Anatomy of Calliphora vicina (Diptera: Calliphoridae) larva and evaluation of Roundup Full II® effect on the development and morphology of this forensic species

Anatomía de la larva de Calliphora vicina (Diptera: Calliphoridae) y evaluación del efecto de Roundup Full II® sobre el desarrollo y morfología de esta especie forense

One of the approaches of forensic entomotoxicology is to study the effect that different xenobiotics have on the development of insects of forensic interest (George et al., 2009; Zanetti et al., 2021; Zanetti & Centeno, 2023). Forensic entomotoxicology can contribute to the estimation of the postmortem interval (PMI), it can determine the cause of death or provide related information, and even verify if the corpse has been moved or its origin (Zanetti et al., 2021; Zanetti & Centeno, 2023).

The Calliphoridae family is capable of being the first colonizer of a corpse (Byrd & Castner, 2009). The genus Calliphora includes the cosmopolitan species Calliphora vicina Robineau-Desvoidy, 1830 which has a wide distribution in Argentina and is very common in the province of Buenos Aires (Mariluis & Mulieri, 2003). It is usually found from autumn to spring in both rural and synanthropic environments (Battan Horenstein et al., 2007), showing a preference for the latter. In addition to its forensic importance, the species also has veterinary and medical value, since it can cause facultative myiasis in vertebrates and can be a mechanical vector of viruses, bacteria, fungi, protozoa and helminths (Maldonado & Centeno, 2003; Byrd & Castner, 2009; Araghi et al., 2015). The blue bottle fly also fulfills an ecological function due to its role in the decomposition of corpses in the environment and in the recycling of nutrients (Byrd & Castner, 2009). Due to its importance, the development of C. vicina has been studied under different variables that could affect its growth rate; one of them are toxic substances or xenobiotics that can cause death (O'Brien & Turner, 2004; Zanetti & Centeno, 2023).

Glyphosate (N-phosphonomethylglycine) is a broad-spectrum post-emergence herbicide. The company Monsanto markets it under the name Roundup® (Agostini et al., 2020) and is currently the most used pesticide worldwide and in Argentina (Mesnage et al., 2015; Anguiano & Ferrari, 2019). There is current evidence of adverse effects of both glyphosate-based herbicides (in addition to the main ingredient, they have other substances such as salts and adjuvants such as ethoxylated alkylamines (POEA), whose function is to facilitate their handling or increase their effectiveness) and glyphosate per se on non-target organisms, such as neurological, teratogenic, mutagenic, carcinogenic, immunological, respiratory and reproductive effects (Mesnage et al., 2015; Cortina et al., 2017; Anguiano & Ferrari, 2019; Agostini et al., 2020). Glyphosate poisoning can be accidental or voluntary (Bradberry et al., 2004). There are reports in Asia, Central and South America that indicate that the greatest poisonings with pesticides as a suicidal method occur mainly in countries dedicated to agricultural activities (Ajdacic-Gross et al., 2008; Lee et al., 2015; Anchía-Jiménez et al., 2021). The symptoms caused by poisoning are very diverse such as vomiting, abdominal pain, gastrointestinal infections, etc., and as pointed above mortality (Chen et al., 2009). It must be taken into account that information on suicides is scarce and/or difficult to access and therefore the real number of cases may be underestimated (Arias & Blanco, 2010; Fritschy, 2012; UNICEF, 2019).

Some works have studied the effects of glyphosate on arthropods. Among them, the chronic and acute toxicity of glyphosate and/or its formulations in crustaceans was evaluated (Gill et al., 2018). Schneider et al. (2009) analyzed in Chrysoperla externa Hagen, 1861 (Neuroptera: Chrysopidae) the alterations that glyphosate (at the maximum nominal concentration recorded in the field) could cause in population, reproductive and developmental parameters. In another work, the possible consequences of glyphosate on the life table of Metopolophium dirhodum Walker, 1849 (Hemiptera: Aphididae) were studied (Saska et al., 2016). Zanetti & Centeno (2024) studied the effect of glyphosate, using Roundup Full® II (RFII) formulation, on the development of Dermestes maculatus DeGeer, 1774 (Coleoptera: Dermestidae) and Lucilia sericata Meigen, 1826 (Diptera: Calliphoridae) under controlled conditions. Then, the anatomy of hide beetles under RFII was evaluated by Zanetti (2024).

