pupa, it also reduces the larval growth rate and increases the duration of the larval stage; therefore, the estimation of the PMImin, with this species when malathion is present in tissues, could be affected.

A new species of Procambarus (Decapoda, Cambaridae) from the State of Querétaro, Mexico.

With a Nearctic distribution, the family Cambaridae harbors a high species richness in Mexico, which is also evident along the Pánuco River catchment. A series of surveys carried on in five populations from the Sierra Gorda Biosphere Reserve in the State of Querétaro resulted in localizing a putative new species for science. A molecular phylogenetic study and species delimitation analyses including all the known Procambarus species from the Pánuco River catchment were conducted based on three mitochondrial genes (16S rDNA, 12S rDNA, and COI; 2,462 bp in total). Phylogeny recovered all species as monophyletic, including the populations under study. All delimitation results based on barcoding, ABGD, GMYC, bPTP, and gonopod differentiation agree in the recognition of a new taxon, to which the name Procambarus xihui sp. nov. is given, and its diagnosis and description are provided. The new species can be distinguished from the remaining species in the genus, among other characters, by a unique configuration of the terminal elements of the first pleopod of form I male, which includes a central projection lamellate, hood-like, forming a concave blade-like structure mesially directed, as well as a caudal process crest-like, mesiodistally directed, forming a lateral side of the concavity.

Species Delimitation of Scavenger Flies in the Valley of Mexico.

Identification of species involved in cadaveric decomposition, such as scavenger Diptera, is a fundamental step for the use of entomological evidence in court. Identification based on morphology is widely used in forensic cases; however, taxonomic knowledge of scavenger fauna is poor for many groups and for many countries, particularly Neotropical ones. A number of studies have documented the utility of a DNA barcoding strategy to assist in the identification of poorly known and diverse groups, particularly in cases involving immature states or fragmented organisms. To provide baseline knowledge of the diversity of scavenger Diptera in the Valley of Mexico, we generated a DNA barcode collection comprised of sequences of the cytochrome c oxidase subunit 1 (COI) gene for all families sampled at a nature reserve located in this region. We collected and identified specimens on the basis of morphology and a species delimitation analysis. Our analyses of 339 individuals delineated 42 species distributed across nine families of Diptera. The richest families were Calliphoridae (9 species), Sarcophagidae (7 species), and Phoridae (6 species). We found many of the species previously recorded for the Valley of Mexico, plus 18 new records for the region. Our study highlights the utility of DNA barcoding as a first-step strategy to assess species richness of poorly studied scavenger fly taxa.

Dysregulation of miR-381-3p and miR-23b-3p in skeletal muscle could be a possible estimator of early post-mortem interval in rats.

BACKGROUND: The post-mortem interval (PMI) is the time elapsed since the dead of an individual until the body is found, which is relevant for forensic purposes. The miRNAs regulate the expression of some genes; and due to their small size, they can better support degradation, which makes them suitable for forensic analysis. In the present work, we evaluated the gene expression of miR-381-3p, miR-23b-3p, and miR-144-3p in skeletal muscle in a murine model at the early PMI. METHODS: We designed a rat model to evaluate the early PMI under controlled conditions. This model consisted in 25 rats divided into five groups of rats, that correspond to the 0, 3, 6, 12 and 24 hours of PMI. The 0 h-PMI was considered as the control group. Muscle samples were taken from each rat to analyze the expression of miR-381-3p, miR-23b-3p, and miR-144-3p by quantitative RT-PCR. The gene expression of each miRNA was expressed as Fold Change (FC) and compared among groups. To find the targets of these miRNAs and the pathways where they participate, we performed an in-silico analysis. From the gene targets of miR-381-3p identified in the silico analysis, the EPC1 gene was selected for gene expression analysis by quantitative RT-PCR in these samples. Also, to evaluate if miR-381-3p could predict the early PMI, a mixed effects model was calculated using its gene expression. RESULTS: An upregulation of miR-381-3p was found at 24 h-PMI compared with the control group of 0 h-PMI and (FC = 1.02 vs. FC = 1.96; p = 0.0079). This was the opposite for miR-23b-3p, which had a down-regulation at 24 h-PMI compared to 0 h-PMI (FC = 1.22 vs. FC = 0.13; p = 0.0079). Moreover, the gene expression of miR-381-3p increased throughout the first 24 h of PMI, contrary to miR-23b-3p. The targets of these two miRNAs, participate in biological pathways related to hypoxia, apoptosis, and RNA metabolism. The gene expression of EPC1 was found downregulated at 3 and 12 h of PMI, whereas it remained unchanged at 6 h and 24 h of PMI. Using a multivariate analysis, it was possible to predict the FC of miR-381-3p of all but 6 h-PMI analyzed PMIs. DISCUSSION: The present results suggest that miR-23b-3p and miR-381-3p participate at the early PMI, probably regulating the expression of some genes related to the autolysis process as EPC1 gene. Although the miR-381-3p gene expression is a potential estimator of PMI, further studies will be required to obtain better estimates.

