In-depth study of spermatophores and reproductive aspects of the copepod Notodiaptomus spinuliferus (Dussart & Matsumura-Tundisi, 1986) using diverse imaging and molecular biology techniques.

• Grant number: 24/21625-3
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: May 01, 2025
• End date: August 22, 2025
• Field of knowledge: Biological Sciences - Zoology - Applied Zoology
• Principal Investigator: Gilmar Perbiche Neves
• Grantee: Paulo Henrique Cardilli Hor
• Supervisor: Nancy Fabiola Mercado Salas
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Institution abroad: Leibniz Institute For The Analysis Of Biodiversity Change, Hamburg, Germany
• Associated to the scholarship: 24/08354-0 - Understanding the morphology and ultrastructure of spermatophores from the copepod Notodiaptomus spinuliferus (Dussart & Matsumura-Tundisi, 1986), BP.IC

Abstract

The project aims to investigate the morphology, ultrastructure, and reproductive aspects of the spermatophores of the species Notodiaptomus spinuliferus, a freshwater copepod widely distributed in the state of São Paulo, Brazil. Although there are many studies on copepod morphology, there is a significant scarcity of research focused on reproductive structure, especially in freshwater environments. To fill this gap, the project adopts a multidisciplinary approach that combines advanced microscopy techniques and molecular analyses.Samples of N. spinuliferus will be collected from various aquatic environments in São Paulo state, such as lakes and reservoirs, with the geographical coordinates of collection sites being recorded to ensure traceability. The samples will be prepared for analysis using different fixation solutions depending on the technique to be applied. For micro-computed tomography (MicroCT), samples will be fixed in 96% alcohol, while absolute alcohol will be used for molecular analyses. For laser confocal microscopy, samples will be fixed in formalin or glycerin with alcohol.External and internal morphological analyses of the spermatophores will be conducted using common optical microscopy and transmission electron microscopy to observe the anatomy and ultrastructure of the spermatophores. Micro-computed tomography will be employed to obtain three-dimensional images of specimens without damaging them, facilitating the understanding of the mechanism of spermatophore fixation in females. Additionally, images will also be obtained using synchrotron X-ray microtomography in partnership with the Karlsruhe Institute of Technology (KIT), where specialists will assist in obtaining and analyzing images.The laser confocal microscopy technique will be utilized to study the adhesion mechanism of the spermatophore to the female's genital segment by employing various dyes to facilitate identification of different structures. Molecular analyses will seek to verify whether there is interspecific reproductive behavior among copepods and catalog genetic material from the species that has not yet been recorded. To this end, ribosomal genes 18S and 28S as well as COI (mitochondrial DNA) will be analyzed. Genetic material will be extracted using specific kits and amplified through Polymerase Chain Reaction (PCR) technique, with results organized for sequencing and deposited in GenBank.The results obtained will be analyzed integratively and comparatively by gathering information from various applied techniques. Three-dimensional models generated from scanning histological slides, MicroCT data, and synchrotron X-rays will be compared to ensure a comprehensive view of the morphological characteristics of specimens. Special focus will be given to the genital segment and spermatophores while molecular data will undergo statistical treatment to verify compatibility of found DNA with N. spinuliferus, also checking for interespecific reproductive patterns.