Morphology is the branch of biology that studies the form and composition of body parts, and is the bases of various fields in biology, such as taxonomy and evolutionary studies (Rocha et al., 2020). Morphology has different divisions and one of them is anatomy. The anatomy of tissues and organs of organisms is part of the species description. Several studies highlight the importance of having detailed knowledge of immature stages in order to correctly identify them, particularly for use in forensic entomology (Grzywacz, 2013; Mendonça et al., 2014), given the applicability of the area or discipline (to use them as the entomological evidence). Moreover, the anatomical study allows evaluating the toxic effect of xenobiotics on the structures (Latif et al., 2013), as well as it is important to be able to understand physiology and carry out studies on it.

The objectives of this work were to characterize the macroscopic and microscopic anatomy of fat bodies, the alimentary canal and the excretory systems of C. vicina larva, as well as the effect of RFII on these structures. Also, possible alterations on the development and external morphology of this species under RFII exposure were evaluated.

It is relevant to claim the use and study of C. vicina as a species of forensic interest. Calliphora vicina larvae have been collected from corpses resulting from drug-related suicides (Sadler et al., 1995). Half of the collected larvae were used as such and the other half were incubated until pupation, to carry out toxicological studies that provided scientific information that also served to resolve forensic cases (Introna et al., 2001).

The microscopic anatomical characteristics of C. vicina presented similarities with those of Lucilia cuprina Wiedemann, 1830 and Chrysomya megacephala Fabricius, 1794 (Diptera: Calliphoridae). In these species four gastric caeca were found at the base of the proventriculus and the beginning of the midgut (Waterhouse & Stay, 1955; Boonswrong et al., 2011; Yasmeen & Amir, 2018). This digestive region in these species is formed by a layer of large cells with a notable basal membrane and a brush border on its surface surrounded by an internal layer of circular muscle and an external layer of longitudinal muscle. A peritrophic membrane was also found in the midgut of L. cuprina and Ch. megacephala (Waterhouse & Stay, 1955; Boonswrong et al., 2011; Yasmeen & Amir, 2018). The mentioned characteristics were also recorded in C. vicina. Also, in this species, small cells were also found alone or in clusters along the midgut at the base of the epithelium. These were also observed in L. cuprina by Waterhouse & Stay (1955) who suggested that those cells made up the imaginal midgut, although no regenerative cells were found (Waterhouse & Stay, 1955). Contrary to what was reported by these authors for L. cuprina, in C. vicina no difference was observed in the cell types or in the characteristics of such cells (presence of vacuoles, arrangement of the nucleus, the border in brush, etc.) depending on the midgut region. Yasmeen & Amir (2018) described the epithelium as cuboidal, with vacuolization and a large nucleus in the center of the cytoplasm. Hobson (1931) reported the absence of vacuoles in the midgut; they were only found in this intestine in the transition zone to the hindgut. The difference observed could also be a result of the methodology used by the latter author.

Regarding the foregut and hindgut, large highly vacuolated cells were also observed in L. sericata (Hobson, 1931), as was observed in C. vicina. According to Hobson (1931), the presence of fat coincided with vacuolization and only occurred in the anterior and posterior regions of fed larvae and could be a temporary storage product. Hobson (1931) also reported that in Lucilia the cells in the gastric diverticula are similar to those in the anterior segment but without vacuoles; however, the cells immediately adjoining the opening into the lumen of the gut are vacuolated. These results matched with what was observed in C. vicina. Salivary glands of C. vicina are similar to those in Ch. megacephala which are large long tubes (Boonswrong et al., 2011).

The excretory system in L. cuprina and Ch. megacephala is made up of two pairs of Malpighian tubules near the end of the midgut (Waterhouse & Stay, 1955; Boonswrong et al., 2011; Yasmeen & Amir, 2018). The same was found in C. vicina. Likewise, Vieira & Lello (1996) indicated that the rosary appearance of the tubules is due to the alternating arrangement of giant cells that are located on the monolayer wall of cobblestones. In L. cuprina, the tubules have a series of circular and longitudinal muscles and the epithelial cells have microvilli and a notable basement membrane (Begum et al., 2012). These characteristics were also recorded in C. vicina.

