Las 4

Ten-day-old adult female cockroaches were dissected under an Olympus SZ61 stereo microscope. Individuals were anesthetized using a CO₂ stream, and cleaned with different solutions: bleach 10%, ethanol 70%, and two passages in the type II sterile water. Then, they were fixed with pins in a dorsal position on a silicone plate. They were ventrally opened via a longitudinal incision in the abdomen, and hindguts and fat bodies were extracted and gently washed with Krebs–Ringer Bicarbonate Buffer (Sigma-Aldrich, Burlington, MA, USA). Hindguts were immediately fixed for electron microscopy, and fat body samples were frozen in liquid nitrogen and stored at −80 °C until use for DNA extraction and acid uric determination. For in vivo visualization, ventrally-opened individuals were photographed using a Leica Z16 microscope equipped with a CF500 camera and LAS 4.9 (Leica, Wetzlar, Germany) software. Z-stacks, followed by extended depth of field application, were extensively used to produce final images.

Individuals preserved in 70% ethanol were cleared using KOH and acetic acid and mounted in slides with Canada balsam. The morphological identification of the specimens was done using a Leica Z16 microscope. We measured structures with taxonomic value and used the keys from Miller and Halbert (2014) (link) and Blackman and Eastop (2019) to identify species of Neophyllaphis. The photographs were taken with a Leica Z16 microscope, equipped with a CF500 camera and LAS 4.9 (Leica) image capture. Mounted specimens were deposited at the aphid collection of the Instituto de Biología Integrativa de Sistemas (Centro Mixto Universidad de Valencia-CSIC, Spain) and in the Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica.

The initial sampling was conducted after the acclimatization period (22 dph), prior to the beginning of the experiment. From each tank, 25 larvae were randomly collected and sacrificed with ice-cold water. A subsample of twenty larvae was then measured for total length under a microscope (Leica DMC2900, Wetzlar, Germany) using the LAS 4.5 software (Leica, Wetzlar, Germany). After measurements, a subsample of 15 larvae was placed in 3 groups of 5 larvae on cover glasses previously weighed to determine the wet weight. The glasses were then placed in a laboratory oven at 60 °C for 48 h to determine the dry weight. In addition, 5 larvae were also placed in RNAlater solution, stored at 4 °C for one week, and then frozen at −80 °C until gene expression analyses.
The same procedure was also used to sample larvae at 29 dph (intermediate sampling) and 36 dph (final sampling). At the latter sampling point, a pool of 17 larvae from each tank was also collected for biochemical analyses, frozen at −80 °C, and subsequently lyophilized until analyses could be conducted.
Furthermore, 3 different samples of enriched rotifers and Artemia sp. metanauplii were collected, frozen at −80 °C, and lyophilized until biochemical analyses. Samples of the inert diets were also stored at −80 °C until analyses. The biochemical and fatty acid composition of the diets is presented in Table 1.