Population genetics of wild and captive Trachemysvenusta (Gray, 1856) (Reptilia: Emydidae) in the Usumacinta river basin in Mexico.

The Mesoamerican slider Trachemysvenusta is endemic to Central America and Southern Mexico. Several human-mediated disturbances, including habitat degradation and illegal hunting for food, have impacted its populations along the Usumacinta river basin. The extent to which these disturbances have affected the genetic diversity and population structure of T. venusta inhabiting the basin remains unresolved. To this end, we analyzed eight microsatellite markers in five wild populations of T. venusta from the middle and lower reaches of the basin as well as one captive population. Our results show high levels of genetic diversity for all analyzed populations, low F ST values, high gene flow and no genetic structure, indicating an absence of genetic differentiation across sites and, thus, a single panmictic population for the basin. Evidence of a genetic bottleneck was observed in two of the wild populations (and the captive one), indicating some impact from disturbances, whether from poaching or habitat fragmentation, despite the seemingly high connectivity of most populations. Results are discussed in terms of the relative importance of genetic parameters for the conservation of T. venusta, particularly in light of the importance of demographic stochasticity in local conditions undergoing rapid changes.

Six new epigean species of Caecidotea/ (Isopoda: Asellidae) distributed along the Trans-Mexican Volcanic Belt in Central Mexico.

Six new epigean freshwater species of the genus Caecidotea in Mexico are described. These species were collected in waterbodies located along the Trans-Mexican Volcanic Belt (TMVB) and the eastern slope of the country: Caecidotea buzwilsoni sp. nov., Caecidotea chicoensis sp. nov., Caecidotea alvarezi sp. nov., Caecidotea mintzita sp. nov., Caecidotea zacapuensis sp. nov., and one additional species Caecidotea villalobosi sp. nov. is described from the Papaloapan basin at the Atlantic Slope of Mexico. The taxonomic treatment for each species includes a detailed description, figures of characters of diagnostic importance and comments. We also provide an updated map of Caecidotea records in Mexico. This work substantially increases the number of epigean Caecidotea known to occur in Mexico to 11 species, a new total of 99 species described to North America.

Standardization of a Culture Medium for Megaselia scalaris (Diptera: Phoridae) for Entomotoxicological Studies.

One of the problems faced by entomotoxicological studies is the large variability of experimental set-ups and the absence of harmonized protocols to compare the data and results obtained by different research groups. Among the wide range of influencing factors on the development and growth of insects, food substrates are remarkably relevant. This article proposes a standardized growth medium to be employed in future entomotoxicological studies on the scuttle fly Megaselia scalaris (Loew, 1866), (Diptera: Phoridae). This species plays an important role in forensic cases related to the decomposition of human remains found indoors and/or in concealed environments, because of their small size and ability to enter these locations at an earlier time than many other insects. Because of these traits, it can be used for the estimation of the minimum postmortem interval (mPMI). We achieved the formulation of a medium that overcomes two traditional disadvantages of culture media used to raise necrophagous Diptera, the unknown media composition and different growth rates from those reared in tissue. The proposed medium is a known composition formulation, free of xenobiotics, in which M. scalaris shows growth rates and development times similar to those obtained when it is reared in tissue. This new diet might be used in future studies to test the effect of substances of forensic interest (prescription or illicit drugs, poisons, etc.) on the larval development time, helping to adjust the estimation of mPMI based on the presence of such substances. Additionally, the formulation revealed some interesting data about nutritional requirements of this species.

