Midgut proteins released by microapocrine secretion in Spodoptera frugiperda

Abstract

Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

Insect midgut carboxypeptidases with emphasis on S10 hemipteran and M14 lepidopteran carboxypeptidases

“…5A is very rare. In accordance with this conclusion, the protein coded by SfCPA is absent from the list of proteins secreted by microapocrine secretion by Silva et al (2013). Table 2 lists the features of the carboxypeptidases in family S28.…”

“…3 shows that all carboxypeptidase As of S. frugiperda are expressed only in the midgut. Two contigs (Sf3308, Sf3681) correspond to carboxypeptidases found in highly purified midgut microvillar membranes from S. frugiperda (Silva et al, 2013), shown to be coded by contigs there named 418 and 492. As only contig Sf3308 has a predicted GPI-anchor (Table 1) and none of them has a predicted transmembrane helix (Table 1), it is not known how the protein coded by contig Sf3681 is so strongly associated with the microvillar membrane.…”

The roles of mucus‐forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut

“…The amount of immobilized enzymes on the PM is significant in S. frugiperda : 13 and 18% of the midgut luminal activity of amylase and trypsin, respectively (Ferreira et al ., ). The enzymes preferentially retained in the PM jelly (now proposed to be a mucus layer) are those released by the microapocrine process (details in Silva et al ., ), especially if still bound to membranes (Bolognesi et al ., ).…”

Gelsolin role in microapocrine secretion

“…Also recently, the immunoscreening of a Spodoptera frugiperda expression midgut cDNA library with antibodies against isolated microapocrine vesicle proteins was performed (Silva et al ., ). The sequences obtained, together with data obtained by pyrosequencing S. frugiperda midgut RNA, were used to identify the proteins secreted and those putatively involved in the secretory machinery.…”

“…26077, 05513-970, Brazil. Fax: 1 55 11 3091 2186; e-mail: clfterra@iq.usp.br performed (Silva et al, 2013). The sequences obtained, together with data obtained by pyrosequencing S. frugiperda midgut RNA, were used to identify the proteins secreted and those putatively involved in the secretory machinery.…”