Costa, M. M. | bioRxiv Science

bioRxiv2024

Cryopreservation Enables Long-Term Studies of Octopus vulgaris Hemocyte Immune Functions

As with their nervous system and other physiological traits, the immune system of cephalopods, in general, and of the common octopus, Octopus vulgaris, in particular, could also present highly evolved characteristics compared to other classes of molluscs. However, to date, there is not much information about it, and studying the defense mechanisms is a key step in understanding their response system to external aggressions and, thus, having the tools to anticipate animal health problems and ensure their welfare. The lack of cell cultures in molluscs is a major problem when carrying out in vitro assays that help to deepen the knowledge of the main immune cells of this species. Cryopreservation becomes an alternative to maintain viable and functional cells after freezing/thawing processes, allowing a larger repertoire of studies to be performed with the same sample or to perform time courses. Having access to good quality cells for long periods will allow to cover a larger repertoire of studies with the same sample or to perform time courses, avoiding logistic bottlenecks that could arise due to the loss of viability and/or functionality of the cells on the lab bench, the lack of time to develop all the desired assays in the same day or the possible losses derived from transporting the samples between different laboratories. Additionally, high-quality suspensions of viable and functional cells are required for successful massive sequencing studies, such as single-cell analysis, where these aspects are the key to an optimal identification. We show here the first functional results from cryopreserved octopus hemocytes, where the cells were able to maintain viability above 80% after two months post cryopreservation and storage at -80{degrees}C and their functional ability to phagocytose bacteria similar to fresh cells. Our data revealed that the acclimation process after thawing was essential to recovering the functional activity of the cells. The results presented here will facilitate the study of the functions of the most important immune cells of this species and will provide tools for cell preservation in other molluscs species.

immunology↗

bioRxiv2023

Potential of MALDI-TOF MS biotyping to detect deltamethrin resistance in the dengue vector Aedes aegypti.

Insecticide resistance in mosquitoes is spreading worldwide and represents a growing threat to vector control. Insecticide resistance is caused by different mechanisms including higher metabolic detoxication, target-site modification, reduced penetration and behavioral changes that are not easily detectable with simple diagnostic methods. Indeed, most molecular resistance diagnostic tools are costly and labor intensive and then difficult to use for routine monitoring of insecticide resistance. The present study aims to determine whether mosquito susceptibility status against the pyrethroid insecticides (mostly used for mosquito control) could be established by the protein signatures of legs and/or thoraxes submitted to MALDI-TOF Mass Spectrometry (MS). The quality of MS spectra for both body parts was controlled to avoid any bias due to unconformity protein profiling. The comparison of MS profiles from three inbreeds Ae. aegypti lines from French Guiana (IRF, IR03, IR13), with distinct deltamethrin resistance genotype / phenotype and the susceptible reference laboratory line BORA (French Polynesia), showed different protein signatures. On both body parts, the analysis of whole protein profiles revealed a singularity of BORA line compared to the three inbreeding lines from French Guiana origin, suggesting that the first criteria of differentiation is the geographical origin and/or the breeding history rather than the insecticide susceptibility profile. However, a deeper analysis of the protein profiles allowed to identify 10 and 11 discriminating peaks from leg and thorax spectra, respectively. Among them, a specific peak around 4870 Da was detected in legs and thoraxes of pyrethroid resistant lines compared to the susceptible counterparts hence suggesting that MS profiling may be promising to rapidly distinguish resistant and susceptible phenotypes. Further work is needed to confirm the nature of this peak as a deltamethrin resistant marker and to validate the routine use of MS profiling to track insecticide resistance in Ae. aegypti field populations. Author SummaryThe monitoring of mosquito insecticide resistance in local populations is essential to guide the choice of the vector control strategy. Current methods for resistance monitoring rely on biological, biochemical and molecular assays that all have their weakness. To circumvent these limitations, alternative methods have to be explored. In previous studies, MALDI-TOF MS profiling have proved it performance to classify mosquitoes at the species and sub-species levels. The present work aim was to assess whether MALDI-TOF MS profiling strategy could be useful for determination of mosquito susceptibility to the most used pyrethroid insecticide. In this way, four mosquito lines with distinct deltamethrin resistance genotype / phenotype were submitted to MS analysis. The accurate comparison of MS spectra showed different peak intensities between mosquitoes exhibiting different insecticide resistance profiles. Among discriminant peaks, one may be promising to detect insecticide-resistance mechanisms in public health mosquitoes. A better characterization of mosquito life traits will help countries to implement timely and locally adapted vector control interventions.

bioengineering↗