Caecidotea camaxtli (Isopoda: Asellidae) a new species from the Tlaxcala valley, Mexico.

A new freshwater isopod, Caecidotea camaxtli sp. nov. (Isopoda, Asellidae) is described from Mexico. The new species can be distinguished from all other species of Caecidotea by the presence of cuticular scales on the caudal process, as well as a short and simple cannula in the apex of the endopodite of pleopod II. [Zoobank URL: urn:lsid:zoobank.org:act:082CF870-ABC3-4E25-B5F2-8CA59359C2B0].

Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead-end.

Caves are perceived as isolated, extreme habitats with a uniquely specialized biota, which long ago led to the idea that caves are "evolutionary dead-ends." This implies that cave-adapted taxa may be doomed for extinction before they can diversify or transition to a more stable state. However, this hypothesis has not been explicitly tested in a phylogenetic framework with multiple independently evolved cave-dwelling groups. Here, we use the freshwater crayfish, a group with dozens of cave-dwelling species in multiple lineages, as a system to test this hypothesis. We consider historical patterns of lineage diversification and habitat transition as well as current patterns of geographic range size. We find that while cave-dwelling lineages have small relative range sizes and rarely transition back to the surface, they exhibit remarkably similar diversification patterns to those of other habitat types and appear to be able to maintain a diversity of lineages through time. This suggests that cave adaptation is not a "dead-end" for freshwater crayfish, which has positive implications for our understanding of biodiversity and conservation in cave habitats.

Sergey gen. n., a new doryctine genus from temperate forests of Mexico and Cuba (Hymenoptera, Braconidae).

The new doryctine genus Sergey gen. n. is described with four new species (Sergey cubaensis Zaldívar-Riverón & Martínez, sp. n., Sergey coahuilensis Zaldívar-Riverón & Martínez, sp. n., Sergey tzeltal Martínez & Zalídivar-Riverón, sp. n., Sergey tzotzil Martínez & Zalídivar-Riverón, sp. n.) from temperate forests of Mexico and Cuba. Similar to many other doryctine taxa, the new genus has a considerably elongated, petiolate basal sternal plate of the first metasomal tergite, although it can be distinguished from these by having the mesoscutum sharply declivous anteriorly with sharp anterolateral edges. The described species have been characterised molecularly based on two mitochondrial (COI, cyt b) and one nuclear (28S) gene markers. Based on the mitochondrial gene genealogies reconstructed, the evidence suggests the existence of incomplete lineage sorting or hybridization in the populations from Chiapas and Oaxaca assigned to Sergey tzeltal sp. n.

A new species of dwarf crayfish (Decapoda: Cambaridae) from central México, as supported by morphological and genetic evidence.

Dwarf crayfish are a subfamily of freshwater decapods distributed along the southeastern coast of United States and the Central Plateau of México. Recent phylogenetic studies have found that diversity of dwarf crayfish in México could be currently underestimated, and suggested the existence of possible new species for a number of clades for which taxonomic identity was uncertain, including one from Zacapu, a small lake in the Trans-Mexican Volcanic Belt. Here, a congruence criterion between morphological and molecular evidence is used to test if this previously detected clade should be considered as a new species. A set of morphometric variables was taken to characterize variation from this population (including some newly proposed traits possibly valuable for species discrimination) and to compare it statistically to its closest relative, C. chapalanus. Also, additional individuals to those previously sequenced were included using a set of molecular characters, including 5 molecular markers (three mitochondrial and two nuclear fragments) and all extant species described to date from México. Principal Component Analysis, Mann-Whitney paired test and Discriminant Factor Analysis support morphological differentiation of the Zacapu population. Phylogenetic analyses are congruent with morphology and confirm that this population constitutes an exclusive monophyletic clade with high support values. Additionally, genetic Cytochrome Oxidase subunit I (cox1) distance between Zacapu and the closest related species is above the average between species distance in crayfish (ML=3.6%). Congruence between morphology and molecular evidence support the hypothesis that the population from lake Zacapu should be considered a new species, to which the name C. zacapuensis sp. nov. is given and its description provided. With respect to its closest relatives, C. zacapuensis sp. nov. is diagnosed according to the following set of morphological characters: a wider cephalotorax (5.10-5.70 vs. 4.40-4.70), wider abdomen (4.52-4.84 vs. 3.94-4.35) a more robust chela (2.12-2.48 vs. 1.72-1.96) and a shorter merus (3.04-4.20 vs. 4.26-4.71) and mesial process of first pleopod of the form I male reaching distally well beyond the other elements. This study emphasizes the utility of using an integrative framework for species recognition in crayfish.