The duration of the development stages was not affected by RFII, similar results were reported in a previous work where the effect of RFII on L. sericata was evaluated (Zanetti & Centeno, 2024). On the contrary, the duration of dermestids exposed to RFII showed a reduction in larval development that translated into a reduction in total development (Zanetti & Centeno, 2024). A study performed on a Chrysopidae species found that development from third larval instar to pupae was shorter in glyphosate-treatment than in the control (Schneider et al., 2009). In contrast, other organophosphates that have been studied in various insects of relevance in forensic cases can delay their life cycle. Abd Al Galil et al. (2021a, b) reported that dimethoate presents a significant negative correlation with the larval and pupal development rates of Chrysomya megacephala Fabricius, C. saffranea Bigot, C. rufifacies Macquart, Sarcophaga peregrine Robineau-Desvoidy, S. dux Thomson, and S. ruficornis Fabricius. Other authors obtained similar results when working with malathion and C. megacephala larvae and pupae (Rashid et al., 2008; Liu et al., 2009). This organophosphate also affected the larval development rate in a species of dipteran of the Phoridae family (Castillo-Alanis et al., 2022). When studying the effect of glyphosate on a species of Chrysopidae, it was observed that the duration of the third larval stage to pupa was shorter in the presence of the herbicide than in the control (Schneider et al., 2009). There are different factors such as the animal species, the concentration of the xenobiotic, dose, stage of development, variability between individuals, pharmacokinetics, etc., which may be the cause of the differences mentioned above (Introna et al., 2001; Avigliano et al., 2014; Zanetti et al., 2021; Castillo-Alanis et al., 2022; Zanetti & Centeno, 2024).

There were also no changes in size due to the RFII in C. vicina. The same was found in D. maculatus (Zanetti & Centeno, 2024). Likewise, there were no external morphological changes in these species caused by RFII. In contrast, in eggs of Crysoperla externa Hagen, 1861 treated with glyphosate abnormalities were evident and adults also developed tumors (Schneider et al., 2009). An explanation similar to that given for cycle length can be applied to size.

When comparing with Schneider et al. (2009), in addition to the fact that the species evaluated are distinct, the preparation of the herbicide was different since authors made solutions considering the maximum nominal concentration recorded in the field, with which they sprayed the food source or diet. In our work, no solutions were prepared but the RFII was purely added to the meat and water mixture. The concentration and doses used were also different from those of Schneider et al. (2009) since we calculated doses taking into account forensic cases (doses that could cause the death of a person, for example a suicide). Furthermore, some aims differed between studies, as well as the behavior of the evaluated insect, its habitat, etc.

Regarding the glyphosate detoxification process in C. vicina, it is likely to occur through the Malpighian Tubules. Some studies that analyzed larvae of Calliphoridae fed on corpses resulting from suicides with different drugs such as amitriptyline, temazepam, among others or a combination of them, indicated that the drugs are efficiently eliminated through the Malphigian Tubules of the dipteran larvae (Introna et al., 2001). Likewise, Farina et al. (2022) indicated that Malphigian Tubules can mitigate the toxic activity of insecticides. Another possible way of drug elimination is through the intestine (Gosselin et al., 2011). Maybe this also occurs with glyphosate.

In conclusion, this work provides novel anatomical data on these forensically relevant fly species and demonstrates that exposure to the RFII compound does not affect lifespan or alter morphological structures in these organisms. Understanding internal morphology is not only important for studying physiological and evolutionary aspects of the species, but also highlights the potential of these flies as model organisms for testing other chemical compounds and exploring toxicological responses in Diptera.

redalyc-journal-id: 3220

This study was funded by the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación [PICT 02992], the Consejo Nacional de Investigaciones Científicas y Técnicas [PIBAA 28720210100376CO], Universidad Nacional de Quilmes, and the Universidad Nacional del Sur. The authors acknowledge to Lic. Ricardo Camina for helping with the statistical analysis and Professor Dr. Natalia Bulnes (Universidad Nacional del Sur, Province of Buenos Aires) for providing the equipment for the macroscopic analysis