Multiple drivers of decline in the global status of freshwater crayfish (Decapoda: Astacidea).

Rates of biodiversity loss are higher in freshwater ecosystems than in most terrestrial or marine ecosystems, making freshwater conservation a priority. However, prioritization methods are impeded by insufficient knowledge on the distribution and conservation status of freshwater taxa, particularly invertebrates. We evaluated the extinction risk of the world's 590 freshwater crayfish species using the IUCN Categories and Criteria and found 32% of all species are threatened with extinction. The level of extinction risk differed between families, with proportionally more threatened species in the Parastacidae and Astacidae than in the Cambaridae. Four described species were Extinct and 21% were assessed as Data Deficient. There was geographical variation in the dominant threats affecting the main centres of crayfish diversity. The majority of threatened US and Mexican species face threats associated with urban development, pollution, damming and water management. Conversely, the majority of Australian threatened species are affected by climate change, harvesting, agriculture and invasive species. Only a small proportion of crayfish are found within the boundaries of protected areas, suggesting that alternative means of long-term protection will be required. Our study highlights many of the significant challenges yet to come for freshwater biodiversity unless conservation planning shifts from a reactive to proactive approach.

Montane and coastal species diversification in the economically important Mexican grasshopper genus Sphenarium (Orthoptera: Pyrgomorphidae).

The genus Sphenarium (Pyrgomorphidae) is a small group of grasshoppers endemic to México and Guatemala that are economically and culturally important both as a food source and as agricultural pests. However, its taxonomy has been largely neglected mainly due to its conserved interspecific external morphology and the considerable intraspecific variation in colour pattern of some taxa. Here we examined morphological as well as mitochondrial and nuclear DNA sequence data to assess the species boundaries and evolutionary history in Sphenarium. Our morphological identification and DNA sequence-based species delimitation, carried out with three different approaches (DNA barcoding, general mixed Yule-coalescent model, Bayesian species delimitation), all recovered a higher number of putative species of Sphenarium than previously recognised. We unambiguously delimit seven species, and between five and ten additional species depending on the data/method analysed. Phylogenetic relationships within the genus strongly support two main clades, one exclusively montane, the other coastal. Divergence time estimates suggest late Miocene to Pliocene ages for the origin and most of the early diversification events in the genus, which were probably influenced by the formation of the Trans-Mexican Volcanic Belt. A series of Pleistocene events could have led to the current species diversification in both montane and coastal regions. This study not only reveals an overlooked species richness for the most popular edible insect in Mexico, but also highlights the influence of the dynamic geological and climatic history of the region in shaping its current diversity.

Genetic diversity and population history of the endangered killifish Aphanius baeticus.

The secondary freshwater fish fauna of the western-Iberian Peninsula basin is primarily restricted to local coastal streams, and man-made salt evaporation ponds, etc., which are susceptible to periodical flood and drought events. Despite its uniqueness in ecological adaptation to high saltwater tolerance, very little is known about this fauna's population dynamics and evolutionary history. The killifish, Aphanius baeticus (Cyprinodontidae) is an endemic species restricted to river basins on Spain's southern Atlantic coastline, considered as "Endangered." In this study, the genetic structure, diversity and historical demography of A. baeticus were analyzed using mitochondrial (cytochrome b, N=131) and nuclear (4 out of 19 microsatellites tested, N=288) markers across its distribution range. The phylogenetic and networking reconstruction revealed subtle phylogeographic structuring. A scattered expansion at the beginning of the interglacial periods, coupled with posterior events of extinction and colonization caused by periodical cycles of flooding, could explain the absence of well-defined phylogenetic relationships among populations. Moreover, very low genetic diversity values and a weak population differentiation were detected. We proposed that dispersals allowed by periodic floods connecting river drainages may have promoted a wide genetic exchange among populations and could have contributed to the current genetic relatedness of these populations.